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Western University Western University
Scholarship@Western Scholarship@Western
Electronic Thesis and Dissertation Repository
9-25-2018 2:00 PM
The Role of the HIV-1 Accessory Proteins Nef and Vpu in The Role of the HIV-1 Accessory Proteins Nef and Vpu in
Subverting Host Cellular Immunity Subverting Host Cellular Immunity
Emily N. Pawlak, The University of Western Ontario
Supervisor: Dikeakos, Jimmy D., The University of Western Ontario
A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree
in Microbiology and Immunology
© Emily N. Pawlak 2018
Follow this and additional works at: https://ir.lib.uwo.ca/etd
Part of the Virology Commons
Recommended Citation Recommended Citation Pawlak, Emily N., "The Role of the HIV-1 Accessory Proteins Nef and Vpu in Subverting Host Cellular Immunity" (2018). Electronic Thesis and Dissertation Repository. 5751. https://ir.lib.uwo.ca/etd/5751
This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected].
i
Abstract
The HIV-1 accessory proteins Nef and Vpu have no known enzymatic activity, yet these viral
proteins are critical for HIV-1 viral spread and pathogenicity. To mediate this, these viral
proteins bind to and hijack host cellular proteins, including proteins implicated in the
membrane trafficking machinery. Key to the effects of these proteins is their ability to alter the
subcellular localization of a plethora of cell surface receptors, including the key immune cell
receptors CD28 and MHC-I. However, the mechanism utilized by Nef and Vpu to modulate
these proteins is not fully understood. Herein, we demonstrated that Nef and Vpu mediate a
decrease in both cell surface and total CD28 protein levels in CD4+ T cells. We have implicated
the endosomal degradation machinery in this process, as we observed that in the presence of
these viral proteins CD28 localizes to LAMP-1 positive compartments in an endosomal
acidification-dependent manner. Moreover, in investigating the mechanisms utilized by Nef
and Vpu to mediate CD28 downregulation we implicated the Nef LL165 and DD175 and Vpu
S52/56 motifs, which interact with specific cellular membrane trafficking machinery, in CD28
downregulation. Furthermore, upon infection with viruses encoding or lacking Nef and Vpu,
we observed differential effects on cellular activation upon stimulation. In seeking to
understand the mechanism of MHC-I downregulation by Nef, we examined the roles of
membrane trafficking mediators in the sorting nexin family of proteins. Namely, we identified
a novel direct Nef binding partner, SNX18, and mapped this interaction with Nef to the SH3
domain of SNX18 and a polyproline PxxP75 motif on Nef. Additionally, using shRNA knock-
downs we have implicated SNX18 in Nef-mediated MHC-I downregulation, a key mechanism
for HIV-1 viral immune evasion. Overall, this work sheds light on the mechanism of Nef- and
Vpu-mediated mis-localization of host cellular proteins, processes key to the pathogenic
effects of these viral proteins.
Keywords
Human immunodeficiency virus type 1, Nef, Vpu, immune evasion, viral infection
ii
Co-Authorship Statement
All studies were completed by Emily N. Pawlak, with the exception of the work outlined
below. Jimmy Dikeakos contributed to the genesis, design, implementation and interpretation
of all experiments, as well as writing and manuscript preparation.
Chapter 2
Sections of Chapter 2 were previously published in:
The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+
T cells. Pawlak EN, Dirk BS, Jacob RA, Johnson AL and Dikeakos JD. Retrovirology 2018
15:6
Copyright of the work presented in this manuscript was retained by the authors as detailed in
Appendix 1.
Co-authors contributed to the writing of this manuscript as well as experimental work as
follows: The microscopy and corresponding analyses presented in Figure 2.9 A, B and Figure
2.10 B, C were completed and analyzed by Brennan S. Dirk. The experiments presented in
Figure 2.8 D, E; Figure 2.21 E, F, G; Figure 2.23 were completed and analyzed by Rajesh A.
Jacob.
Chapter 3
The work presented in Chapter 3 was completed by Emily N. Pawlak, with the exception of
the microscopy experiments presented in Figure 3.4 A, B and Figure 3.6 A, B which were
performed and analyzed by Logan R. Van Nynattan.
iii
Acknowledgments
First and foremost, I would like to acknowledge my supervisor Dr. Jimmy Dikeakos, without
whom this thesis would not have been possible. I will always be tremendously grateful you
took a chance on me as a clueless fourth-year student and gave me the opportunity to work in
your lab for all these years. Thank you for your guidance in the lab; from teaching me how to
run Western blots, to the use of a French Press, this has served me well. In addition to all that
you have taught me about science, I will also take away with me a wealth of knowledge
regarding life in general.
Secondly, I would like to recognize my advisory committee members, Drs. Joe Mymryk and
Lackshman Gunaratnam, for giving me their time and constructive feedback. This has been
critical in helping me produce this thesis.
Finally, to all the Dikeakos lab members, I could not have asked for a better group of people
to work with! Additionally, I would like to thank Aaron, Brennan, Logan and Rajesh, who all
contributed to the work within this thesis - your respective expertise is what made this possible.
iv
Table of Contents
Abstract ................................................................................................................................ i
Co-Authorship Statement.................................................................................................... ii
Acknowledgments.............................................................................................................. iii
Table of Contents ............................................................................................................... iv
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
List of Appendices ............................................................................................................ xii
List of Abbreviations ....................................................................................................... xiii
Chapter 1 ............................................................................................................................. 1
1 Introduction .................................................................................................................... 1
1.1 General thesis overview .......................................................................................... 1
1.2 Introduction to lentiviruses ..................................................................................... 1
1.3 Biology of HIV-1 .................................................................................................... 4
1.3.1 Clinical course of infection ......................................................................... 4
1.3.2 HIV-1 virions and replication cycle ............................................................ 5
1.4 HIV-1 accessory proteins ........................................................................................ 8
1.4.1 HIV-1 Vpr and Vif ...................................................................................... 8
1.4.2 HIV-1 Nef and Vpu .................................................................................... 9
1.5 Biological effects of HIV-1 Nef and Vpu ............................................................... 9
1.5.1 Evasion of cell-mediated adaptive immune responses ............................. 13
1.5.2 Evasion of humoral and innate immune responses ................................... 14
1.5.3 Alterations in immune cell homing........................................................... 15
1.5.4 Counteraction of host cell restriction factors ............................................ 16
1.5.5 Modulation of immune cell activation ...................................................... 17
v
1.5.6 Modulation of cell death pathways ........................................................... 22
1.5.7 Functional redundancy of HIV-1 Nef and Vpu ........................................ 23
1.5.8 The importance of Nef and Vpu in vivo.................................................... 25
1.6 How do HIV-1 Nef and Vpu mediate their functions? ......................................... 25
1.6.1 Nef: host cell protein interactions ............................................................. 33
1.6.2 Nef oligomerization .................................................................................. 38
1.6.3 Vpu oligomerization ................................................................................. 39
1.6.4 Vpu: host cell protein interactions ............................................................ 39
1.7 Thesis overview .................................................................................................... 43
1.7.1 Chapter 2 ................................................................................................... 44
1.7.2 Chapter 3 ................................................................................................... 44
1.7.3 Significance............................................................................................... 45
1.8 References ............................................................................................................. 45
Chapter 2 ........................................................................................................................... 73
2 The downregulation of CD28 by HIV-1 Nef and Vpu ................................................ 73
2.1 Introduction ........................................................................................................... 73
2.2 Results ................................................................................................................... 75
2.2.1 The HIV-1 accessory proteins Nef and Vpu downregulate cell surface and
total CD28 protein levels .......................................................................... 75
2.2.2 Nef and Vpu-mediated effects on CD28 are dependent on the cellular
degradation machinery .............................................................................. 91
2.2.3 Motifs ascribed to specific Nef: host cell protein interactions are critical for
Nef-mediated CD28 downregulation ........................................................ 96
2.2.4 Motifs in Vpu implicated in interactions with specific host cellular proteins
are critical for CD28 downregulation ..................................................... 102
2.2.5 Implicating host cell proteins in Nef- and Vpu-mediated CD28
downregulation ....................................................................................... 107
2.2.6 Patient-derived Vpu proteins mediate CD28 downregulation ................ 114
vi
2.2.7 Nef and Vpu differentially alter responsiveness to CD28-mediated
stimulation............................................................................................... 119
2.3 Discussion ........................................................................................................... 122
2.4 Materials and Methods ........................................................................................ 130
2.4.1 DNA constructs ....................................................................................... 130
2.4.2 Cell culture .............................................................................................. 132
2.4.3 Transfections and Infections ................................................................... 133
2.4.4 Flow cytometry analysis of cell surface receptors .................................. 135
2.4.5 Protein analysis ....................................................................................... 136
2.4.6 Microscopy ............................................................................................. 137
2.4.7 Cell activation analysis ........................................................................... 138
2.4.8 Data and Statistical analysis.................................................................... 139
2.5 References ........................................................................................................... 140
Chapter 3 ......................................................................................................................... 149
3 Defining the role of the novel interaction between HIV-1 Nef and the membrane
trafficking protein SNX18.......................................................................................... 149
3.1 Introduction ......................................................................................................... 149
3.2 Results ................................................................................................................. 151
3.2.1 SNX18 and Nef interact in vitro through a SNX18 SH3 domain: Nef PxxP
motif interaction ...................................................................................... 151
3.2.2 SNX18 and Nef interact in cells ............................................................. 158
3.2.3 SNX18 is required for Nef-dependent reductions in cell surface MHC-I
................................................................................................................. 161
3.2.4 SNX18 is unlikely to be required for Nef-mediated secretion in extracellular
vesicles .................................................................................................... 164
3.3 Discussion ........................................................................................................... 170
3.4 Material and Methods ......................................................................................... 175
3.4.1 DNA vectors ........................................................................................... 175
vii
3.4.2 Bacterial protein expression and purification ......................................... 176
3.4.3 Protein capture assay............................................................................... 177
3.4.4 Cell culture .............................................................................................. 178
3.4.5 Transfections and virus production ......................................................... 178
3.4.6 Western blot analysis .............................................................................. 178
3.4.7 Microscopy ............................................................................................. 179
3.4.8 Extracellular vesicle purification ............................................................ 180
3.4.9 Transient shRNA knock-down experiments ........................................... 181
3.4.10 Flow cytometry ....................................................................................... 181
3.4.11 Data analysis ........................................................................................... 181
3.5 References ........................................................................................................... 183
Chapter 4 ......................................................................................................................... 188
4 General Discussion..................................................................................................... 188
4.1 Thesis overview .................................................................................................. 188
4.1.1 Chapter 2: Findings, limitations and future work ................................... 188
4.1.2 Chapter 3: Findings, limitations and future work ................................... 196
4.2 Significance......................................................................................................... 200
4.2.1 Implications for HIV-1 treatment ........................................................... 200
4.2.2 Concluding remarks ................................................................................ 202
4.3 References ........................................................................................................... 203
Curriculum Vitae ............................................................................................................ 210
viii
List of Tables
Table 1 The HIV-1 genes and their encoded proteins .............................................................. 7
ix
List of Figures
Figure 1.1 HIV-1 genome organization .................................................................................... 6
Figure 1.2 Immune cell surface proteins that are targeted by the HIV-1 accessory proteins Nef
and Vpu ................................................................................................................................... 11
Figure 1.3 Schematic of select molecules found in the immunological synapse .................... 19
Figure 1.4 Structure of HIV-1 Nef and Vpu and motifs that interact with host cell proteins . 28
Figure 1.5 The endosomal trafficking network....................................................................... 30
Figure 1.6 General mechanism of vesicle transport................................................................ 31
Figure 2.1 HIV-1 Nef and Vpu downregulate cell surface CD28 protein levels .................... 76
Figure 2.2 Gating of live infected Sup-T1 cells infected with Gag-Pol truncated VSV-G
pseudotyped NL4.3 ................................................................................................................. 78
Figure 2.3 Analysis of primary CD4+ T cells infected with replication competent virus ...... 79
Figure 2.4 Schematic of CD28-FLAG construct utilized to overexpress CD28 in cells ........ 81
Figure 2.5 Decreased CD28-FLAG protein levels are detected in the presence of Nef in HeLa
cells ......................................................................................................................................... 82
Figure 2.6 HIV-1 lentiviral vector utilized to express epitope tagged CD28 in the presence or
absence of Nef......................................................................................................................... 83
Figure 2.7 CD28-FLAG protein levels are reduced in the presence of Nef in CD4+ Sup-T1 T
cells ......................................................................................................................................... 84
Figure 2.8 HIV-1 Nef and Vpu downregulate total CD28 protein levels ............................... 86
Figure 2.9 The subcellular localization of CD28 is altered in the presence of Nef or Vpu .... 89
x
Figure 2.10 Inhibition of the degradation machinery increases CD28 protein levels in infected
cells ......................................................................................................................................... 93
Figure 2.11 Ammonium chloride treatment increases total CD4 levels in infected cells ...... 95
Figure 2.12 Gating of Sup-T1 cells infected with VSV-G pseudotyped NL4.3 encoding various
Nef mutants ............................................................................................................................. 98
Figure 2.13 Nef: host protein interaction motifs are critical for Nef-mediated CD28
downregulation in the presence of Vpu .................................................................................. 99
Figure 2.14 Nef: host cell protein interaction motifs are critical for Nef-mediated CD28
downregulation ..................................................................................................................... 101
Figure 2.15 Motifs in Vpu are critical for downregulation of CD28 .................................... 105
Figure 2.16 Specific motifs in Vpu are critical for downregulation of CD4 ........................ 106
Figure 2.17 Downregulation of cell surface CD28 by Nef and Vpu in the presence of shRNA
targeting AP-2 ....................................................................................................................... 108
Figure 2.18 Nef-mediated cell surface downregulation of CD28 in the presence of
overexpressed wild-type or dominant negative non-functional SNX9 ................................. 110
Figure 2.19 HeLa cells with decreased SNX9 expression exhibit reductions in Nef-mediated
decreases in total CD28-FLAG ............................................................................................. 112
Figure 2.20 SNX9-targeting shRNA reduces Nef-mediated decreases in total CD28 in Sup-T1
cells ....................................................................................................................................... 113
Figure 2.21 Non-NL4.3 Vpu proteins downregulate cell surface CD28 .............................. 116
Figure 2.22 Gating of CD4+ peripheral blood mononuclear cells infected with VSV-G
pseudotyped NL4.3 ............................................................................................................... 118
Figure 2.23 Infected cell response to CD28-stimulation changes in the absence of Nef or Vpu
............................................................................................................................................... 120
xi
Figure 2.24 Protein sequence alignment of the Vpu proteins which were tested for their ability
to downregulate CD28 .......................................................................................................... 127
Figure 3.1 GST-SNX18, but not GST-SNX9, pulls down His6-Nef .................................... 152
Figure 3.2 The direct Nef: SNX18 interaction is dependent on the SH3 domain ................ 154
Figure 3.3 In vitro protein capture of His6-Nef and a Nef mutant, His6-Nef AxxA75, by the SH3
domain of SNX18 ................................................................................................................. 157
Figure 3.4 Mutation of the Nef PxxP75 motif reduces SNX18: Nef BiFC intensity ............. 159
Figure 3.5 Knock-down of SNX18 reduces Nef-mediated MHC-I downregulation ............ 162
Figure 3.6 The SNX18: Nef BiFC complex co-localizes with CD63................................... 165
Figure 3.7 Extracellular vesicles purified from Nef-expressing SNX18 knock-down cells . 168
Figure 3.8 Proposed model for the role of SNX18 in Nef-mediated MHC-I trafficking ..... 174
Figure 4.1 Models of Nef- and Vpu-mediated CD28 downregulation ................................. 194
Figure 4.2 Model of Nef-mediated evasion of cytotoxic T cell responses ........................... 199
xii
List of Appendices
Appendix 1 Copyright permission for published work ......................................................... 209
Appendix 2 Ethics statement ................................................................................................ 209
xiii
List of Abbreviations
ADAM A disintegrin and
metalloprotease
ADCC Antibody-dependent
cellular cytotoxicity
AIDS Acquired immune
deficiency syndrome
AP-1 Adaptor protein 1
AP-2 Adaptor protein 2
APCs Antigen presenting cells
APOBEC3 Apolipoprotein B mRNA
editing complex 3
Arf1 ADP-ribosylation factor 1
ATCC American Type Culture
Collection
BiFC Bimolecular fluorescence
complementation
BSA Bovine serum albumin
BST-2 Bone marrow stromal cell
antigen 2
COPI Coatomer protein I
CTL Cytotoxic T lymphocyte
DCs Dendritic cells
DMEM Dulbecco’s modified
Eagle’s medium
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic
acid
ER Endoplasmic reticulum
ERAD ER-associated degradation
ESCRT Endosomal sorting
complexes required for
transport
FBS Fetal bovine serum
GAPDH glyceraldehyde 3-
phosphate dehydrogenase
GFP Green fluorescent protein
HAART Highly active antiretroviral
therapy
xiv
HIV Human immunodeficiency
virus
HIV-1 Human immunodeficiency
virus type 1
HIV-2 Human immunodeficiency
virus type 2
HRS Hepatocyte growth factor-
regulated tyrosine kinase
substrate
HTLV Human T-leukemia virus
ICAM-1 Intracellular adhesion
molecule 1
IPTG Isopropyl β-D-1-
thiogalactopyranoside
LAMP-1 lysosomal-associated
membrane protein 1
LAP LC3-associated
phagocytosis
LAV Lymphadenopathy-
associated virus
LTRs Long terminal repeats
MFI Mean fluoresence intensity
MHC-I Major histocompatibility
complex class I
MHC-II Major histocompatibility
complex class II
MVBs Multivesicular bodies
N-WASP Neural Wiskott-Aldrich
syndrome protein
NAK Nef-associated kinase
NK Natural killer
PACS Phosphofurin acidic cluster
sorting
PAK2 p21 activated kinase
PBMCs peripheral blood
mononuclear cells
PBS Phosphate buffered saline
PHA Phytohemagglutinin
PI3K Phosphoinositide 3-kinase
RPMI Roswell Park Memorial
Institute media
SCF Skp Cullin F-box
containing
SD Standard deviation
SE Standard error
SERINC Serine incorporator
xv
SFKs Src family kinases
SIV Simian immunodeficiency
virus
SNX18 Sorting nexin 18
SNX9 Sorting nexin 9
TCR T cell receptor
TNFα Tumor necrosis factor-
alpha
v-ATPase vacuolar ATPase
Vif Viral infectivity factor
Vpu Viral protein U
VSV-G Vesicular stomatitis virus
envelope glycoprotein
β-TrCP β-transducin-repeat
containing protein
1
Chapter 1
1 Introduction
1.1 General thesis overview
The Oxford English Dictionary defines a virus as “an infectious, often pathogenic agent or
biological entity which is typically smaller than a bacterium, which is able to function only
within the living cells of a host animal, plant, or microorganism, and which consists of a
nucleic acid molecule (either DNA or RNA) surrounded by a protein coat, often with an
outer lipid membrane”[1]. Indeed, viruses require host cells they can infect, as they are
obligate intracellular parasites and require eukaryotic or bacterial host cells to replicate.
Thus, as will be highlighted in this thesis, viruses have evolved ingenious mechanisms to
make use of host cellular machinery to turn their hosts into efficient virus production
factories. Moreover, given their generally minute size, many viruses have small genomes
with limited coding capacity. Therefore, they must encode proteins which function in
numerous different ways to make the host a prime location for replication.
1.2 Introduction to lentiviruses
In the early 1980’s a new illness appeared in North America which baffled medical
professionals. This ailment was characterized by an increased incidence of opportunistic
infections and rare malignancies that resulted in a rapid death [2]. This illness was later
recognized as being caused by a severe immune deficiency, thus it was termed acquired
immune deficiency syndrome (AIDS) by the Centers for Disease Control. Originally, AIDS
was falsely believed to only affect men who have sex with men, however it was later proven
that this illness could be acquired through exposure to contaminated bodily fluids through
transfusion of blood products, heterosexual sex, and mother to child transmission during
childbirth or breastfeeding [3-7].
In 1983, Dr. Luc Montagnier’s laboratory at the Institut Pasteur in France isolated a virus
from the lymph node of a patient exhibiting lymphadenopathy. This patient was considered
high risk for developing the above mentioned novel immune deficiency syndrome [8].
Montagnier’s group named the isolated virus lymphadenopathy-associated virus (LAV)
2
and classified it as a retrovirus belonging to the human T-leukemia virus (HTLV) family.
These findings were published in Science concurrently with an article by Dr. Robert Gallo
and colleagues at the National Cancer Institute in Bethesda, Maryland. In the latter
publication, Gallo’s group identified a virus within an AIDS patient they believed to be
HTLV, thus they termed this virus HTLV-III [9].
The following year, Gallo and colleagues published a series of articles illustrating that this
novel virus was the causative agent of the acute immune deficiency termed AIDS [10-12].
By 1986, it was widely recognized that LAV and HTLV-III were the same virus, hence
this newfound consensus in the virology community resulted in the creation of a new virus
name: human immunodeficiency virus (HIV) [13]. In the three decades since its initial
discovery, this deadly virus has spread to millions of people globally [14], making it a
major area of research interest.
Extensive phylogenetic analysis of HIV viral genomes has demonstrated that the
transmission of HIV to humans occurred many years prior to the first documented cases of
AIDS, most likely around the 1930’s in what is now known as Kinshasa in the Democratic
Republic of the Congo [15-17]. Specifically, the epidemic is believed to have arisen from
transmission of the primate simian immunodeficiency virus (SIV) from an infected
chimpanzee to a human, likely through the act of bush meat hunting [18-20]. Interestingly,
phylogenetic studies suggest that there have been multiple transmission events of SIV from
infected primates to humans, however a single transmission event led to the group of HIV-
1 viruses that is responsible for the vast majority of infections world-wide. Specifically,
the human immunodeficiency virus type 1 (HIV-1) is classified into four phylogenetically
related clusters termed groups M, N, O and P [21-23]. Group M is so named as it is the
“major” group of HIV-1, having caused the majority of HIV cases in the global epidemic.
This group is believed to have arisen through transmission from an SIV infected
chimpanzee [24, 25]. Similar to Group M, Group N likely arose from a single transmission
event of SIV from an infected chimpanzee [25], while HIV-1 groups O and P are thought
to have arisen from two independent transmission events from SIV infected gorillas [23,
26]. In addition to HIV-1, there is also human immunodeficiency virus type 2 (HIV-2),
which is distinct from HIV-1 and is derived from transmission of SIV from infected sooty
3
mangabeys [27-29]. Similar to HIV-1, HIV-2 is separated into numerous groups which
derived from independent transmission events [19]. However, in contrast to HIV-1, HIV-
2 exhibits decreased transmissibility [30, 31], and only the minority of HIV-2 infected
individuals develop immunodeficiency [32-34]. Since HIV-1 Group M is responsible for
the vast majority of HIV infections globally, it is the primary focus of most current HIV
research and much of what we know about HIV is from experiments using Group M
viruses.
As a result of a highly error prone viral replication apparatus, HIV viruses are highly
diverse. Indeed, HIV-1 group M itself can be classified into numerous phylogenetically
related subtypes or clades [35-37]. However, most HIV research has been performed using
HIV-1 group M subtype B viruses as a result of these being the most prominent viruses in
the developed world [38]. However, it is becoming increasingly clear that there are
functional differences between group M subtypes, specifically in relation to transmission
and disease progression [39-42]. Therefore, while the majority of this thesis will describe
work primarily completed using HIV-1 Group M subtype B viruses, it must be
acknowledged that there may be differences between HIV-1 viruses of different subtypes.
HIV-1, and the related HIV-2 and SIV, are all classified as retroviruses in the genus
Lentivirus. The family Retroviridae are enveloped viruses, which carry positive sense RNA
in their virions [43]. These viruses are unique in that they encode a viral reverse
transcriptase enzyme, which transcribes a double stranded DNA copy of the viral RNA
genome [43]. The viral genome encoding DNA, known as pro-viral DNA, becomes
permanently integrated into the host cell genome, a characteristic which has driven interest
in the use of retroviral vectors in gene therapy [44]. From the integrated pro-viral DNA,
viral RNAs and their encoded proteins can be produced to facilitate the production of
progeny viruses and all retroviruses encode the structural and enzymatic proteins required
to make virions [43]. However, the retroviruses in the genus Lentivirus are generally quite
complex, as they have evolved a larger repertoire of proteins to ensure optimal virus
replication [45, 46]. Specifically, lentiviruses have evolved regulatory proteins, such as
HIV-1 Tat and Rev, which ensure enhanced gene expression [47]. Moreover, select
lentiviruses, in particular lentiviruses that infect primates, have evolved multiple accessory
4
proteins which enhance viral spread and persistence within the host [48]. As such, primate
lentiviruses are highly successful at hijacking host cells to facilitate viral spread.
1.3 Biology of HIV-1
1.3.1 Clinical course of infection
Unlike in the 1980’s, when the virus was first discovered, infection with HIV-1 no longer
presages certain death. Typically, when an individual becomes infected with HIV-1, an
immune response is mounted against this foreign invasion. Patients experience flu-like
symptoms during the first few weeks of the acute infection, as the virus disseminates,
primarily infecting the CD4+ T cell component of the immune system [49, 50].
Unfortunately, unlike for many other viruses, the immune system in unable to clear HIV-
1. If left untreated, the virus continues to replicate and this chronic infection slowly
depletes the CD4+ T cells of infected individuals. During this period, which can last up to
a decade, infection often lacks visible symptoms [51, 52]. Eventually, infected individuals
experience a drastic and catastrophic loss in immune system function, which leads to
opportunistic infections and malignancies. Clinically, AIDS is defined in adults as a CD4+
T cell count lower than 200 cells per microliter of blood [53]. However, since the mid-
1990’s, with the implementation of a combination therapy called highly active anti-
retroviral therapy (HAART) that targets different aspects of the viral life cycle, progression
of HIV-1 infection to the immunocompromised state can be prevented [54]. Yet, it is
important to note that HIV-1 infected individuals must remain on therapy for the remainder
of their lives, as a pool of latently infected cells branded the “latent reservoir” remains
present during HAART treatment. This latent reservoir can re-populate the body with virus
upon cessation of anti-retroviral treatment [55]. Thus, a significant amount of the current
HIV-1 research is focused on finding an HIV-1 cure through targeting this latent reservoir.
Finally, it is important to acknowledge that while the implementation of effective HAART
means that HIV-1 infection can be treated, access to HAART is not universal and The Joint
United Nations Programme on HIV/AIDS estimates that approximately half of all HIV
infected individuals world-wide are currently not on treatment [56].
5
1.3.2 HIV-1 virions and replication cycle
HIV-1 virions are enveloped and contain two copies of the viral genome which is
comprised of 9 genes that encode for 15 proteins (Figure 1.1). The HIV-1 proteins are
classified as: structural, enzymatic, regulatory or accessory (Table 1; [43, 45]). Within
virions, the RNA genome is associated with the viral nucleocapsid protein, which is further
surrounded by the capsid protein to form the viral core complex [57]. This core is
surrounded by a host cell derived lipid envelope, which associates with the viral matrix
protein and contains the membrane-embedded viral glycoproteins gp120 and gp41.
To initiate the viral replication cycle (reviewed in [45]), the HIV-1 virion surface
glycoproteins engage the viral entry receptor, CD4, and the co-receptors, CCR5 or CXCR4.
These virus-host interactions facilitate fusion of the virion envelope with the host cell
envelope, thereby ensuring entry [58]. The strict requirement for CD4 and CCR5 or
CXCR4 to enter cells implies that HIV-1 infections are limited to CD4+ cells, namely the
following immune cell types: monocytes, dendritic cells (DCs) and CD4+ T cells [59-62].
Post-entry, the viral core dissociates and the HIV-1 RNA genome is reverse transcribed to
DNA by the viral reverse transcriptase. The resulting double stranded DNA, or pro-viral
DNA, is transported into the nucleus and integrated into the host cell genome via the viral
integrase enzyme. Ultimately, the integrated viral DNA will be transcribed and the
resulting mRNAs generate HIV-1 proteins that fulfill specific functions within infected
cells. Specifically, the HIV-1 regulatory proteins Tat and Rev enhance transcription and
transport of RNA encoding viral proteins out of the nucleus for translation [47]. In this
manner, the RNA and proteins necessary to form new virions are produced and bud from
the host cell plasma membrane, after which the viral protease enzyme mediates proteolytic
cleavage of viral protein precursors to form mature, infective virions. Overall, the process
of replication is promoted by the HIV-1 accessory proteins Nef, Vif, Vpr and Vpu, which
will be discussed in further detail below.
6
Figure 1.1 HIV-1 genome organization
The 5’ and 3’ ends of the HIV-1 genome encode the long terminal repeats (LTRs),
repeating sequences necessary for integration of the viral genome into the host cell
genome. The LTRs also contain host cell transcription factor binding regions that
enhance viral gene transcription [63]. The gag, pol and env genes encode the viral
enzymatic and structural proteins. The rev and tat genes encode regulatory elements that
enhance viral gene expression. The accessory proteins are encoded by the vif, vpr, vpu
and nef genes.
3’ LTR gag pol
tat
rev
env5’ LTR
vif vpu nef
vpr
7
Table 1 The HIV-1 genes and their encoded proteins
Gene Protein Function
Structural
gag p17/matrix Targets viral proteins to sites of viral
assembly at the plasma membrane
p24/capsid Constitutes the major structural
components of the viral core
p7/nucleocapsid Incorporation of viral genomic RNA
into virions and coating of viral RNA
within the virion core
p6 Virion budding and incorporation of
Vpr into budding virions
env gp120/surface Binding to viral entry receptor/co-
receptors
gp41/transmembrane Facilitates fusion of the virion
envelope with the host cell membrane
during viral entry
Enzymatic
pol Integrase Integration of viral genome into host
cell genome
Reverse transcriptase Transcription of pro-viral DNA from
virion-derived viral RNA
Protease Proteolytic cleavage of viral proteins
within virions to form mature virus
Regulatory
rev Rev Regulates viral mRNA expression via
shuttling of viral mRNA from the
nucleus
tat Tat Enhances transcription of viral
genome
Accessory
vpu Vpu Mediates immune evasion and
increases viral release and spread
vpr Vpr Enhances viral replication
vif Vif Prevents viral genome hypermutation
nef Nef Mediates immune evasion and
increases virion infectivity
8
1.4 HIV-1 accessory proteins
Unlike other lentiviruses, HIV and the related SIV encode multiple accessory proteins.
These proteins were originally termed “accessory” as they were not required for viral
replication in vitro. However, the accessory proteins have since been deemed necessary to
facilitate viral spread and to mediate evasion of the immune response during infections in
vivo [48]. Indeed, the importance of HIV-1 accessory proteins for viral dissemination and
pathogenicity has been demonstrated using both mouse and non-human primate infection
models [64-68]. Moreover, these proteins fulfill their functions without possessing any
known enzymatic activity and therefore function solely by interacting with and hijacking
host cell proteins. Specifically, these proteins primarily mediate their effects by co-opting
the host cell’s protein degradation or membrane trafficking machinery (reviewed in [48,
69]). In this way, they sequester host cellular proteins within specific locations in the cell
or target them for degradation via the lysosome or proteasome. Given their reliance on the
host cellular machinery to fulfill their functions, the accessory proteins exemplify viruses’
strict requirement for a host.
1.4.1 HIV-1 Vpr and Vif
HIV-1 Vpr, or viral protein R, is an ~14 kDa protein that is actively incorporated into
budding virions through its association with the HIV-1 p6 protein [70-73]. Multiple effects
have been attributed to Vpr (reviewed in [74]), but this accessory proteins’ functions still
remain poorly defined. Notably, Vpr is critical for efficient viral replication in
macrophages in vitro [75], but it remains scantily understood how this is mediated. It has
been postulated that this effect in macrophages may relate to Vpr’s ability to facilitate
degradation of an as of yet undefined host anti-viral factor which inhibits virion envelope
incorporation [76-78]. Moreover, during infection of lymphoid cells, Vpr inhibits G2 to S-
phase cell cycle progression by inducing G2 cycle arrest, resulting in cells lagging in G2
phase, during which HIV-1 transcription is highly active [79, 80]. In addition, Vpr has been
implicated in modulating cell survival [81]. While the mechanisms that mediate the above
described consequences of Vpr expression remain poorly defined, these effects may be
related to Vpr’s ability to readily hijack the host cellular ubiquitin ligase machinery to
9
mediate the ubiquitination and subsequent degradation of DNA repair pathway proteins
[82-86].
Similar to Vpr, the HIV-1 Viral infectivity factor (Vif) protein contributes to efficient viral
replication by targeting host cellular proteins for ubiquitination. Specifically, Vif mediates
the poly-ubiquitination of host cell restriction factors, cellular apolipoprotein B mRNA
editing complex 3 (APOBEC3) proteins, leading to their degradation [87, 88]. Members of
the APOBEC3 family of proteins limit viral replication by inducing viral genome
hypermutations during synthesis of cDNA from viral RNA. Specifically, APOBEC3
proteins mediate deamination of viral RNA, such that upon reverse transcription the newly
transcribed viral cDNA contains G to A mutations, which impede the viral life cycle [89].
Vif counteracts this by targeting APOBEC3 proteins for degradation, thereby preventing
their incorporation into budding virions [88]. Alternatively, degradation-independent
mechanisms of APOBEC3 protein counteraction by Vif have also been proposed [90].
1.4.2 HIV-1 Nef and Vpu
In contrast to the relatively small number of host cell proteins currently described as targets
for intracellular sequestration or degradation by Vif or Vpr, the accessory proteins Nef and
Vpu modulate the expression, localization, and function of an exorbitant number of host
cellular proteins. Large scale screens have identified >30 host cell proteins that are
modulated by Nef or Vpu [91-93]. Thus, Nef and Vpu have evolved the amazing ability to
mediate numerous functions within host cells. Due to the sheer number of host cellular
proteins that are altered by Nef and Vpu, we have only begun to understand the functions
of Nef and Vpu, how these functions are mediated, and the complex interplay between
these two proteins. The accessory proteins Nef and Vpu will be the focus of this thesis,
with their functions described in more detail below.
1.5 Biological effects of HIV-1 Nef and Vpu
HIV-1 Nef and Vpu are viral proteins that, despite their lack of enzymatic activity, facilitate
the creation of highly efficient virus production factories within their host. These proteins
are especially interesting as they have distinct, but sometimes overlapping functions. HIV-
1 Nef, or negative factor, so named due to initial descriptions of it negatively affecting viral
10
replication [94-96], and viral protein u (Vpu), are small membrane associated proteins of
~27 and ~16 kDa, respectively [97-99]. Vpu is a type-I transmembrane protein [100],
whereas Nef is post-translationally modified through the addition of a lipid myristoyl
group, which mediates Nef’s association with membranes [101]. Due to their membrane
association, it is unsurprising that these proteins primarily usurp host cellular membrane
trafficking pathways in order to alter the host cell environment [69]. As with the accessory
proteins Vpr and Vif, many of these functions are mediated by sequestering host cellular
proteins within the cell or targeting host proteins for degradation, with many of the proteins
targeted by Nef and Vpu being cell surface associated (Figure 1.2). A critical role for HIV-
1 Nef and Vpu during infection is their contributions to viral immune evasion. Both
proteins actively contribute to counteracting the innate and adaptive immune response.
Collectively, these Nef and Vpu functions antagonize immune responses to promote viral
spread and persistence.
11
Figure 1.2 Immune cell surface proteins that are targeted by the HIV-1 accessory
proteins Nef and Vpu
Multiple cell surface associated proteins undergo modifications in their subcellular
localization within HIV-1 infected immune cells through the activities of HIV-1 Nef and
Vpu. These modifications reduce the abundance of these molecules on the cell surface
and can alter immune responses. Molecules can be targeted by Nef (blue), Vpu (purple)
or both Nef and Vpu (green). Modifications include: decreases in cell surface MHC-I
thereby preventing detection by cytotoxic T cells [102-104], Nef-mediated decreases in
the cell surface chemokine receptors CCR5 and CXCR4 [105], Nef and Vpu evasion of
NK cell responses by downregulation of ligands that bind to NK cell activating receptors
[106-109], and decreases in cell surface CD4 to prevent detection of infected cells by
NK cells through inducing exposure of antibody-dependent cellular cytotoxicity
(ADCC)-mediated epitopes on the HIV-1 envelope protein [110-112]. Nef and Vpu
decrease cell surface levels of the host cell restriction factors serine incorporator
(SERINC) 5 and bone marrow stromal cell antigen 2 (BST-2; also called tetherin),
12
respectively, inhibiting incorporation of these anti-viral factors into budding virions
[113-116]. Finally, the activation of infected T cells by antigen presenting cells is
inhibited by the downregulation of activating cell surface molecules in both infected
CD4+ T cells and in infected antigen presenting cells [117-119].
13
1.5.1 Evasion of cell-mediated adaptive immune responses
During viral infections, the primary immune cell type that target infected cells are CD8+
cytotoxic T lymphocytes (CTLs). Specifically, CTLs recognize and eliminate cells which
present virus-derived peptides or antigens on major histocompatibility complex class I
(MHC-I) molecules (reviewed in [120]). Mature MHC-I molecules are heterodimers
composed of a common β2-microglobulin light chain and a variable heavy chain that binds
to 8-10 amino acid peptide antigens [120]. To prevent detection of infected cells by CTLs,
Nef and Vpu mediate decreases in cell surface levels of MHC-I. Specifically, Nef mediates
the endocytosis of cell surface MHC-I, followed by its sequestration in the cellular
paranuclear region [102, 121-123]. In contrast, Vpu disrupts the formation of newly
synthesized MHC-I in the endoplasmic reticulum (ER) [103]. Interestingly, Nef
preferentially targets HLA-A and HLA-B [124], while Vpu targets HLA-C [125].
Ultimately, the downregulation of MHC-I molecules by HIV-1 has been implicated in
reducing CTL killing of infected cells [104].
The second arm of cell-mediated adaptive immune responses is comprised of helper CD4+
T cells, which function during infection to regulate the immune response [126]. CD4+ T
cells are activated comparably to CD8+ CTLs, through the presentation of antigens on
MHC class II (MHC-II) molecules by antigen presenting cells (APCs), including HIV-1
infected monocytes and dendritic cells. Mature MHC-II molecules are presented on the cell
surface subsequent to being loaded with peptide antigen in an endocytic cellular
compartment. Prior to this, MHC-II molecules are complexed with the MHC-II invariant
chain (CD74; Ii), which is degraded when the MHC-II molecule is loaded with a peptide
antigen [127]. Both Nef and Vpu alter the expression of mature MHC-II molecules on the
cell surface [119, 128-130], owing to their ability to interact with the MHC-II invariant
chain. Briefly, Nef induces accumulation of the invariant chain on the cell surface or in
intracellular compartments [129, 131]. Alternatively, Vpu may decrease the levels of cell
surface invariant chain, though there are conflicting reports [92, 128]. Taken together, these
alterations in invariant chain subcellular localization likely contribute to Nef- and Vpu-
mediated decreases in the quantity of peptide-loaded mature MHC-II molecules on the cell
surface [119, 128, 130]. This decrease has been associated with a diminished ability of
14
monocytic cells to activate T cells [128]. Moreover, a correlation has been observed
between rapid disease progression in HIV infected children and increased Nef-mediated
up-regulation of cell surface invariant chain and downregulation of mature MHC-II [132].
1.5.2 Evasion of humoral and innate immune responses
In addition to cell-mediated adaptive immune responses, a large antibody-mediated or
humoral response is developed during HIV-1 infection. Of particular importance is the
humoral response targeting the HIV-1 surface glycoprotein, or envelope, which is present
on the surface of virions and infected cells. Notably, during virion entry the envelope
glycoprotein interacts with the viral entry receptor CD4 and undergoes a conformational
change to allow for virion envelope fusion with the host cellular membrane [133-135]. We
have recently begun to appreciate that the envelope glycoprotein conformational change
induced upon CD4 binding exposes epitopes that are targeted by antibodies that can be
detected by innate immune cells to facilitate antibody-dependent cellular cytotoxicity
(ADCC) [111, 112, 136, 137]. During ADCC, innate immune cells, such as natural killer
(NK) cells, detect infected cells that have been bound by specific antibodies and
subsequently mediate their lysis. Importantly, Nef and Vpu counteract this effect through
their ability to decrease cell surface and total cellular CD4 levels [111, 112, 138], via
targeting CD4 for lysosomal or proteasomal degradation [139-141]. This is believed to
limit the interactions between the viral envelope and CD4 on the surface of infected cells,
thereby reducing the exposure of CD4-induced epitopes that are targeted by ADCC-
mediating antibodies. Ultimately, this leads to a reduction in ADCC in the presence of Nef
and Vpu [111, 112], with lower levels of cell surface CD4 correlating with a decreased
susceptibility to ADCC [138].
The ability of NK cells to target and eliminate HIV-1 infected cells is not solely dependent
on ADCC-mediating antibodies binding to infected cells. Indeed, viral infections can alter
the cell surface levels of NK cell activating or inhibitory receptors, allowing for NK cells
to detect and lyse virally infected cells [142]. However, viruses have evolved the ability to
counteract this innate immune mechanism by directly altering these receptors. Briefly,
upon infection with HIV-1 lacking a functional nef gene, Jurkat T cells exhibit increases in
receptors that are detected by the NK cell activating receptor, NKG2D [106]. However,
15
upon infection with viruses encoding Nef, Nef mediates the downregulation of NKG2D
ligands, such that infection does not increase the abundance of these receptors on the cell
surface [106]. This activity can prevent NK cell-mediated targeting of infected cells, as a
correlation exists between the increased ability of Nef alleles to downregulate NKG2D
ligands and decreases in NK cell mediated-ADCC [110]. Nef has also been implicated
through an undefined mechanism in the downregulation of NKp44L, the ligand for the NK
cell activating receptor NKp44, with Nef expression reducing NKp44L-mediated killing
of infected CD4+ T cells [107].
The modulation of NK activating receptors is also attributable to Vpu. Specifically, Vpu
mediates decreases in cell surface levels of the NK cell activating receptor NTB-A through
altering the processing and glycosylation of newly translated NTB-A [143]. This activity
decreases NK cell-mediated effects against virally infected cells [108]. Moreover,
reductions in NK cell lysis of infected T cells has also been linked to the ability of Vpu to
decrease surface levels of the adhesion molecule intracellular adhesion molecule 1 (ICAM-
1), which limits the formation of adhesions between T cells and NK cells that are necessary
for NK cell-mediated killing [144]. Finally, both Nef and Vpu target the NK cell activating
receptor PVR (CD155), limiting cell surface levels to decrease susceptibility to NK cell
killing [109, 145]. Overall, the presence of a functional Nef and Vpu protein has been
shown to reduce NK cell-mediated killing of HIV infected CD4+ T cells [106-109].
1.5.3 Alterations in immune cell homing
Critical to immune responses in vivo is the ability of immune cells to survey an organism
for infections and be recruited to sites where immune cell responses are established.
Adhesion molecules are essential for homing of immune cells to the secondary lymphoid
organs, namely the lymph nodes, where immune responses are developed (reviewed in
[146]). Nef and Vpu independently contribute to decreases in cell surface levels of the
adhesion molecule L-selectin (CD62L; [147]), which mediates entry of T cells into lymph
nodes. This is accomplished by interactions between the viral proteins and L-selectin,
which facilitates the inhibition of L-selectin transport to the cell surface via Nef-mediated
decreases in total L-selectin protein levels and Vpu-mediated intracellular sequestration of
L-selectin [147]. Viral protein induced reductions in cell surface L-selectin results in
16
decreased adhesion of infected CD4+ T cells to the L-selectin ligand fibronectin and
reduces L-selectin-mediated immune cell activating signaling [147]. Moreover,
recruitment of immune cells to sites of infection is often mediated by migration in response
to various stimuli, including chemokines. Nef and Vpu can limit this migration in response
to chemokines by decreasing cell surface levels of chemokine receptors, including the HIV-
1 viral entry co-receptors CCR5 and CXCR4, or by altering the ability of cells to migrate
in response to chemokines [105, 148-153]. Notably, Nef inhibits migration of infected T
cells in response to the CXCR4 ligand SDF1 alpha [152, 153]. Additionally, Vpu reduces
cell surface levels of CCR7 on HIV-1 infected cells, reducing migration in response to the
CCR7 ligand [154]. The above-mentioned alterations in the migration and homing of HIV-
1 infected immune cells may alter the ability of infected cells to be detected by the immune
system and may increase viral dissemination by promoting continued circulation of
infected cells throughout the body.
1.5.4 Counteraction of host cell restriction factors
In addition to the aforementioned mechanisms mediated by HIV-1 Nef and Vpu to evade
adaptive and innate immune responses, these viral proteins also mediate evasion of intrinsic
antiviral proteins known as restriction factors. Restriction factors are key components of
innate immunity and function to impede viral replication [155]. Specifically, Vpu’s most
recognized function is undoubtedly its ability to counteract BST-2, an interferon induced
protein. BST-2 inhibits release of enveloped viruses by incorporating into the budding
virions’ envelope to tether these virions to the surface of infected cells [156]. Indeed, in
cells expressing endogenous BST-2 the presence of Vpu can increase HIV-1 virion release
by 20-fold [113, 115]. Vpu mediates increased virion release through interacting with BST-
2 and preventing its incorporation into budding virions. This is facilitated through
numerous proposed mechanisms, including removing BST-2 from sites of viral assembly
[157, 158], and decreasing cell surface levels of BST-2 [113]. Decreases in the abundance
of cell surface BST-2 are achieved through inhibiting transport of newly synthesized BST-
2 to the cell surface [159], sequestering BST-2 away from the plasma membrane [160],
and degrading BST-2 in lysosomes [161-163]. Moreover, it was recently shown that
decreases in cell surface BST-2 are important for evasion of ADCC, as the tethering of
17
virions on the cell surface increases the exposure of CD4-induced envelope epitopes to
which ADCC-mediating antibodies may bind [164]. Finally, the counteraction of BST-2
has been implicated in facilitating viral dissemination early in infection [165].
Similar to Vpu, Nef also counteracts restriction factors, specifically the newly identified
restriction factors belonging to the SERINC family of proteins, SERINC3 and SERINC5
[114, 116]. Nef has long been known for its’ ability to increase virion infectivity [166,
167], but it was not until 2015 that it was discovered that this is primarily due to the ability
of Nef to counteract SERINC5 [114, 116]. Specifically, Nef limits the incorporation of
SERINC5 into budding virions [114, 116, 168]. This action is facilitated by binding to
SERINC5, which results in the redirection of SERINC5 from viral assembly sites at the
plasma membrane to the lysosome where it is degraded [168, 169]. How SERINC5 limits
infectivity of virions is not yet clear, but it may be related to effects on the virion envelope
proteins as some HIV-1 envelopes appear to confer increased resistance, or alternatively
sensitivity, to the effects of SERINC5 [170, 171].
1.5.5 Modulation of immune cell activation
CD4+ T cells, the primary cell type infected by HIV-1 [172], are classically activated
through signaling provided by APCs, including B cells, dendritic cells and monocytic cells.
Specifically, the T cell receptor (TCR) is engaged by binding to antigen presented in the
context of MHC-II molecules on the surface of APCs. Subsequent to initial TCR
engagement, additional cell surface receptors and intracellular signaling molecules are
recruited to the site of the TCR: MHC-II complex. This forms the immunological synapse,
the sum of interactions between T cells and APCs that mediate T cell activation (Figure
1.3; reviewed in [173]). When the immunological synapse is formed, intracellular signaling
pathways and transcription factors are activated, leading to increased cytokine production.
These effects can result in immune cell proliferation, which is essential for amplification
of the immune response. Nef and Vpu can influence CD4+ T cell activation, which is
particularly important for HIV-1 as viral gene transcription and subsequent virion
production can be induced by transcription factors that are upregulated upon T cell
activation [174-176]. Therefore, HIV-1 propagation may be optimized by promoting an
18
environment in which activation is sufficient to mediate viral replication and
dissemination, whilst also preventing anti-viral immune responses and cell death.
19
Figure 1.3 Schematic of select molecules found in the immunological synapse
Immunological synapses form between T cells and antigen presenting cells to mediate
sustained T cell activating signaling (reviewed in [173]). In CD4+ T cells, these
interactions include TCR binding to antigen presented in the context of MHC-II
molecules, which is facilitated by the co-receptor CD4. TCR-mediated signaling is also
enhanced or inhibited by binding of the co-stimulatory or co-inhibitory receptors CD28
and CTLA-4, respectively, to B7.1/B7.2 on antigen presenting cells. Engagement of the
TCR allows for recruitment of downstream signaling molecules, including Zap70 and
Lck. Moreover, to ensure sustained interactions between T cells and antigen presenting
cells, adhesion molecules, such as ICAM-1, bind to their ligands on the cognate cell.
20
1.5.5.1 Disruption of the immunological synapse
Alterations in immune cell activation may be facilitated by changes in the cell surface
architecture of HIV infected CD4+ T cells, as cell surface associated molecules can
influence the formation, maintenance, and downstream signaling of the immunological
synapse. Indeed, these processes are thwarted by HIV-1 Nef. Specifically, Nef reduces the
formation of immunological synapse-like complexes between T cells and peptide loaded
APCs [177]. This is facilitated by changes to immunological synapse-associated molecules
that are mediated by HIV-1 Nef and these changes are briefly described below.
Cell surface co-stimulatory or co-inhibitory receptors and their cognate ligands are key
components of the immunological synapse. Thus, they are commonly targeted by
intracellular parasites to evade immune responses [178]. Specifically, T lymphocytes
express cell surface CD28, which functions as a co-stimulatory molecule to initiate a
signaling cascade that acts in concert with TCR signaling to promote T cell activation
[179]. This is facilitated by the binding of CD28 on T cells to the ligands B7.1/B7.2
(CD80/CD86) on the surface of APCs (Figure 1.3). However, following T cell activation,
the co-inhibitory molecule CTLA-4 is also upregulated to prevent over-activation of T
cells. Consequentially, CTLA-4 competes with CD28 for binding to B7.1/B7.2 on APCs.
However, HIV-1 alters these responses, as Nef reduces T cell surface levels of CTLA-4
and CD28 [118, 180], as well as B7.1/B7.2 on monocytic cells [117]. Specifically, HIV-1
Nef interacts with CD28 and decrease its abundance on the cell surface [118, 181]. In
addition, Nef decreases total cellular CTLA-4 protein levels, presumably by mediating
CTLA-4 degradation [180]. Finally, Nef also subverts the immunological synapse by
sequestering B7.1/B7.2 within an intracellular compartment to limit availability for binding
to CD28 or CTLA-4 [117, 182, 183]. Moreover, cellular receptors outside of the
immunological synapse can act as co-stimulatory molecules by mediating signaling upon
ligand binding. This includes L-selectin, which is downregulated from the cell surface by
Nef and Vpu, a function that is associated with decreased T cell proliferation [147].
In addition to altering cell surface receptors that modulate T cell activation, Nef can alter
molecules that facilitate signaling downstream of these receptors. Specifically, Nef
sequesters Lck, a kinase that plays a key role in signaling downstream of the TCR, in the
21
trans-Golgi network to prevents its recruitment to the immunological synapse [177, 184-
186]. Moreover, Nef impedes the immunological synapse recruitment of phosphoproteins
that facilitate downstream signaling. Namely, Nef inhibits the clustering of phosphorylated
neural Wiskott-Aldrich syndrome protein (N-WASP), an actin cytoskeleton regulator, near
the immunological synapse, which may contribute to Nef’s ability to reduce actin ring
formation and spreading subsequent to TCR stimulation [184, 187]. This process has been
linked to the ability of Nef to form a Nef:kinase complex, called the Nef-associated kinase
(NAK) complex, which contains the Nef-binding serine-threonine kinase p21 activated
kinase 2 (PAK2) [188]. These Nef-induced changes to the mediators that potentiate
signaling upon TCR engagement likely limit the signaling pathways that classically occur
upon TCR activation. Overall, limiting the association of the aforementioned molecules
with the immunological synapse may inhibit the formation of fully competent
immunological synapses, and thus inhibit the activation of T cells by external stimuli [189].
1.5.5.1 Inhibition of immune cell proliferation
HIV-1 can also modulate immune cell activation independently of its ability to alter
immunological synapses or signaling pathways. Namely, HIV-1 can inhibit the key
downstream consequence of T cell activation, T cell proliferation, which serves to amplify
the immune response. These effects are mediated through HIV-1 altering amino acid
metabolism. Specifically, a proteomic screen for host cell plasma membrane proteins that
are modulated by Nef and Vpu identified the amino acid transporter SNAT1 as being
downregulated in the presence of Vpu [91]. This is mediated by the ability of Vpu to bind
to SNAT1 and facilitate its degradation in lysosomes [91]. SNAT1 depletion results in
decreased alanine uptake and reduced proliferation upon T cell stimulation [91].
1.5.5.2 Alterations in transcription factor activation
Despite the ability of Nef to limit T cell activation by APCs in a classical TCR-dependent
manner, Nef is paradoxically known to facilitate increases in T cell activation. Specifically,
Nef lowers the threshold for activation of pathways that lead to stimulation of the activities
of transcription factors NF-κB and NFAT [185, 190-193]. This function induces expression
of antiviral mediators and cytokines, including IL-2 [185, 190-193]. The exact mechanism
22
by which Nef alters the threshold for activation of these pathways remains enigmatic, but
is likely through Nef’s ability to modulate intracellular kinases and actin cytoskeleton
regulators [186, 187, 194]. Moreover, it has been proposed that Nef reduces the threshold
for activation to stimulate these specific pathways while alternatively limiting the
activation of others [195].
In contrast to the effects of Nef, Vpu inhibits the immune cell activating transcription factor
NF-κB. Briefly, HIV-1 Vpu counteracts NF-κB through its’ ability to downregulate BST-
2, thereby preventing clustering of virion-bound BST-2 on the cell surface [196].
Otherwise, BST-2 would initiate a signaling cascade activating NF-κB. Moreover, Vpu has
BST-2-independent effects on NF-κB, whereby it prevents the nuclear translocation of
functional NF-κB in part through inhibiting degradation of the NF-κB inhibitor IκB [197,
198]. It has been proposed that the seemingly opposing effects of Nef and Vpu are due to
differences in Nef and Vpu expression patterns during infection. Indeed, Nef is expressed
early in infection, while Vpu is expressed at later time points [199-201]. In this way, early
in infection Nef can activate NF-κB, which facilitates HIV-1 viral genome transcription,
while later in infection Vpu reverses this Nef-mediated effect to mitigate hyper activation
[202, 203].
1.5.6 Modulation of cell death pathways
While HIV-1 may increase immune cell activation in order to enhance viral gene
transcription, the virus ensures that cellular activation is controlled to guarantee viral
immune evasion and to prevent excessive activation which may lead to programmed cell
death, or apoptosis [189]. Nef and Vpu can regulate this balance between activation and
cell death through their effects on cellular activation, as well as their described pro- and
anti-apoptotic functions. Indeed, both Nef and Vpu have ascribed pro- and anti-apoptotic
effects, rendering it difficult to conclude if these proteins are pro- versus anti-apoptotic.
However these differential effects may be related to differences that occur in cell types and
time points post infection [204]. Namely, it was proposed that these viral proteins may act
in an anti-apoptotic manner early in infection to allow for viral replication, followed by
acting in a pro-apoptotic manner later in infection [204]. Both Nef and Vpu are implicated
in influencing the tumour suppressor p53 which activates apoptosis. Specifically, Vpu was
23
shown to increase p53 protein levels [205], while Nef is reported to bind to p53 and prevent
p53-induced apoptosis [206]. Nef also inhibits apoptosis through its ability to bind to the
kinase PAK2, as this induces PAK-mediated phosphorylation of BAD, inhibiting this pro-
apoptotic protein [207]. Moreover, Nef binds to and inhibits the apoptosis signal-regulating
kinase 1, a kinase that functions to induce apoptosis [208]. Finally, Nef inhibits apoptosis
by binding to the membrane trafficking adaptor protein AP-2 [209]. In contrast to these
anti-apoptotic effects, both Nef and Vpu contribute to increases in Fas/Fas ligand mediated
apoptosis, whereby Vpu induces increased sensitivity to Fas-mediated apoptosis [210],
while Nef increases Fas ligand expression to induce apoptosis in neighbouring cells [211].
Interestingly, AIDS disease progression is correlated with increased Fas expression on T
cells [212]. Finally, Nef inhibits the anti-apoptotic proteins Bcl-2 and Bcl-XL to promote
apoptosis [213].
The effects of Nef on the secretion of the cytokine transcription necrosis factor-alpha
(TNFα) is a prime example of HIV-1 balancing cellular activation with induction of
apoptosis. Specifically, through formation of the Nef-associated kinase complex, Nef can
induce the secretion of extracellular vesicles containing Nef, Vpu, TNFα and A disintegrin
and metalloprotease (ADAM)-17 [214], the metalloproteinase which cleaves pro-TNFα to
facilitate TNFα shedding [215, 216]. Surprisingly, vesicles containing these components
are detectable in the plasma of HIV-1 positive individuals who are on anti-retroviral
therapy [217]. Nef-induced secretion of these vesicles is associated with TNFα-mediated
cell activation, which promotes viral transcription and enhances HIV-1 replication [214].
Nef thus induces TNFα production to enhance viral replication, despite TNFα being a pro-
apoptotic mediator [218].
1.5.7 Functional redundancy of HIV-1 Nef and Vpu
The relationship between HIV-1 Vpu and Nef is complex and dynamic. Clear examples
exist of these viral proteins functioning together to achieve the same effect. Specifically,
Nef and Vpu both contribute to decreasing levels of cell surface MHC-I, CD4, MHC-II,
PVR and L-selectin [102, 103, 109, 119, 124, 125, 128-130, 139-141, 147]. This
redundancy may be due to the sheer importance of these modifications for successful
24
dissemination and persistence of the virus. In this way, Nef and Vpu could compensate for
each other should defects in the other protein arise.
Given the ability of Nef and Vpu to perform similar or related functions, there are
interesting evolutionary dynamics between them. Specifically, in SIVs closely related to
HIV-1, Nef counteracts the effects of the restriction factor BST-2 [219]. However, small
differences between the primate and human BST-2 protein prevent chimpanzee SIV Nef
proteins from counteracting human BST-2 [219-221]. Therefore, HIV-1 Vpu took over this
SIV Nef function by evolving the ability to counteract human BST-2 [219], a function
which was likely essential for the zoonotic transmission of SIV from chimpanzees to
humans. Moreover, while most HIV-1 Group M viruses encode Vpu proteins that
counteract this restriction factor, there have been viruses identified in which Vpu has lost
the ability to counteract BST-2 and the Nef protein from these viruses performs this
function [222]. Moreover, in the case of HIV-1 Groups O and N, viruses have been
identified that encode either Nef or Vpu proteins that counteract BST-2 [219, 223-225].
Thus, the counteraction of BST-2 by Nef and Vpu clearly demonstrate the close
relationship between these proteins’ functions and their ability to compensate for each
other to facilitate key tasks.
Another example of compensation is through the control of cellular activation. Specifically,
SIV viruses encode Nef proteins capable of downregulating cell surface levels of CD3, a
protein which functions to stabilize the TCR [226, 227]. The ability of SIV Nef proteins to
downregulate CD3 is key to limiting extensive cell activation and may contribute to the
decreased pathogenicity associated with SIV infections, relative to HIV-1 infections [202,
228, 229]. Interestingly, Nef-mediated downregulation of CD3 is a function that was often
lost when SIV viruses gained a vpu gene [229-231]. With the recent demonstration that
HIV-1 Vpu can reduce T cell activation through its ability to suppress NF-κB activity, it
was proposed that Vpu may be compensating for the loss of CD3 downregulation ability
in HIV-1 Nef proteins to prevent overly pathogenic effects [202, 203]. Ultimately, the
control of cellular activation may represent another example of the functional
interrelatedness of Nef and Vpu.
25
1.5.8 The importance of Nef and Vpu in vivo
Overall, the HIV-1 accessory proteins Nef and Vpu mediate a plethora of effects in HIV-1
infected cells. These effects can in turn impact pathogenicity and viral spread in vivo.
Evidence for the importance of Nef in HIV-1 pathogenicity first came from a cohort of
individuals who were infected with HIV-1 via transfusion of blood products obtained from
an HIV-1 positive donor who was infected with a virus lacking a functional nef gene.
Interestingly, individuals infected with Nef defective virus exhibited extremely slow
disease progression [232]. Moreover, transgenic mice that were developed to express Nef
in CD4+ cells in the absence of all other HIV-1 proteins exhibited an AIDS-like phenotype
[233], illustrating the pathogenic potential of this protein in the absence of infection. A
more recently developed mouse model for studying the effects of HIV-1 was also utilized
to demonstrate that nef-deficient viruses replicate less rapidly and induce loss of a lower
number of T cells [234]. In addition, the importance of Nef has been illustrated through the
use of SIV infections in non-human primate models, as nef genes encoding a premature
stop codon readily revert to nef genes that encode a functional Nef protein [235].
In the case of Vpu, multiple groups have illustrated the importance of Vpu for viral
replication and dissemination using humanized mouse models [68, 236, 237]. A recent
study suggested that this effect is primarily mediated though the ability of Vpu to
counteract the restriction factor BST-2 [165]. Moreover, in infections of pig-tailed
macaques with chimeric simian-human immunodeficiency viruses, the inhibition of Vpu
protein production, the scrambling of the amino acids in the Vpu transmembrane domain,
or the mutation of functionally important Vpu residues, all resulted in decreased viral
pathogenicity [66, 67, 238]. Thus, despite Nef and Vpu being classified as “accessory”
proteins, they are critical for infection and pathogenicity of the virus in vivo.
1.6 How do HIV-1 Nef and Vpu mediate their functions?
HIV-1 Nef and Vpu mediate their multiple effects without possessing their own enzymatic
activity. This is amazing given their relatively small size, with the prototypical HIV-1
laboratory strain NL4.3 encoding Nef and Vpu proteins containing 206 and 81 amino acids,
respectively. This capacity to facilitate many modifications within host cells is due to their
26
ability to co-opt ubiquitous trafficking pathways. The net result of subverting trafficking
pathways is typically the modification of the subcellular localization of many host cell
proteins, which in turn alters their function.
Nef and Vpu can co-opt host trafficking pathways due to the presence of a number of motifs
that allow them to bind cellular proteins by mimicking signals utilized by host proteins
(Figure 1.4). Importantly, despite the high diversity observed within the HIV-1 Group M
viruses, these Nef and Vpu motifs are often very well conserved [239-243]. Additionally,
both proteins contain regions that lack structure, and this intrinsic disorder may facilitate
an increased number of host cell protein interactions.
28
Figure 1.4 Structure of HIV-1 Nef and Vpu and motifs that interact with host cell
proteins
Left: Cartoon illustrations of the Nef (A; blue) and Vpu (B; purple) proteins. Right:
schematics illustrating motifs within NL4.3 Nef (A) or Vpu (B). The location of the
protein motifs and their binding partners or functions are indicated. (A) NL4.3 Nef is a
206 amino acid cytoplasmic protein which is associated with membranes through
myristoylation of G2 [99, 244-246]. Nef associates with the membrane trafficking
adaptor proteins AP-1 and AP-2 through interactions mediated by the
W13/V16/M20/EEEE65 and ExxxLL165/DD175 motifs, respectively [247-249]. The
vesicular coat protein β-COP interacts with Nef R17/19 [181, 250, 251]. The membrane
trafficking regulators phosphofurin acidic cluster sorting (PACS)-1 and -2 bind to the
Nef acidic cluster, EEEE65, and W113/Y120 [122, 123, 252]. The v-ATPase mediates
interactions with DD175 [253, 254]. The Nef-targeted cargo protein CD4 interacts with
Nef via WL58 [255]. The kinases PAK2 and SFKs bind to H89/F191 and PxxP75,
respectively [256-262]. Nef dimerizes via an interface comprised of
R105/I109/L112/Y115/F121/D123 [257, 263]. (B) The NL4.3 Vpu protein is an 81 amino acid
transmembrane protein containing a short luminal domain and two cytoplasmic helixes.
The transmembrane domain amino acids A10/A14/A18/W22 mediate interactions with the
Vpu targeted cargo BST-2 [264, 265]. The RL45 and ExxxLV64 motifs mediate binding
to the adaptor proteins AP-1 and AP-2 [266]. The E3 ubiquitin ligase component β-TrCP
binds to the Vpu DSGpS56 motif upon serine phosphorylation [267].
29
The host cell machinery that Nef and Vpu hijack are primarily involved in membrane
trafficking. This fundamental cellular process can be defined as the movement of cargo
molecules from one location to another in membrane bound vesicles. Nef and Vpu may
have a specific preference for these cellular processes as they themselves are membrane
associated proteins [246, 268]. Specifically, the host cell proteins to which Nef and Vpu
bind are proteins which facilitate trafficking through the endosomal trafficking network
(Figure 1.5), or the ubiquitin ligase machinery which modifies cargo proteins to target them
for trafficking to specific locations [69]. The movement of vesicle-associated molecules
around the cell requires a large number of trafficking proteins which not only facilitate the
physical movement of cargo along the cellular cytoskeleton, but also dictate directionality
and specificity. These processes unambiguously determine where vesicles from one
compartment fuse with vesicles from another compartment (Figure 1.6; [269]). Notably,
this process is regulated in part by sequences within proteins or post translational
modifications, such as ubiquitination, that serve to direct where cargo go by giving
specificity to interactions with cellular trafficking proteins. The importance of regulating
vesicular trafficking led to the 2013 Nobel prize in Physiology or Medicine being award to
Drs. James Rothman, Randy Schekman and Thomas Südhof, who were recognized for their
invaluable contributions to understanding how these membrane trafficking processes occur
[270]. Importantly, viruses and viral proteins, including HIV-1 Nef and Vpu, have evolved
the ability to mimic or alter these trafficking signals and thereby alter the localization of
their target proteins to fulfill their functions [271].
30
Figure 1.5 The endosomal trafficking network
Proteins destined for the cell surface or compartments in the endocytic network are
translated on the rough ER and trafficked through the Golgi complex to the trans-Golgi
network wherein proteins are processed. From here, proteins can be shuttled in
membrane enclosed vesicles to the plasma membrane and various intracellular
compartments, including: early endosomes, recycling endosomes, late endosomes or
multivesicular bodies (MVBs), and lysosomes. Arrows indicate the pathways through
which vesicle transport can occur.
31
Figure 1.6 General mechanism of vesicle transport
(1) Signals in the cytoplasmic tail of cargo proteins recruit adaptor proteins to specific
membrane regions. This process can be further regulated by changes in membrane
phospholipids. Other proteins necessary for subsequent stages of transport may also be
recruited, including v-SNAREs. (2) Adaptor proteins contain specific sequences
mediating recruitment of vesicular coat proteins which oligomerize at sites of vesicle
budding. This oligomerization can be regulated by the ADP-ribosylation factor family
of GTPases. (3) Membrane bending ensues and the scission of the vesicle membranes
from the donor membrane occurs, often assisted by host cell enzymes that polymerize
along the neck of budding vesicles and through regulated actin polymerization. Once a
transport vesicle buds, the associated coat and adaptor proteins disassociate and recycle
back to the donor membrane to be used in future vesicle budding events. (4/5) At
32
acceptor membranes, various tethering factors bring the vesicle into proximity of the
acceptor membrane and proteins known as t-SNAREs interact with vesicle incorporated
v-SNAREs to reduce the energy barrier needed to fuse membranes. Tethering factors or
protein complexes, SNARE proteins, and enzymes in the Rab GTPase family all function
to mediate specificity of transport, such that they ensure vesicle fusion occurs with the
correct target membranes. Figure is modified from [269].
33
1.6.1 Nef: host cell protein interactions
In order for Nef to mediate its effects it associates with host cell membranes. This is
facilitated by the post-translational modification of Nef on glycine residue two, which is
modified by the addition of a lipid myristoyl group derived from myristic acid and
effectively functions as a lipid anchor [99, 244-246]. When HIV-1 Nef myristoylation is
inhibited through mutation of glycine residue two to alanine, virtually all of Nef’s functions
are inhibited, including downregulation of CD4, MHC-I, mature MHC-II, NK cell
activating receptors, and SERINC5 [106, 109, 114, 119]. The association of Nef with
membranes presumably brings this protein within close proximity of host cell membrane-
associated proteins, to which Nef must bind to facilitate its functions.
1.6.1.1 Vesicular adaptor proteins: AP-1 and AP-2
Key membrane-associated proteins to which Nef binds are the membrane trafficking
adaptor proteins adaptor protein 1 (AP-1) and adaptor protein 2 (AP-2). AP-1 and AP-2
are heterotetrameric protein complexes that function as vesicular adaptors to connect cargo
to vesicular coat proteins [272]. These proteins are recruited to specific cargo based on
sorting signals present in the cytoplasmic tail of cargo proteins. Two prototypical sorting
signals recognized by AP-1 and AP-2 are the tyrosine (YxxØ, where x is any amino acid
and Ø is any amino acid with a bulky hydrophobic amino acid) and dileucine
([D/E]xxxL[L/I], where x is any amino acid) based sorting signals (reviewed in [272]).
These distinct signals are bound by different subunits of the adaptor protein complexes,
with the μ subunits typically recognizing tyrosine based motifs and the combination of γ –
σ (AP-1) or α– σ (AP-2) subunits recognizing dileucine motifs. Nef has evolved the ability
to hijack these signals to forms complexes with specific cargo and adaptor proteins.
Namely, Nef can bind the cytoplasmic tail of MHC-I molecules by facilitating recognition
of an unconventional tyrosine-based sorting signal (Y320SQA) in the MHC-I cytoplasmic
tail by the AP-1 μ subunit [247]. Specifically, a crystal structure containing Nef, the AP-1
μ subunit, and the cytoplasmic tail of MHC-I revealed that these proteins form a ternary
complex which allows for AP-1 to bind the MHC-I Y320SQA motif despite it lacking the
hydrophobic residue normally found in canonical tyrosine sorting signals [247]. Formation
of the ternary complex is facilitated through the creation of a hydrophobic pocket
34
containing Nef W13, V16, M20 and an acidic cluster, Nef EEEE65, which together mediate
interactions with AP-1 [247, 248]. Moreover, mutation of Nef M20 and EEEE65 inhibit
MHC-I downregulation [247, 248, 273, 274], indicating that the ability of Nef to form this
Nef: AP-1: MHC-I ternary complex is essential for the ability of Nef to downregulate
MHC-I.
In addition to hijacking AP-1 complexes, Nef hijacks AP-2 in a similar manner.
Specifically, Nef forms a ternary complex with AP-2 and the cytoplasmic tail of the host
cell receptor CD4 [275]. Unlike the Nef: AP-1: MHC-I ternary complex, the Nef: AP-2:
CD4 ternary complex can be formed through the recognition of a dileucine based motif in
Nef (ExxxLL165) by the AP-2 α and σ subunits, as defined by a crystal structure of Nef and
AP-2 α and σ [249]. The Nef: AP-2 structure also revealed a surface wherein CD4 could
bind to form a Nef: CD4: AP-2 ternary complex [249]. This predicted CD4 binding site
contained Nef WL58 [249], which has been previously implicated in the interaction between
Nef and the cytoplasmic tail of CD4 [255]. The recruitment of AP-2 to the CD4
cytoplasmic tail by Nef is believed to facilitate the recruitment of the vesicular coat protein
clathrin to mediate endocytosis of CD4-containing vesicles from the cell surface, thereby
decreasing cell surface levels of CD4. Indeed, mutation of the Nef AP-2 binding motif
(LL165) or CD4 binding motif (WL58) abolishes the ability of Nef to downregulate cell
surface CD4 [276, 277].
The Nef: AP-2 α and σ structure also suggested that the Nef diacidic motif (DD175), which
takes the form of aspartic or glutamic acid residues depending on the Nef protein, functions
to stabilize the interaction between Nef and AP-2 [249]. This may explain, to some extent,
the requirement of this motif in the downregulation of CD4 [278]. However, the Nef
diacidic motif has also been implicated in the binding of Nef to the vacuolar ATPase (v-
ATPase) [253, 254], a proton pump which acidifies endosomes [279]. It has been proposed
that since v-ATPase binds to both Nef and the μ subunit of AP-2 it connects Nef to AP-2
to mediate CD4 downregulation [253]. Interestingly, the mechanisms utilized by Nef to
hijack AP-2-mediated trafficking to downregulate CD4 may also be co-opted to
downregulate other cell surface receptors. Indeed, mutation of the Nef dileucine motif
(LL165) and the Nef diacidic motif (DD175) have been shown to abolish the ability of Nef
35
to alter the subcellular localization of other cell surface proteins including: the MHC-II
invariant chain [131, 280], CD28 [118, 280], CTLA-4 [180], and the restriction factor
SERINC5 [114, 281]. Overall, the ability of Nef to co-opt classical membrane trafficking
adaptors, namely AP-1 and AP-2, are key to Nef’s functions.
1.6.1.2 Vesicular coat proteins and their regulators: Arf1 and β-COP
The oligomerization of adaptor protein-containing vesicular coats is a highly regulated
process. Indeed, the GTPase ADP-ribosylation factor 1 (Arf1) regulates the formation of
AP-1 containing coats by interacting with AP-1 and promoting formation of an “open
complex” wherein the cargo-binding regions are exposed [282]. Interestingly, a functional
Arf1 protein is necessary for Nef-mediated MHC-I downregulation in T cells [283].
Moreover, the expression of a constitutively active Arf1 increased the ability of MHC-I to
co-immunoprecipitate with AP-1 in the presence of Nef [283]. A more recent cryo-electron
microscopy study revealed that Arf1 may be contributing to Nef-mediated MHC-I
downregulation [284]. This study illustrated that a Nef: MHC-I cytoplasmic tail fusion
protein could form a trimeric complex with Arf1 and AP-1, implicating Arf1 in promoting
the formation of AP-1 coated vesicles [284]. Arf1 is also known to regulate the formation
of vesicular coats containing the coat protein coatomer protein I (COPI) [285, 286], which
Nef has also been implicated in hijacking. Indeed, it was demonstrated that
immunoprecipitation of Nef can pull down the COPI subunit β-COP from CHO cell lysate
[287]. While this Nef: β-COP interaction was initially mapped to the Nef diacidic motif
[287], this was later challenged and the interaction mapped to Nef R17/19 [181, 250, 251].
Through the use of short hairpin RNAs to deplete β-COP and the overexpression of Nef
R17/19 mutants, β-COP was implicated in Nef-mediated degradation of MHC-I and CD4,
presumably in lysosomes [181, 251]. Generally, Nef may utilize components of the COPI
trafficking pathway, including Arf1 and β-COP to mediate its distinct functions.
1.6.1.3 Vesicular trafficking regulators: PACS-1 and PACS-2
There are many proteins that regulate membrane trafficking by promoting or inhibiting the
association of specific cargo with trafficking adaptors or coat proteins. A prime example
of trafficking regulators are PACS-1 and PACS-2, which link acidic cluster containing
36
cargo molecules with membrane trafficking machinery [288]. Specifically, PACS-1 and
PACS-2 link cargo to the vesicular adaptor proteins AP-1 and COPI, respectively [289,
290]. Interestingly, Nef contains an acidic cluster, EEEE65, which in conjunction with
residues W113 and Y120 facilitates binding to PACS-1 and PACS-2 [122, 123, 252]. Given
that PACS-1 forms ternary complexes with AP-1 and cargo proteins [289], and that PACS-
1 binds to Nef and AP-1 [123, 289], PACS-1 may facilitate the formation of multi-protein
complexes that include Nef, AP-1 and cargo molecules [291]. Indeed, the PACS proteins
have been implicated in the ability of Nef to downregulate cell surface MHC-I, with
mutation of the PACS-1/2 binding acidic cluster (EEEE64) or overexpression of dominant
negative forms of PACS-1/2, inhibiting Nef-mediated MHC-I downregulation [122, 252].
Finally, the interaction between the PACS proteins and Nef may be important for mediating
additional Nef functions, as mutation of the Nef EEEE65 acidic cluster also inhibits the
ability of Nef to downregulate cell surface MHC-II [119], CD28 [280], and a variety of
chemokine receptors [105].
1.6.1.4 Vesicular trafficking sorting signals: ubiquitin
In addition to mediating recruitment of the membrane trafficking machinery through
specific protein motifs, Nef can alter the localization of target proteins through pathways
that are regulated by post-translational modifications. One key protein modification is the
covalent addition of 8 kDa ubiquitin peptides in a process known as ubiquitination.
Ubiquitination is classically thought of as functioning to mark proteins destined for
degradation in the cytosolic proteasome, however ubiquitination also plays a role during
membrane trafficking (reviewed in [292]). Specifically, ubiquitination functions as a signal
for endocytosis from the cell surface by interacting with vesicular adaptor proteins.
Moreover, endocytosed ubiquitinated proteins present in early endosomes are detected by
components of the endosomal sorting complexes required for transport (ESCRT)
machinery and are subsequently sorted into intraluminal vesicles that are degraded in the
lysosome. HIV-1 Nef can hijack ubiquitin-dependent pathways in order to downregulate
various cell surface receptors. Specifically, Nef hijacks the ESCRT associated protein
ALIX, which binds to ubiquitinated cargo [293], by binding directly to ALIX to link un-
ubiquitinated CD4 to the ESCRT machinery [294]. This interaction mediates CD4’s sorting
37
into intraluminal vesicles and subsequent lysosomal degradation [294, 295]. Nef also
mediates the accelerated trafficking of ubiquitinated SERINC5 to lysosomes where this
restriction factor is degraded [169]. Additionally, Nef induces ubiquitination of the
chemokine receptor CXCR4 to promote its downregulation from the cell surface [148].
Nef also enhances the ubiquitination and degradation of Giα2 which functions in signaling
downstream of chemokine receptors [296]. Finally, the Nef protein itself can be
ubiquitinated at K144 and mutation of this residue inhibits the ability of Nef to downregulate
both CD4 and MHC-I [297, 298]. However, it is unknown if the ubiquitination of Nef is
required for CD4 and MHC-I downregulation.
1.6.1.5 Kinases: SFKs and PAK2
Membrane trafficking pathways and proteins may be altered by signaling molecules, such
as intracellular kinases. Nef may therefore modulate trafficking though its ability to bind
to specific kinases, namely members of the Src family kinases (SFKs) and PAK2.
Specifically, Nef binds to and mediates activation of the tyrosine kinases Hck, Lyn, and c-
Src, members of the SFKs [299]. The details of this interaction have been elucidated using
mutational analyses, crystal structures, and solution NMR structures. These studies
indicate that the RT loop of the SH3 domain in SFKs interacts with the Nef type II
polyproline helix, specifically the P69xRPxxPxRP78 encoding region [256-260]. Similar
SH3: polyproline helix interaction interfaces are commonly used by host cellular proteins
[300]. Moreover, the Nef PxxP75 motif is critical for this interaction, as a Nef AxxA75
mutant is unable to activate SFKs [299]. The Nef: SFK interaction has been implicated in
the formation of a multi-kinase signaling complex which activates a phosphoinositide 3-
kinase (PI3K)-signaling pathway that induces the endocytosis of cell surface MHC-I [121,
122, 301]. Therefore, mutation of the Nef PxxP75 motif or inhibition of Nef: SFK binding
using small molecule inhibitors can block Nef-mediated downregulation of cell surface
MHC-I [121, 301]. Interestingly, inhibition of the Nef: SFK interaction has also been
shown to negatively affect the ability of Nef to increase virion infectivity and alter cellular
activation [302, 303]. In addition, mutation of the Nef PxxP75 motif can inhibit Nef-
mediated downregulation of MHC-II [119, 304], NK cell activating receptors [109, 305],
and chemokine receptors [105, 149-151]. While these effects may be due to the inability
38
of Nef to bind SFKs, the Nef PxxP75 motif is also implicated in facilitating the binding of
Nef to the serine-threonine kinase PAK2 [306, 307].
When Nef-associated serine-threonine kinase activity was first observed in T cells, the
identity of the Nef-associated kinase was unknown. Thus, this complex was initially termed
the Nef-associated kinase (NAK; [307, 308]). It was later recognized that the Nef-
associated kinase is PAK2 [309-311]. Unlike the Nef-binding SFKs, PAK2 lacks a SH3
domain to interact with the Nef PxxP75 motif. Rather, mutational analysis and protein
modeling suggests that PAK2 interacts with a hydrophobic patch on Nef containing H89
and F191 [261, 262]. Thus, the requirement of the Nef PxxP75 motif for PAK2 binding has
been suggested to be due to PAK2 forming a complex with Nef concomitantly with other
associated proteins, including the SH3-domain containing protein Vav, which may act to
stabilize the interaction between Nef and PAK2 [312]. The association of Nef with PAK2
may facilitate PAK2 activation in cells [311]. The Nef: PAK2 interaction is primarily
associated with the ability of Nef to alter T cell activation [187, 188, 194, 313], modulate
T cell survival [207], and to mediate the secretion of extracellular vesicles [314]. Overall,
Nef-mediated interactions with cellular kinases, including SFKs and PAK2, are key to
many alterations in the cellular environment facilitated by Nef.
1.6.2 Nef oligomerization
In order for Nef to mediate its functions, including activating cellular kinases, protein
dimerization may be required. Indeed, Nef dimers and trimers were observed in vitro [263,
315]. Based on crystal structures and mutational analyses, a dimer interface was proposed
which consists of a hydrophobic patch made up of Nef residues I109, L112, Y115 and F121
flanked by electrostatic interactions between R105 on one monomer and D123 on the other
monomer [257, 263]. Studies suggest that in cells Nef can oligomerize and that Nef
dimerization requires the Nef I109/L112/Y115/F121 hydrophobic patch [316-318]. Moreover,
the significance of this dimerization interface was supported by findings that mutation of
D123 or the hydrophobic patch inhibits various Nef functions, including CD4
downregulation, MHC-I downregulation and enhancement of virion infectivity [316, 319].
However, more recent work suggests that the Nef dimer interface may be altered when Nef
interacts with various binding partners [260, 320], and the importance of Nef D123 for
39
dimerization has been questioned [247, 260, 321]. Nonetheless, it is believed that Nef’s
ability to dimerize is critical for its function and the creation of small molecule inhibitors
to block Nef’s effects via targeting dimerization have been proposed [318, 322].
1.6.3 Vpu oligomerization
The HIV-1 Vpu protein, like Nef, is membrane associated given that it contains a
transmembrane domain. Specifically, Vpu is a type I integral membrane protein with a
single alpha helical transmembrane domain [268]. Interestingly, Vpu oligomerizes and is
classified as a class IA viroporin, similar to the unrelated influenza A’s matrix 2 protein
[323]. Computer modeling and NMR spectroscopy suggest Vpu forms pentameric
oligomers [268, 324-326], thereby forming a pore within membranes which can mediate
selective transport of cations [327, 328]. The functional significance of ion channel
formation by Vpu remains enigmatic, though pore formation has been correlated with the
ability of Vpu to downregulate CD4 and enhance virion release [328, 329]. In contrast, it
has been suggested that the active form of Vpu for CD4 downregulation is monomeric, as
mutation of Vpu residue W22 increases Vpu oligomerization and decreases the ability of
Vpu to downregulate CD4, without inhibiting Vpu: CD4 interactions [330]. Moreover, a
Vpu transmembrane domain mutation (S23A) which inhibits Vpu ion channel activity did
not inhibit the ability of Vpu to counteract BST-2 [331]. Finally, the effects of Vpu ion
channel formation may be restricted to specific subcellular locations given that live cell
imaging indicates that Vpu does not oligomerize in the ER [326].
1.6.4 Vpu: host cell protein interactions
1.6.4.1 Target protein binding
In addition to mediating oligomerization, the transmembrane domain of Vpu is implicated
in forming key interactions with host cellular proteins that Vpu targets for downregulation.
Indeed, Vpu counteracts BST-2 by binding to BST-2 via an interaction interface that is
mediated by the transmembrane domains of these proteins. This interface has been mapped
using mutational analysis and NMR spectroscopy to Vpu residues A10, A14, A18 and W22,
mutation of which inhibit Vpu: BST-2 interactions and the ability of Vpu to antagonize
BST-2 [264, 265]. Moreover, structural modeling of Vpu suggests that the transmembrane
40
domain forms a helix with A10, A14, A18 and W22 located on same face of the helix [264,
325]. This model implies that these residues could constitute an interaction interface. The
transmembrane domain of Vpu has also been implicated in Vpu’s ability to downregulate
cell surface CD4. Specifically, while a number of studies suggest that Vpu and CD4
interact via their cytoplasmic domains [332-336], mutation of transmembrane domain
residues or replacement of the Vpu transmembrane domain with that of other proteins have
been shown to inhibit the ability of Vpu to degrade or bind CD4 [330, 337-339].
Additionally, combinations of mutations within the Vpu transmembrane domain residues
A19, A14, A18 and W22 or scrambling of the Vpu transmembrane domain sequence inhibit
the ability Vpu to downregulate NK cell activating receptors [108, 145, 305] and the
chemokine receptor CCR7 [154]. These findings demonstrate that the Vpu transmembrane
domain is critical for Vpu to mediate its function, likely through facilitating binding to
target proteins.
1.6.4.2 Vesicular adaptor proteins: AP-1 and AP-2
The interactions between Vpu and host cell proteins enable Vpu to alter the subcellular
localization of target proteins by linking them to cellular machinery, similar to the
functions of Nef. Recently, it was demonstrated that Vpu can hijack membrane trafficking
adaptor proteins. Specifically, the cytoplasmic tail of Vpu contains a dileucine-like motif
(ExxxLV64; Figure 1.4), similar to the conventional adaptor protein-binding dileucine
motif ([D/E]xxxL[L/I/V]). Using chromatography this motif in Vpu was shown to mediate
a direct interaction with AP-1 and AP-2 [340]. Moreover, the formation of a fusion protein
containing the cytoplasmic tails of BST-2 and Vpu exhibited enhanced AP-1 binding,
compared to BST-2 or Vpu alone [340]. The latter BST-2: Vpu fusion was successfully co-
crystalized with AP-1 [340]. This structure indicated that Vpu forms a ternary complex
with BST-2 and AP-1 whereby an atypical tyrosine binding motif, YxYxxØ (where Ø,
where x is any amino acid and Ø is any amino acid with a bulky hydrophobic amino acid),
in BST-2 binds to AP-1 μ. This structure also demonstrated that the Vpu dileucine-like
motif binds to the AP-1 γ and σ subunits, while Vpu residues RL45 interact with AP-1 at
the γ-σ interface [340]. The formation of a Vpu: BST-2: AP-1 complex may link BST-2 to
the endocytic trafficking network to facilitate Vpu-mediated exclusion of BST-2 from viral
41
assembly sites and BST-2 lysosomal degradation. This possibility is supported by mutation
of the Vpu dileucine-like (ExxxLV64) or RL45 motifs, which inhibits both Vpu-mediated
decreases in cell surface BST-2 and increases in virion release [340-342]. Moreover, AP-
2 may form a ternary complex with Vpu and BST-2 similar to AP-1, as AP-2 was co-
immunoprecipitated with Vpu in BST-2 expressing HEK293T cells in a manner dependent
on the Vpu ExxxLV64 motif [266]. Interestingly, the recruitment of AP-1 or AP-2 to BST-
2 to form a ternary complex may be regulated by phosphorylation of Vpu, specifically at
serine residues S52/56 [266], which are phosphorylated by casein kinase II [343].
1.6.4.3 LC3-associated phagocytosis mediators: LC3C
In addition to mediating interactions with host cellular adaptor proteins, the Vpu ExxxLV64
motif mediates an interaction with the protein LC3C [344]. LC3C is a member of the Atg8
family of ubiquitin-like proteins that are conjugated to phospholipids. This conjugation
leads to membrane association of cargo and facilitates selective transport of LC3
conjugated membranes to either autophagosomes or a non-canonical phagocytic pathway
termed LC3-associated phagocytosis (LAP) [345]. Interestingly, depletion of autophagy or
LAP proteins revealed that proteins specifically facilitating LAP are required for Vpu to
enhance virion release in a BST-2 dependent manner [344]. Therefore, formation of a Vpu:
LC3C complex may contribute to the downregulation of BST-2 by connecting BST-2 to
LAP machinery [344].
1.6.4.4 Ubiquitin ligase machinery: β-TrCP
Vpu mediates some of its effects by hijacking the cellular ubiquitin ligase machinery.
Specifically, Vpu links target host cell proteins CD4 and BST-2 to β-transducin-repeat
containing protein (β-TrCP) [162, 267, 346], a component of the Skp Cullin F-box
containing (SCF) E3 ubiquitin ligase complex. Beta-TrCP functions as an adaptor molecule
that binds phosphorylated cargo to connect it to the ubiquitin conjugating machinery [347].
Vpu usurps this machinery through its casein kinase phosphorylated serine residues which
are part of the DSGNES56 motif in NL4.3 Vpu (Figure 1.4). This sequence partially
matches the canonical β-TrCP recognition motif, DpSGøxpS, where x is any amino acid, ø
is a hydrophobic residue, and the serine residues are phosphorylated [347]. Binding of Vpu
42
to β-TrCP facilitates formation of a ternary complex between Vpu, β-TrCP, and CD4 or
BST-2 [162, 267, 346]. In the case of CD4, the formation of Vpu: CD4: β-TrCP complexes
in the ER allows for β-TrCP to recruit additional components of the SCF E3 ubiquitin
ligase machinery which mediates polyubiquitination of the CD4 cytoplasmic tail [139, 267,
348]. Subsequently, components of the cellular ER associated degradation (ERAD)
pathway mediate the translocation of ubiquitinated CD4 from the ER to the cytoplasm
where CD4 is degraded by the proteasome [140, 337, 348]. Key to this process is the initial
Vpu: β-TrCP interaction, as mutation of the DSGNES56 motif serine residues to non-
phosphorylatable residues inhibits β-TrCP binding to Vpu and Vpu-mediated CD4
degradation [267, 335, 349, 350].
In contrast to CD4 downregulation, Vpu binds to BST-2 on endosomes or at the trans-
Golgi network [161, 351]. Formation of Vpu: BST-2: β-TrCP ternary complexes can
subsequently mediate lysosomal degradation of BST-2 [161, 351]. Specifically, binding of
β-TrCP to Vpu that is bound to BST-2 may facilitate recruitment of E3 ubiquitin ligases
which enhance the ubiquitination of BST-2 [351]. The ubiquitination of BST-2 serves as a
signal to sort BST-2 into degradative lysosomes through detection by components of the
ESCRT machinery. Namely, this activity is regulated by the ESCRT-0 protein hepatocyte
growth factor-regulated tyrosine kinase substrate (HRS) which binds ubiquitinated cargo
and links them to ESCRT machinery that mediates trafficking to lysosomes [292]. Indeed,
HRS co-immunoprecipitated with BST-2 and Vpu while siRNA knock-down of HRS
inhibited the ability of Vpu to downregulate cell surface and total BST-2 protein levels
[352]. Moreover, inhibition of endosomal acidification or the use of lysosomal inhibitors
reduced Vpu-mediated BST-2 downregulation [161, 163, 353]. Furthermore, siRNA- or
shRNA-mediated knock-downs implicated β-TrCP in Vpu-mediated decreases of cell
surface and total BST-2 protein levels [161, 353, 354]. However, mutation of the β-TrCP
binding site does not completely abolish the ability of Vpu to enhance virion release [161,
351, 353]. Thus, β-TrCP binding may only constitute one element of the mechanism
through which Vpu counteracts BST-2.
In addition to mediating the downregulation of CD4 and BST-2, the binding of β-TrCP to
Vpu may have additional effects within infected cells. Specifically, the Vpu: β-TrCP
43
interaction may inhibit the ability of β-TrCP to fulfill its normal cellular functions. Briefly,
when the NF-κB signaling pathway is initiated, the NF-κB inhibitor IκB is phosphorylated
and subsequently bound by β-TrCP (reviewed in [355]). This interaction facilitates IκB
ubiquitination and proteasomal degradation to allow for NF-κB to translocate into the
nucleus and function as a transcription factor. However, binding of Vpu to β-TrCP may
sequester β-TrCP away from this canonical target and prevent IκB degradation. This
dissociation may contribute to Vpu’s ability to suppress NF-κB activation [197, 198, 356].
Similar to IκB, the pro-apoptotic protein p53 is also phosphorylated and subsequently
degraded by β-TrCP [357]. Accordingly, expression of Vpu was shown to inhibit the
degradation of p53 leading to increased p53-induced apoptosis [205]. Overall, Vpu
mediates hijacking of the ubiquitin ligase machinery by connecting it to specific host
proteins and may prevent β-TrCP from facilitating its normal functions.
1.7 Thesis overview
It is established that HIV-1 Nef and Vpu are essential for viral spread and contribute to
AIDS pathogenesis. This is due to the ability of these viral proteins to alter and evade
immune responses. Many specific functions of Nef and Vpu have been described that
contribute to creating an optimal host cell environment for HIV. This is facilitated by the
hijacking of host cell membrane trafficking machinery to alter the sub-cellular localization
of target proteins. However, our understanding of how HIV-1 Nef and Vpu create the
optimal host cell environment is not yet complete.
Our general hypothesis is that these undefined pathways are mediated by Nef and Vpu
commandeering host cell proteins that are implicated in membrane trafficking events. The
work within this thesis involves investigating the mechanisms by which HIV-1 Nef and
Vpu alter the host cellular environment, which may facilitate evasion of the immune system
and support viral spread. Specifically, we examine the mechanisms by which HIV-1 Nef
and Vpu reduce cell surface levels of the co-stimulatory molecule CD28 (Chapter 2) and
the mechanism by which Nef reduces cell surface levels of MHC-I (Chapter 3).
44
1.7.1 Chapter 2
Previous studies illustrated that the HIV-1 Nef protein can decrease cell surface levels of
the T cell co-stimulatory molecule CD28 [118, 181]. However, these studies did not define
the mechanistic details of CD28 downregulation during an HIV-1 infection. Therefore, our
objectives were to:
• Determine the subcellular fate of CD28 upon HIV-1 infection
• Describe the HIV-1 viral proteins and their host cell binding motifs implicated in
CD28 downregulation
• Examine the functional consequences of CD28 downregulation
In undertaking our investigation into the mechanism by which HIV-1 Nef reduces
endogenous levels of cell surface CD28 on CD4+ T cells, we also observed downregulation
of cell surface CD28 mediated by HIV-1 Vpu. Therefore, we examined the mechanism of
CD28 downregulation mediated by both HIV-1 Nef and Vpu. We found that both Nef and
Vpu decrease total CD28 protein levels and facilitate transport of CD28 to LAMP-1
positive lysosomal compartments. Moreover, we identified viral protein motifs that were
essential for this function, thereby demonstrating that specific membrane trafficking
proteins known to interact with these motifs may be important for downregulating CD28.
Finally, we postulate that the downregulation of CD28 is playing a role in the broad
mechanisms through which Nef and Vpu modulate T cell activation.
1.7.2 Chapter 3
It is known that HIV-1 Nef reduces cell surface levels of MHC-I through its ability to
facilitate trafficking of MHC-I to an intracellular compartment wherein this receptor is
sequestered. A number of Nef-interacting host cell membrane trafficking proteins have
been implicated in MHC-I downregulation, however given the sheer complexity of moving
cargo within the cell, there are undoubtedly many additional host cell proteins that take
part in this process. We hypothesized that the sorting nexin proteins SNX9 and SNX18
were potential trafficking mediators that could facilitate this Nef function. The rationale
for this being that SNX18 was previously shown to form a complex with Nef that localizes
to AP-1 positive compartments, subcellular locales through which MHC-I traffics during
45
Nef-mediated downregulation [358]. Moreover, both SNX18 and SNX9 interact with Nef-
binding proteins, namely AP-1 and AP-2, respectively. Our objectives in this study were
to:
• Determine if Nef directly binds SNX18 and SNX9, and map this interaction
• Determine if the Nef: SNX18 complex plays a role during MHC-I downregulation
In this study, we showed that SNX18, but not SNX9, is a Nef-binding protein. We mapped
this interaction to the SH3 domain of SNX18 and the Nef PxxP75 motif. Moreover, we
demonstrated that reducing SNX18 protein levels reduces the ability of Nef to
downregulate cell surface levels of SNX18. Overall, we have identified a novel Nef: host
protein interaction which mediates MHC-I downregulation, a Nef function that is key to
HIV-1 pathogenesis [359, 360].
1.7.3 Significance
The work presented in this thesis details mechanisms through which the HIV-1 viral
proteins Nef and Vpu subvert host cellular machinery to alter the sub-cellular localization
of the immune cell receptors CD28 and MHC-I. The ability of viruses, like HIV-1, to hijack
host cell machinery is key to their ability to replicate, as viruses cannot produce progeny
virions without the host cell. Therefore, viruses have evolved ingenious ways to usurp host
cellular pathways. Understanding how viruses take control of cellular pathways and the
cellular machinery not only informs us about how viruses mediate their effects, but also
about normal cellular function.
1.8 References
1. Oxford English Dictionary: Oxford Univeristy Press; [cited 2018 26 March 2018].
Available from: http://www.oed.com/.
2. Kaposi's sarcoma and Pneumocystis pneumonia among homosexual men - New
York City and California. MMWR Morbidity and mortality weekly report.
1981;30(25):305-8.
3. Greene WC. A history of AIDS: looking back to see ahead. Eur J Immunol. 2007;37
Suppl 1:S94-102.
4. Immunodeficiency among female sexual partners of males with acquired immune
deficiency syndrome (AIDS) - New York. MMWR Morbidity and mortality weekly
report. 1983;31(52):697-8.
46
5. Centers for Disease C. Possible transfusion-associated acquired immune deficiency
syndrome (AIDS) - California. MMWR Morbidity and mortality weekly report.
1982;31(48):652-4.
6. Serwadda D, Mugerwa RD, Sewankambo NK, Lwegaba A, Carswell JW, Kirya
GB, et al. Slim disease: a new disease in Uganda and its association with HTLV-
III infection. Lancet. 1985;2(8460):849-52.
7. Newell ML, Peckham C. Risk factors for vertical transmission of HIV-1 and early
markers of HIV-1 infection in children. AIDS. 1993;7 Suppl 1:S91-7.
8. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, et al.
Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune
deficiency syndrome (AIDS). Science. 1983;220(4599):868-71.
9. Gallo RC, Sarin PS, Gelmann EP, Robert-Guroff M, Richardson E, Kalyanaraman
VS, et al. Isolation of human T-cell leukemia virus in acquired immune deficiency
syndrome (AIDS). Science. 1983;220(4599):865-7.
10. Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, Kaplan M, Haynes BF, et al.
Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from
patients with AIDS and at risk for AIDS. Science. 1984;224(4648):500-3.
11. Popovic M, Sarngadharan MG, Read E, Gallo RC. Detection, isolation, and
continuous production of cytopathic retroviruses (HTLV-III) from patients with
AIDS and pre-AIDS. Science. 1984;224(4648):497-500.
12. Schupbach J, Popovic M, Gilden RV, Gonda MA, Sarngadharan MG, Gallo RC.
Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-
III) associated with AIDS. Science. 1984;224(4648):503-5.
13. Coffin J, Haase A, Levy JA, Montagnier L, Oroszlan S, Teich N, et al. What to call
the AIDS virus? Nature. 1986;321(6065):10.
14. HIV/AIDS JUNPo. UNAIDS DATA 2017. 2017.
15. Korber B, Muldoon M, Theiler J, Gao F, Gupta R, Lapedes A, et al. Timing the
ancestor of the HIV-1 pandemic strains. Science. 2000;288(5472):1789-96.
16. Vidal N, Peeters M, Mulanga-Kabeya C, Nzilambi N, Robertson D, Ilunga W, et
al. Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group
M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1
pandemic originated in Central Africa. J Virol. 2000;74(22):10498-507.
17. Worobey M, Santiago ML, Keele BF, Ndjango JB, Joy JB, Labama BL, et al.
Origin of AIDS: contaminated polio vaccine theory refuted. Nature.
2004;428(6985):820.
18. Peeters M, Courgnaud V, Abela B, Auzel P, Pourrut X, Bibollet-Ruche F, et al.
Risk to human health from a plethora of simian immunodeficiency viruses in
primate bushmeat. Emerg Infect Dis. 2002;8(5):451-7.
19. Sharp PM, Hahn BH. Origins of HIV and the AIDS pandemic. Cold Spring Harb
Perspect Med. 2011;1(1):a006841.
47
20. Sharp PM, Robertson DL, Hahn BH. Cross-species transmission and recombination
of 'AIDS' viruses. Philos Trans R Soc Lond B Biol Sci. 1995;349(1327):41-7.
21. Simon F, Mauclere P, Roques P, Loussert-Ajaka I, Muller-Trutwin MC, Saragosti
S, et al. Identification of a new human immunodeficiency virus type 1 distinct from
group M and group O. Nat Med. 1998;4(9):1032-7.
22. Gurtler LG, Hauser PH, Eberle J, von Brunn A, Knapp S, Zekeng L, et al. A new
subtype of human immunodeficiency virus type 1 (MVP-5180) from Cameroon. J
Virol. 1994;68(3):1581-5.
23. Plantier JC, Leoz M, Dickerson JE, De Oliveira F, Cordonnier F, Lemee V, et al.
A new human immunodeficiency virus derived from gorillas. Nat Med.
2009;15(8):871-2.
24. Gao F, Bailes E, Robertson DL, Chen Y, Rodenburg CM, Michael SF, et al. Origin
of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature.
1999;397(6718):436-41.
25. Keele BF, Van Heuverswyn F, Li Y, Bailes E, Takehisa J, Santiago ML, et al.
Chimpanzee reservoirs of pandemic and nonpandemic HIV-1. Science.
2006;313(5786):523-6.
26. D'Arc M, Ayouba A, Esteban A, Learn GH, Boue V, Liegeois F, et al. Origin of
the HIV-1 group O epidemic in western lowland gorillas. Proc Natl Acad Sci U S
A. 2015;112(11):E1343-52.
27. Chen Z, Luckay A, Sodora DL, Telfer P, Reed P, Gettie A, et al. Human
immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a
distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency
virus-infected sooty mangabeys. J Virol. 1997;71(5):3953-60.
28. Gao F, Yue L, White AT, Pappas PG, Barchue J, Hanson AP, et al. Human infection
by genetically diverse SIVSM-related HIV-2 in west Africa. Nature.
1992;358(6386):495-9.
29. Clavel F, Guetard D, Brun-Vezinet F, Chamaret S, Rey MA, Santos-Ferreira MO,
et al. Isolation of a new human retrovirus from West African patients with AIDS.
Science. 1986;233(4761):343-6.
30. Donnelly C, Leisenring W, Kanki P, Awerbuch T, Sandberg S. Comparison of
transmission rates of HIV-1 and HIV-2 in a cohort of prostitutes in Senegal. Bull
Math Biol. 1993;55(4):731-43.
31. Kanki PJ, Travers KU, S MB, Hsieh CC, Marlink RG, Gueye NA, et al. Slower
heterosexual spread of HIV-2 than HIV-1. Lancet. 1994;343(8903):943-6.
32. Poulsen AG, Aaby P, Larsen O, Jensen H, Naucler A, Lisse IM, et al. 9-year HIV-
2-associated mortality in an urban community in Bissau, west Africa. Lancet.
1997;349(9056):911-4.
33. Poulsen AG, Kvinesdal B, Aaby P, Molbak K, Frederiksen K, Dias F, et al.
Prevalence of and mortality from human immunodeficiency virus type 2 in Bissau,
West Africa. Lancet. 1989;1(8642):827-31.
48
34. Marlink R, Kanki P, Thior I, Travers K, Eisen G, Siby T, et al. Reduced rate of
disease development after HIV-2 infection as compared to HIV-1. Science.
1994;265(5178):1587-90.
35. Hemelaar J. Implications of HIV diversity for the HIV-1 pandemic. J Infect.
2013;66(5):391-400.
36. Hemelaar J. The origin and diversity of the HIV-1 pandemic. Trends Mol Med.
2012;18(3):182-92.
37. Louwagie J, McCutchan FE, Peeters M, Brennan TP, Sanders-Buell E, Eddy GA,
et al. Phylogenetic analysis of gag genes from 70 international HIV-1 isolates
provides evidence for multiple genotypes. AIDS. 1993;7(6):769-80.
38. Buonaguro L, Tornesello ML, Buonaguro FM. Human immunodeficiency virus
type 1 subtype distribution in the worldwide epidemic: pathogenetic and
therapeutic implications. J Virol. 2007;81(19):10209-19.
39. Renjifo B, Gilbert P, Chaplin B, Msamanga G, Mwakagile D, Fawzi W, et al.
Preferential in-utero transmission of HIV-1 subtype C as compared to HIV-1
subtype A or D. AIDS. 2004;18(12):1629-36.
40. John-Stewart GC, Nduati RW, Rousseau CM, Mbori-Ngacha DA, Richardson BA,
Rainwater S, et al. Subtype C Is associated with increased vaginal shedding of HIV-
1. J Infect Dis. 2005;192(3):492-6.
41. Kaleebu P, French N, Mahe C, Yirrell D, Watera C, Lyagoba F, et al. Effect of
human immunodeficiency virus (HIV) type 1 envelope subtypes A and D on
disease progression in a large cohort of HIV-1-positive persons in Uganda. The
Journal of infectious diseases. 2002;185(9):1244-50.
42. Baeten JM, Chohan B, Lavreys L, Chohan V, McClelland RS, Certain L, et al. HIV-
1 subtype D infection is associated with faster disease progression than subtype A
in spite of similar plasma HIV-1 loads. J Infect Dis. 2007;195(8):1177-80.
43. Coffin JM HS, Varmus HE. Retroviruses. Cold Spring Harbor (NY): Cold Spring
Harbor Laboratory Press; 1997.
44. Kotterman MA, Chalberg TW, Schaffer DV. Viral Vectors for Gene Therapy:
Translational and Clinical Outlook. Annu Rev Biomed Eng. 2015;17:63-89.
45. Frankel AD, Young JA. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem.
1998;67:1-25.
46. Tang H, Kuhen KL, Wong-Staal F. Lentivirus replication and regulation. Annu Rev
Genet. 1999;33:133-70.
47. Rosen CA, Pavlakis GN. Tat and Rev: positive regulators of HIV gene expression.
AIDS. 1990;4(6):499-509.
48. Malim MH, Emerman M. HIV-1 accessory proteins - ensuring viral survival in a
hostile environment. Cell Host Microbe. 2008;3(6):388-98.
49. Kahn JO, Walker BD. Acute Human Immunodeficiency Virus Type 1 infection. N
Engl J Med. 1998;339(1):33-9.
49
50. Schacker T, Collier AC, Hughes J, Shea T, Corey L. Clinical and epidemiologic
features of primary HIV infection. Ann Intern Med. 1996;125(4):257-64.
51. Moss AR, Bacchetti P. Natural history of HIV infection. AIDS. 1989;3(2):55-61.
52. Maartens G, Celum C, Lewin SR. HIV infection: epidemiology, pathogenesis,
treatment, and prevention. Lancet. 2014;384(9939):258-71.
53. Centers for Disease C, Prevention. Revised surveillance case definition for HIV
infection - United States, 2014. MMWR Recomm Rep. 2014;63(RR-03):1-10.
54. Palella FJ, Jr., Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, et
al. Declining morbidity and mortality among patients with advanced human
immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J
Med. 1998;338(13):853-60.
55. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al.
Identification of a reservoir for HIV-1 in patients on highly active antiretroviral
therapy. Science. 1997;278(5341):1295-300.
56. HIV/AIDS JUNPo. 2016 Global AIDS Update. 2016.
57. Freed EO. HIV-1 gag proteins: diverse functions in the virus life cycle. Virology.
1998;251(1):1-15.
58. Klasse PJ. The molecular basis of HIV entry. Cell Microbiol. 2012;14(8):1183-92.
59. Klatzmann D, Champagne E, Chamaret S, Gruest J, Guetard D, Hercend T, et al.
T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV.
Nature. 1984;312(5996):767-8.
60. Dalgleish AG, Beverley PC, Clapham PR, Crawford DH, Greaves MF, Weiss RA.
The CD4 (T4) antigen is an essential component of the receptor for the AIDS
retrovirus. Nature. 1984;312(5996):763-7.
61. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification
of a major co-receptor for primary isolates of HIV-1. Nature. 1996;381(6584):661-
6.
62. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional
cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science.
1996;272(5263):872-7.
63. Pereira LA, Bentley K, Peeters A, Churchill MJ, Deacon NJ. A compilation of
cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic
Acids Res. 2000;28(3):663-8.
64. Desrosiers RC, Lifson JD, Gibbs JS, Czajak SC, Howe AY, Arthur LO, et al.
Identification of highly attenuated mutants of simian immunodeficiency virus. J
Virol. 1998;72(2):1431-7.
65. Lang SM, Weeger M, Stahl-Hennig C, Coulibaly C, Hunsmann G, Muller J, et al.
Importance of vpr for infection of rhesus monkeys with simian immunodeficiency
virus. J Virol. 1993;67(2):902-12.
50
66. Stephens EB, McCormick C, Pacyniak E, Griffin D, Pinson DM, Sun F, et al.
Deletion of the vpu sequences prior to the env in a simian-human
immunodeficiency virus results in enhanced Env precursor synthesis but is less
pathogenic for pig-tailed macaques. Virology. 2002;293(2):252-61.
67. Hout DR, Gomez ML, Pacyniak E, Gomez LM, Inbody SH, Mulcahy ER, et al.
Scrambling of the amino acids within the transmembrane domain of Vpu results in
a simian-human immunodeficiency virus (SHIVTM) that is less pathogenic for pig-
tailed macaques. Virology. 2005;339(1):56-69.
68. Dave VP, Hajjar F, Dieng MM, Haddad E, Cohen EA. Efficient BST2 antagonism
by Vpu is critical for early HIV-1 dissemination in humanized mice. Retrovirology.
2013;10:128.
69. Tokarev A, Guatelli J. Misdirection of membrane trafficking by HIV-1 Vpu and
Nef: Keys to viral virulence and persistence. Cell Logist. 2011;1(3):90-102.
70. Ogawa K, Shibata R, Kiyomasu T, Higuchi I, Kishida Y, Ishimoto A, et al.
Mutational analysis of the human immunodeficiency virus vpr open reading frame.
J Virol. 1989;63(9):4110-4.
71. Wong-Staal F, Chanda PK, Ghrayeb J. Human immunodeficiency virus: the eighth
gene. AIDS Res Hum Retroviruses. 1987;3(1):33-9.
72. Bachand F, Yao XJ, Hrimech M, Rougeau N, Cohen EA. Incorporation of Vpr into
human immunodeficiency virus type 1 requires a direct interaction with the p6
domain of the p55 gag precursor. J Biol Chem. 1999;274(13):9083-91.
73. Cohen EA, Dehni G, Sodroski JG, Haseltine WA. Human immunodeficiency virus
vpr product is a virion-associated regulatory protein. J Virol. 1990;64(6):3097-9.
74. Guenzel CA, Herate C, Benichou S. HIV-1 Vpr - a still "enigmatic multitasker".
Front Microbiol. 2014;5:127.
75. Eckstein DA, Sherman MP, Penn ML, Chin PS, De Noronha CM, Greene WC, et
al. HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages
but not nondividing CD4+ T cells. J Exp Med. 2001;194(10):1407-19.
76. Mashiba M, Collins DR, Terry VH, Collins KL. Vpr overcomes macrophage-
specific restriction of HIV-1 Env expression and virion production. Cell Host
Microbe. 2014;16(6):722-35.
77. Collins DR, Lubow J, Lukic Z, Mashiba M, Collins KL. Vpr Promotes
Macrophage-Dependent HIV-1 Infection of CD4+ T Lymphocytes. PLoS Pathog.
2015;11(7):e1005054.
78. Zhang X, Zhou T, Frabutt DA, Zheng YH. HIV-1 Vpr increases Env expression by
preventing Env from endoplasmic reticulum-associated protein degradation
(ERAD). Virology. 2016;496:194-202.
79. He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR. Human
immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase
of the cell cycle by inhibiting p34cdc2 activity. J Virol. 1995;69(11):6705-11.
51
80. Rogel ME, Wu LI, Emerman M. The human immunodeficiency virus type 1 vpr
gene prevents cell proliferation during chronic infection. J Virol. 1995;69(2):882-
8.
81. Muthumani K, Choo AY, Premkumar A, Hwang DS, Thieu KP, Desai BM, et al.
Human immunodeficiency virus type 1 (HIV-1) Vpr-regulated cell death: insights
into mechanism. Cell Death Differ. 2005;12 Suppl 1:962-70.
82. Wu Y, Zhou X, Barnes CO, DeLucia M, Cohen AE, Gronenborn AM, et al. The
DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human
UNG2 toward destruction. Nat Struct Mol Biol. 2016;23(10):933-40.
83. Lahouassa H, Blondot ML, Chauveau L, Chougui G, Morel M, Leduc M, et al.
HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages. Proc
Natl Acad Sci U S A. 2016;113(19):5311-6.
84. Zhou X, DeLucia M, Hao C, Hrecka K, Monnie C, Skowronski J, et al. HIV-1 Vpr
protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-
DCAF1 E3 ubiquitin ligase. J Biol Chem. 2017;292(51):21117-27.
85. Laguette N, Bregnard C, Hue P, Basbous J, Yatim A, Larroque M, et al. Premature
activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from
innate immune sensing. Cell. 2014;156(1-2):134-45.
86. Fregoso OI, Emerman M. Activation of the DNA Damage Response Is a Conserved
Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment.
MBio. 2016;7(5).
87. Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that
inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature.
2002;418(6898):646-50.
88. Sheehy AM, Gaddis NC, Malim MH. The antiretroviral enzyme APOBEC3G is
degraded by the proteasome in response to HIV-1 Vif. Nat Med. 2003;9(11):1404-
7.
89. Stavrou S, Ross SR. APOBEC3 Proteins in Viral Immunity. J Immunol.
2015;195(10):4565-70.
90. Feng Y, Baig TT, Love RP, Chelico L. Suppression of APOBEC3-mediated
restriction of HIV-1 by Vif. Front Microbiol. 2014;5:450.
91. Matheson NJ, Sumner J, Wals K, Rapiteanu R, Weekes MP, Vigan R, et al. Cell
Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid
Metabolism by Vpu and Nef. Cell Host Microbe. 2015;18(4):409-23.
92. Haller C, Muller B, Fritz JV, Lamas-Murua M, Stolp B, Pujol FM, et al. HIV-1 Nef
and Vpu are functionally redundant broad-spectrum modulators of cell surface
receptors, including tetraspanins. J Virol. 2014;88(24):14241-57.
93. Saxena R, Gupta S, Singh K, Mitra K, Tripathi AK, Tripathi RK. Proteomic
profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.
PLoS One. 2015;10(4):e0122994.
52
94. Terwilliger E, Sodroski JG, Rosen CA, Haseltine WA. Effects of mutations within
the 3' orf open reading frame region of human T-cell lymphotropic virus type III
(HTLV-III/LAV) on replication and cytopathogenicity. J Virol. 1986;60(2):754-
60.
95. Luciw PA, Cheng-Mayer C, Levy JA. Mutational analysis of the human
immunodeficiency virus: the orf-B region down-regulates virus replication. Proc
Natl Acad Sci U S A. 1987;84(5):1434-8.
96. Cheng-Mayer C, Iannello P, Shaw K, Luciw PA, Levy JA. Differential effects of
nef on HIV replication: implications for viral pathogenesis in the host. Science.
1989;246(4937):1629-32.
97. Cohen EA, Terwilliger EF, Sodroski JG, Haseltine WA. Identification of a protein
encoded by the vpu gene of HIV-1. Nature. 1988;334(6182):532-4.
98. Strebel K, Klimkait T, Martin MA. A novel gene of HIV-1, vpu, and its 16-
kilodalton product. Science. 1988;241(4870):1221-3.
99. Allan JS, Coligan JE, Lee TH, McLane MF, Kanki PJ, Groopman JE, et al. A new
HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients.
Science. 1985;230(4727):810-3.
100. Maldarelli F, Chen MY, Willey RL, Strebel K. Human immunodeficiency virus
type 1 Vpu protein is an oligomeric type I integral membrane protein. J Virol.
1993;67(8):5056-61.
101. Yu G, Felsted RL. Effect of myristoylation on p27 nef subcellular distribution and
suppression of HIV-LTR transcription. Virology. 1992;187(1):46-55.
102. Schwartz O, Marechal V, Le Gall S, Lemonnier F, Heard JM. Endocytosis of major
histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.
Nat Med. 1996;2(3):338-42.
103. Kerkau T, Bacik I, Bennink JR, Yewdell JW, Hunig T, Schimpl A, et al. The human
immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step
in the biosynthesis of major histocompatibility complex (MHC) class I molecules.
J Exp Med. 1997;185(7):1295-305.
104. Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D. HIV-1 Nef protein
protects infected primary cells against killing by cytotoxic T lymphocytes. Nature.
1998;391(6665):397-401.
105. Michel N, Ganter K, Venzke S, Bitzegeio J, Fackler OT, Keppler OT. The Nef
protein of human immunodeficiency virus is a broad-spectrum modulator of
chemokine receptor cell surface levels that acts independently of classical motifs
for receptor endocytosis and Galphai signaling. Mol Biol Cell. 2006;17(8):3578-
90.
106. Cerboni C, Neri F, Casartelli N, Zingoni A, Cosman D, Rossi P, et al. Human
immunodeficiency virus 1 Nef protein downmodulates the ligands of the activating
receptor NKG2D and inhibits natural killer cell-mediated cytotoxicity. J Gen Virol.
2007;88(Pt 1):242-50.
53
107. Fausther-Bovendo H, Sol-Foulon N, Candotti D, Agut H, Schwartz O, Debre P, et
al. HIV escape from natural killer cytotoxicity: nef inhibits NKp44L expression on
CD4+ T cells. AIDS. 2009;23(9):1077-87.
108. Shah AH, Sowrirajan B, Davis ZB, Ward JP, Campbell EM, Planelles V, et al.
Degranulation of natural killer cells following interaction with HIV-1-infected cells
is hindered by downmodulation of NTB-A by Vpu. Cell Host Microbe.
2010;8(5):397-409.
109. Matusali G, Potesta M, Santoni A, Cerboni C, Doria M. The human
immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural
killer cell-activating ligand PVR. J Virol. 2012;86(8):4496-504.
110. Alsahafi N, Richard J, Prevost J, Coutu M, Brassard N, Parsons MS, et al. Impaired
Downregulation of NKG2D Ligands by Nef Proteins from Elite Controllers
Sensitizes HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity. J
Virol. 2017;91(16).
111. Veillette M, Desormeaux A, Medjahed H, Gharsallah NE, Coutu M, Baalwa J, et
al. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by
antibody-dependent cell-mediated cytotoxicity. J Virol. 2014;88(5):2633-44.
112. Veillette M, Coutu M, Richard J, Batraville LA, Dagher O, Bernard N, et al. The
HIV-1 gp120 CD4-bound conformation is preferentially targeted by antibody-
dependent cellular cytotoxicity-mediating antibodies in sera from HIV-1-infected
individuals. J Virol. 2015;89(1):545-51.
113. Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, et al.
The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated
from the cell surface by the viral Vpu protein. Cell Host Microbe. 2008;3(4):245-
52.
114. Rosa A, Chande A, Ziglio S, De Sanctis V, Bertorelli R, Goh SL, et al. HIV-1 Nef
promotes infection by excluding SERINC5 from virion incorporation. Nature.
2015;526(7572):212-7.
115. Neil SJ, Zang T, Bieniasz PD. Tetherin inhibits retrovirus release and is
antagonized by HIV-1 Vpu. Nature. 2008;451(7177):425-30.
116. Usami Y, Wu Y, Gottlinger HG. SERINC3 and SERINC5 restrict HIV-1 infectivity
and are counteracted by Nef. Nature. 2015;526(7572):218-23.
117. Chaudhry A, Das SR, Hussain A, Mayor S, George A, Bal V, et al. The Nef protein
of HIV-1 induces loss of cell surface costimulatory molecules CD80 and CD86 in
APCs. J Immunol. 2005;175(7):4566-74.
118. Swigut T, Shohdy N, Skowronski J. Mechanism for down-regulation of CD28 by
Nef. EMBO J. 2001;20(7):1593-604.
119. Stumptner-Cuvelette P, Morchoisne S, Dugast M, Le Gall S, Raposo G, Schwartz
O, et al. HIV-1 Nef impairs MHC class II antigen presentation and surface
expression. Proc Natl Acad Sci U S A. 2001;98(21):12144-9.
54
120. Hewitt EW. The MHC class I antigen presentation pathway: strategies for viral
immune evasion. Immunology. 2003;110(2):163-9.
121. Hung CH, Thomas L, Ruby CE, Atkins KM, MorriS NP, Knight ZA, et al. HIV-1
nef assembles a src family Kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-
surface MHC-I. Cell Host Microbe. 2007;1(2):121-33.
122. Atkins KM, Thomas L, Youker RT, Harriff MJ, Pissani F, You HH, et al. HIV-1
Nef binds PACS-2 to assemble a multikinase cascade that triggers major
histocompatibility complex class I (MHC-I) down-regulation - Analysis using short
interfering RNA and knock-out mice. J Biol Chem. 2008;283(17):11772-84.
123. Dikeakos JD, Thomas L, Kwon G, Elferich J, Shinde U, Thomas G. An interdomain
binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to
down-regulate MHC-I. Mol Biol Cell. 2012;23(11):2184-97.
124. Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, Strominger JL, et
al. The selective downregulation of class I major histocompatibility complex
proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity.
1999;10(6):661-71.
125. Apps R, Del Prete GQ, Chatterjee P, Lara A, Brumme ZL, Brockman MA, et al.
HIV-1 Vpu Mediates HLA-C Downregulation. Cell Host Microbe.
2016;19(5):686-95.
126. Whitmire JK. Induction and function of virus-specific CD4+ T cell responses.
Virology. 2011;411(2):216-28.
127. Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev
Immunol. 2013;31:443-73.
128. Hussain A, Wesley C, Khalid M, Chaudhry A, Jameel S. Human immunodeficiency
virus type 1 Vpu protein interacts with CD74 and modulates major
histocompatibility complex class II presentation. J Virol. 2008;82(2):893-902.
129. Stumptner-Cuvelette P, Jouve M, Helft J, Dugast M, Glouzman AS, Jooss K, et al.
Human immunodeficiency virus-1 Nef expression induces intracellular
accumulation of multivesicular bodies and major histocompatibility complex class
II complexes: potential role of phosphatidylinositol 3-kinase. Mol Biol Cell.
2003;14(12):4857-70.
130. Chaudhry A, Verghese DA, Das SR, Jameel S, George A, Bal V, et al. HIV-1 Nef
promotes endocytosis of cell surface MHC class II molecules via a constitutive
pathway. J Immunol. 2009;183(4):2415-24.
131. Toussaint H, Gobert FX, Schindler M, Banning C, Kozik P, Jouve M, et al. Human
immunodeficiency virus type 1 nef expression prevents AP-2-mediated
internalization of the major histocompatibility complex class II-associated invariant
chain. J Virol. 2008;82(17):8373-82.
132. Schindler M, Wildum S, Casartelli N, Doria M, Kirchhoff F. Nef alleles from
children with non-progressive HIV-1 infection modulate MHC-II expression more
efficiently than those from rapid progressors. AIDS. 2007;21(9):1103-7.
55
133. Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA.
Structure of an HIV gp120 envelope glycoprotein in complex with the CD4
receptor and a neutralizing human antibody. Nature. 1998;393(6686):648-59.
134. Stein BS, Gowda SD, Lifson JD, Penhallow RC, Bensch KG, Engleman EG. pH-
independent HIV entry into CD4-positive T cells via virus envelope fusion to the
plasma membrane. Cell. 1987;49(5):659-68.
135. Doms RW, Moore JP. HIV-1 membrane fusion: targets of opportunity. J Cell Biol.
2000;151(2):F9-14.
136. Richard J, Prevost J, Alsahafi N, Ding S, Finzi A. Impact of HIV-1 Envelope
Conformation on ADCC Responses. Trends Microbiol. 2018;26(4):253-65.
137. Prevost J, Richard J, Medjahed H, Alexander A, Jones J, Kappes JC, et al.
Incomplete Downregulation of CD4 Expression Affects HIV-1 Env Conformation
and Antibody-Dependent Cellular Cytotoxicity Responses. J Virol. 2018;92(13).
138. Alsahafi N, Ding S, Richard J, Markle T, Brassard N, Walker B, et al. Nef Proteins
from HIV-1 Elite Controllers Are Inefficient at Preventing Antibody-Dependent
Cellular Cytotoxicity. J Virol. 2015;90(6):2993-3002.
139. Schubert U, Anton LC, Bacik I, Cox JH, Bour S, Bennink JR, et al. CD4
glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu
protein requires the function of proteasomes and the ubiquitin-conjugating
pathway. J Virol. 1998;72(3):2280-8.
140. Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeficiency virus
type 1 Vpu protein induces rapid degradation of CD4. J Virol. 1992;66(12):7193-
200.
141. Rhee SS, Marsh JW. Human immunodeficiency virus type 1 Nef-induced down-
modulation of CD4 is due to rapid internalization and degradation of surface CD4.
J Virol. 1994;68(8):5156-63.
142. Brandstadter JD, Yang Y. Natural killer cell responses to viral infection. J Innate
Immun. 2011;3(3):274-9.
143. Bolduan S, Hubel P, Reif T, Lodermeyer V, Hohne K, Fritz JV, et al. HIV-1 Vpu
affects the anterograde transport and the glycosylation pattern of NTB-A. Virology.
2013;440(2):190-203.
144. Sugden SM, Pham TN, Cohen EA. HIV-1 Vpu Downmodulates ICAM-1
Expression, Resulting in Decreased Killing of Infected CD4(+) T Cells by NK
Cells. J Virol. 2017;91(8).
145. Bolduan S, Reif T, Schindler M, Schubert U. HIV-1 Vpu mediated downregulation
of CD155 requires alanine residues 10, 14 and 18 of the transmembrane domain.
Virology. 2014;464-465:375-84.
146. von Andrian UH, Mempel TR. Homing and cellular traffic in lymph nodes. Nat
Rev Immunol. 2003;3(11):867-78.
56
147. Vassena L, Giuliani E, Koppensteiner H, Bolduan S, Schindler M, Doria M. HIV-
1 Nef and Vpu Interfere with L-Selectin (CD62L) Cell Surface Expression To
Inhibit Adhesion and Signaling in Infected CD4+ T Lymphocytes. J Virol.
2015;89(10):5687-700.
148. Chandrasekaran P, Moore V, Buckley M, Spurrier J, Kehrl JH, Venkatesan S. HIV-
1 Nef down-modulates C-C and C-X-C chemokine receptors via ubiquitin and
ubiquitin-independent mechanism. PLoS One. 2014;9(1):e86998.
149. Toyoda M, Ogata Y, Mahiti M, Maeda Y, Kuang XT, Miura T, et al. Differential
Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors. J
Virol. 2015;89(18):9639-52.
150. Venzke S, Michel N, Allespach I, Fackler OT, Keppler OT. Expression of Nef
downregulates CXCR4, the major coreceptor of human immunodeficiency virus,
from the surfaces of target cells and thereby enhances resistance to superinfection.
J Virol. 2006;80(22):11141-52.
151. Michel N, Allespach I, Venzke S, Fackler OT, Keppler OT. The Nef protein of
human immunodeficiency virus establishes superinfection immunity by a dual
strategy to downregulate cell-surface CCR5 and CD4. Curr Biol. 2005;15(8):714-
23.
152. Stolp B, Reichman-Fried M, Abraham L, Pan X, Giese SI, Hannemann S, et al.
HIV-1 Nef interferes with host cell motility by deregulation of Cofilin. Cell Host
Microbe. 2009;6(2):174-86.
153. Choe EY, Schoenberger ES, Groopman JE, Park IW. HIV Nef inhibits T cell
migration. J Biol Chem. 2002;277(48):46079-84.
154. Ramirez PW, Famiglietti M, Sowrirajan B, DePaula-Silva AB, Rodesch C, Barker
E, et al. Downmodulation of CCR7 by HIV-1 Vpu results in impaired migration
and chemotactic signaling within CD4(+) T cells. Cell Rep. 2014;7(6):2019-30.
155. Doyle T, Goujon C, Malim MH. HIV-1 and interferons: who's interfering with
whom? Nat Rev Microbiol. 2015;13(7):403-13.
156. Swiecki M, Omattage NS, Brett TJ. BST-2/tetherin: structural biology, viral
antagonism, and immunobiology of a potent host antiviral factor. Mol Immunol.
2013;54(2):132-9.
157. Pujol FM, Laketa V, Schmidt F, Mukenhirn M, Muller B, Boulant S, et al. HIV-1
Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from
Virus Assembly Sites. J Virol. 2016;90(15):6709-23.
158. Lewinski MK, Jafari M, Zhang H, Opella SJ, Guatelli J. Membrane Anchoring by
a C-terminal Tryptophan Enables HIV-1 Vpu to Displace Bone Marrow Stromal
Antigen 2 (BST2) from Sites of Viral Assembly. J Biol Chem.
2015;290(17):10919-33.
159. Dube M, Paquay C, Roy BB, Bego MG, Mercier J, Cohen EA. HIV-1 Vpu
antagonizes BST-2 by interfering mainly with the trafficking of newly synthesized
BST-2 to the cell surface. Traffic. 2011;12(12):1714-29.
57
160. Dube M, Roy BB, Guiot-Guillain P, Binette J, Mercier J, Chiasson A, et al.
Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and
sequestration of the restriction factor in a perinuclear compartment. PLoS Pathog.
2010;6(4):e1000856.
161. Douglas JL, Viswanathan K, McCarroll MN, Gustin JK, Fruh K, Moses AV. Vpu
directs the degradation of the human immunodeficiency virus restriction factor
BST-2/Tetherin via a {beta}TrCP-dependent mechanism. J Virol.
2009;83(16):7931-47.
162. Iwabu Y, Fujita H, Kinomoto M, Kaneko K, Ishizaka Y, Tanaka Y, et al. HIV-1
accessory protein Vpu internalizes cell-surface BST-2/tetherin through
transmembrane interactions leading to lysosomes. J Biol Chem.
2009;284(50):35060-72.
163. Rollason R, Dunstan K, Billcliff PG, Bishop P, Gleeson P, Wise H, et al. Expression
of HIV-1 Vpu leads to loss of the viral restriction factor CD317/Tetherin from lipid
rafts and its enhanced lysosomal degradation. PLoS One. 2013;8(9):e75680.
164. Pham TN, Lukhele S, Dallaire F, Perron G, Cohen EA. Enhancing Virion Tethering
by BST2 Sensitizes Productively and Latently HIV-infected T cells to ADCC
Mediated by Broadly Neutralizing Antibodies. Sci Rep. 2016;6:37225.
165. Yamada E, Nakaoka S, Klein L, Reith E, Langer S, Hopfensperger K, et al. Human-
Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for
Efficient Early HIV-1 Replication In Vivo. Cell Host Microbe. 2018;23(1):110-20
e7.
166. Chowers MY, Spina CA, Kwoh TJ, Fitch NJ, Richman DD, Guatelli JC. Optimal
infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef
gene. J Virol. 1994;68(5):2906-14.
167. Miller MD, Warmerdam MT, Gaston I, Greene WC, Feinberg MB. The human
immunodeficiency virus-1 nef gene product: a positive factor for viral infection and
replication in primary lymphocytes and macrophages. J Exp Med.
1994;179(1):101-13.
168. Trautz B, Pierini V, Wombacher R, Stolp B, Chase AJ, Pizzato M, et al. The
Antagonism of HIV-1 Nef to SERINC5 Particle Infectivity Restriction Involves the
Counteraction of Virion-Associated Pools of the Restriction Factor. J Virol.
2016;90(23):10915-27.
169. Shi J, Xiong R, Zhou T, Su P, Zhang X, Qiu X, et al. HIV-1 Nef antagonizes
SERINC5 restriction by downregulation of SERINC5 via the endosome/lysosome
system. J Virol. 2018.
170. Sood C, Marin M, Chande A, Pizzato M, Melikyan GB. SERINC5 protein inhibits
HIV-1 fusion pore formation by promoting functional inactivation of envelope
glycoproteins. J Biol Chem. 2017;292(14):6014-26.
171. Beitari S, Ding S, Pan Q, Finzi A, Liang C. Effect of HIV-1 Env on SERINC5
Antagonism. J Virol. 2017;91(4).
58
172. McDougal JS, Mawle A, Cort SP, Nicholson JK, Cross GD, Scheppler-Campbell
JA, et al. Cellular tropism of the human retrovirus HTLV-III/LAV. I. Role of T cell
activation and expression of the T4 antigen. J Immunol. 1985;135(5):3151-62.
173. Bromley SK, Burack WR, Johnson KG, Somersalo K, Sims TN, Sumen C, et al.
The immunological synapse. Annu Rev Immunol. 2001;19:375-96.
174. Nabel G, Baltimore D. An inducible transcription factor activates expression of
human immunodeficiency virus in T cells. Nature. 1987;326(6114):711-3.
175. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB. Tumor necrosis factor alpha
activates human immunodeficiency virus type 1 through induction of nuclear factor
binding to the NF-kappa B sites in the long terminal repeat. Proc Natl Acad Sci U
S A. 1989;86(15):5974-8.
176. Tong-Starksen SE, Luciw PA, Peterlin BM. Human immunodeficiency virus long
terminal repeat responds to T-cell activation signals. Proc Natl Acad Sci U S A.
1987;84(19):6845-9.
177. Thoulouze MI, Sol-Foulon N, Blanchet F, Dautry-Varsat A, Schwartz O, Alcover
A. Human immunodeficiency virus type-1 infection impairs the formation of the
immunological synapse. Immunity. 2006;24(5):547-61.
178. Khan N, Gowthaman U, Pahari S, Agrewala JN. Manipulation of costimulatory
molecules by intracellular pathogens: veni, vidi, vici!! PLoS Pathog.
2012;8(6):e1002676.
179. Boomer JS, Green JM. An enigmatic tail of CD28 signaling. Cold Spring Harb
Perspect Biol. 2010;2(8):a002436.
180. El-Far M, Isabelle C, Chomont N, Bourbonniere M, Fonseca S, Ancuta P, et al.
Down-regulation of CTLA-4 by HIV-1 Nef protein. PLoS One. 2013;8(1):e54295.
181. Leonard JA, Filzen T, Carter CC, Schaefer M, Collins KL. HIV-1 Nef disrupts
intracellular trafficking of major histocompatibility complex class I, CD4, CD8,
and CD28 by distinct pathways that share common elements. J Virol.
2011;85(14):6867-81.
182. Chaudhry A, Das SR, Jameel S, George A, Bal V, Mayor S, et al. A two-pronged
mechanism for HIV-1 Nef-mediated endocytosis of immune costimulatory
molecules CD80 and CD86. Cell Host Microbe. 2007;1(1):37-49.
183. Chaudhry A, Das SR, Jameel S, George A, Bal V, Mayor S, et al. HIV-1 Nef
induces a Rab11-dependent routing of endocytosed immune costimulatory proteins
CD80 and CD86 to the Golgi. Traffic. 2008;9(11):1925-35.
184. Rudolph JM, Eickel N, Haller C, Schindler M, Fackler OT. Inhibition of T-cell
receptor-induced actin remodeling and relocalization of Lck are evolutionarily
conserved activities of lentiviral Nef proteins. J Virol. 2009;83(22):11528-39.
185. Neri F, Giolo G, Potesta M, Petrini S, Doria M. The HIV-1 Nef protein has a dual
role in T cell receptor signaling in infected CD4+ T lymphocytes. Virology.
2011;410(2):316-26.
59
186. Pan X, Rudolph JM, Abraham L, Habermann A, Haller C, Krijnse-Locker J, et al.
HIV-1 Nef compensates for disorganization of the immunological synapse by
inducing trans-Golgi network-associated Lck signaling. Blood. 2012;119(3):786-
97.
187. Haller C, Rauch S, Michel N, Hannemann S, Lehmann MJ, Keppler OT, et al. The
HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-
lymphocyte contacts by modulation of N-Wasp activity. J Biol Chem.
2006;281(28):19618-30.
188. Imle A, Abraham L, Tsopoulidis N, Hoflack B, Saksela K, Fackler OT. Association
with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the
Exocyst Complex. MBio. 2015;6(5):e01309-15.
189. Fackler OT, Alcover A, Schwartz O. Modulation of the immunological synapse: a
key to HIV-1 pathogenesis? Nat Rev Immunol. 2007;7(4):310-7.
190. Schrager JA, Marsh JW. HIV-1 Nef increases T cell activation in a stimulus-
dependent manner. Proc Natl Acad Sci U S A. 1999;96(14):8167-72.
191. Wang JK, Kiyokawa E, Verdin E, Trono D. The Nef protein of HIV-1 associates
with rafts and primes T cells for activation. Proc Natl Acad Sci U S A.
2000;97(1):394-9.
192. Schibeci SD, Clegg AO, Biti RA, Sagawa K, Stewart GJ, Williamson P. HIV-Nef
enhances interleukin-2 production and phosphatidylinositol 3-kinase activity in a
human T cell line. AIDS. 2000;14(12):1701-7.
193. Simmons A, Aluvihare V, McMichael A. Nef triggers a transcriptional program in
T cells imitating single-signal T cell activation and inducing HIV virulence
mediators. Immunity. 2001;14(6):763-77.
194. Olivieri KC, Mukerji J, Gabuzda D. Nef-mediated enhancement of cellular
activation and human immunodeficiency virus type 1 replication in primary T cells
is dependent on association with p21-activated kinase 2. Retrovirology. 2011;8:64.
195. Abraham L, Fackler OT. HIV-1 Nef: a multifaceted modulator of T cell receptor
signaling. Cell Commun Signal. 2012;10(1):39.
196. Galao RP, Le Tortorec A, Pickering S, Kueck T, Neil SJ. Innate sensing of HIV-1
assembly by Tetherin induces NFkappaB-dependent proinflammatory responses.
Cell Host Microbe. 2012;12(5):633-44.
197. Bour S, Perrin C, Akari H, Strebel K. The human immunodeficiency virus type 1
Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated
degradation of Ikappa B. J Biol Chem. 2001;276(19):15920-8.
198. Sauter D, Hotter D, Van Driessche B, Sturzel CM, Kluge SF, Wildum S, et al.
Differential regulation of NF-kappaB-mediated proviral and antiviral host gene
expression by primate lentiviral Nef and Vpu proteins. Cell Rep. 2015;10(4):586-
99.
199. Wu Y, Marsh JW. Selective transcription and modulation of resting T cell activity
by preintegrated HIV DNA. Science. 2001;293(5534):1503-6.
60
200. Spina CA, Guatelli JC, Richman DD. Establishment of a stable, inducible form of
human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in
vitro. J Virol. 1995;69(5):2977-88.
201. Arrigo SJ, Chen IS. Rev is necessary for translation but not cytoplasmic
accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 1991;5(5):808-
19.
202. Heusinger E, Kirchhoff F. Primate Lentiviruses Modulate NF-kappaB Activity by
Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression. Front
Microbiol. 2017;8:198.
203. Sauter D, Kirchhoff F. Multilayered and versatile inhibition of cellular antiviral
factors by HIV and SIV accessory proteins. Cytokine Growth Factor Rev. 2018.
204. Abbas W, Herbein G. T-Cell Signaling in HIV-1 Infection. Open Virol J.
2013;7:57-71.
205. Verma S, Ali A, Arora S, Banerjea AC. Inhibition of beta-TrcP-dependent
ubiquitination of p53 by HIV-1 Vpu promotes p53-mediated apoptosis in human T
cells. Blood. 2011;117(24):6600-7.
206. Greenway AL, McPhee DA, Allen K, Johnstone R, Holloway G, Mills J, et al.
Human immunodeficiency virus type 1 Nef binds to tumor suppressor p53 and
protects cells against p53-mediated apoptosis. J Virol. 2002;76(6):2692-702.
207. Wolf D, Witte V, Laffert B, Blume K, Stromer E, Trapp S, et al. HIV-1 Nef
associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation
to induce anti-apoptotic signals. Nat Med. 2001;7(11):1217-24.
208. Geleziunas R, Xu W, Takeda K, Ichijo H, Greene WC. HIV-1 Nef inhibits ASK1-
dependent death signalling providing a potential mechanism for protecting the
infected host cell. Nature. 2001;410(6830):834-8.
209. Jacob RA, Johnson AL, Pawlak EN, Dirk BS, Van Nynatten LR, Haeryfar SMM,
et al. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-
mediated CD4+ T cell apoptosis. Virology. 2017;509:1-10.
210. Casella CR, Rapaport EL, Finkel TH. Vpu increases susceptibility of human
immunodeficiency virus type 1-infected cells to fas killing. J Virol. 1999;73(1):92-
100.
211. Zauli G, Gibellini D, Secchiero P, Dutartre H, Olive D, Capitani S, et al. Human
immunodeficiency virus type 1 Nef protein sensitizes CD4(+) T lymphoid cells to
apoptosis via functional upregulation of the CD95/CD95 ligand pathway. Blood.
1999;93(3):1000-10.
212. Aries SP, Schaaf B, Muller C, Dennin RH, Dalhoff K. Fas (CD95) expression on
CD4+ T cells from HIV-infected patients increases with disease progression. J Mol
Med (Berl). 1995;73(12):591-3.
213. Rasola A, Gramaglia D, Boccaccio C, Comoglio PM. Apoptosis enhancement by
the HIV-1 Nef protein. J Immunol. 2001;166(1):81-8.
61
214. Arenaccio C, Chiozzini C, Columba-Cabezas S, Manfredi F, Affabris E, Baur A, et
al. Exosomes from human immunodeficiency virus type 1 (HIV-1)-infected cells
license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef- and
ADAM17-dependent mechanism. J Virol. 2014;88(19):11529-39.
215. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, et al. A
metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.
Nature. 1997;385(6618):729-33.
216. Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, et al. Cloning
of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-
alpha. Nature. 1997;385(6618):733-6.
217. Lee JH, Schierer S, Blume K, Dindorf J, Wittki S, Xiang W, et al. HIV-Nef and
ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with
Immune Pathogenesis in Chronic HIV Infection. EBioMedicine. 2016;6:103-13.
218. Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives.
Trends Cell Biol. 2001;11(9):372-7.
219. Sauter D, Schindler M, Specht A, Landford WN, Munch J, Kim KA, et al. Tetherin-
driven adaptation of Vpu and Nef function and the evolution of pandemic and
nonpandemic HIV-1 strains. Cell Host Microbe. 2009;6(5):409-21.
220. Jia B, Serra-Moreno R, Neidermyer W, Rahmberg A, Mackey J, Fofana IB, et al.
Species-specific activity of SIV Nef and HIV-1 Vpu in overcoming restriction by
tetherin/BST2. PLoS Pathog. 2009;5(5):e1000429.
221. Zhang F, Wilson SJ, Landford WC, Virgen B, Gregory D, Johnson MC, et al. Nef
proteins from simian immunodeficiency viruses are tetherin antagonists. Cell Host
Microbe. 2009;6(1):54-67.
222. Arias JF, Colomer-Lluch M, von Bredow B, Greene JM, MacDonald J, O'Connor
DH, et al. Tetherin Antagonism by HIV-1 Group M Nef Proteins. J Virol. 2016.
223. Mack K, Starz K, Sauter D, Langer S, Bibollet-Ruche F, Learn GH, et al. Efficient
Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain. J Virol.
2017;91(6).
224. Bego MG, Cong L, Mack K, Kirchhoff F, Cohen EA. Differential Control of BST2
Restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists
Encoded by HIV-1 Group M and O Strains. J Virol. 2016;90(22):10236-46.
225. Kluge SF, Mack K, Iyer SS, Pujol FM, Heigele A, Learn GH, et al. Nef proteins of
epidemic HIV-1 group O strains antagonize human tetherin. Cell Host Microbe.
2014;16(5):639-50.
226. Schaefer TM, Bell I, Pfeifer ME, Ghosh M, Trible RP, Fuller CL, et al. The
conserved process of TCR/CD3 complex down-modulation by SIV Nef is mediated
by the central core, not endocytic motifs. Virology. 2002;302(1):106-22.
227. Swigut T, Greenberg M, Skowronski J. Cooperative interactions of simian
immunodeficiency virus Nef, AP-2, and CD3-zeta mediate the selective induction
of T-cell receptor-CD3 endocytosis. J Virol. 2003;77(14):8116-26.
62
228. Kirchhoff F. Is the high virulence of HIV-1 an unfortunate coincidence of primate
lentiviral evolution? Nat Rev Microbiol. 2009;7(6):467-76.
229. Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, et al. Nef-
mediated suppression of T cell activation was lost in a lentiviral lineage that gave
rise to HIV-1. Cell. 2006;125(6):1055-67.
230. Arhel N, Lehmann M, Clauss K, Nienhaus GU, Piguet V, Kirchhoff F. The inability
to disrupt the immunological synapse between infected human T cells and APCs
distinguishes HIV-1 from most other primate lentiviruses. J Clin Invest.
2009;119(10):2965-75.
231. Schmokel J, Sauter D, Schindler M, Leendertz FH, Bailes E, Dazza MC, et al. The
presence of a vpu gene and the lack of Nef-mediated downmodulation of T cell
receptor-CD3 are not always linked in primate lentiviruses. J Virol.
2011;85(2):742-52.
232. Zaunders J, Dyer WB, Churchill M. The Sydney Blood Bank Cohort: implications
for viral fitness as a cause of elite control. Curr Opin HIV AIDS. 2011;6(3):151-6.
233. Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P. Nef harbors a major
determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in
transgenic mice. Cell. 1998;95(2):163-75.
234. Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, et al. Nef
functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+
thymocytes. Retrovirology. 2012;9:44.
235. Kestler HW, 3rd, Ringler DJ, Mori K, Panicali DL, Sehgal PK, Daniel MD, et al.
Importance of the nef gene for maintenance of high virus loads and for development
of AIDS. Cell. 1991;65(4):651-62.
236. Ikeda H, Nakaoka S, de Boer RJ, Morita S, Misawa N, Koyanagi Y, et al.
Quantifying the effect of Vpu on the promotion of HIV-1 replication in the
humanized mouse model. Retrovirology. 2016;13:23.
237. Sato K, Misawa N, Fukuhara M, Iwami S, An DS, Ito M, et al. Vpu augments the
initial burst phase of HIV-1 propagation and downregulates BST2 and CD4 in
humanized mice. J Virol. 2012;86(9):5000-13.
238. Singh DK, Griffin DM, Pacyniak E, Jackson M, Werle MJ, Wisdom B, et al. The
presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4(+)
T cell loss caused by the simian-human immunodeficiency virus SHIV(KU-
lbMC33) in pig-tailed macaques. Virology. 2003;313(2):435-51.
239. Kirchhoff F, Easterbrook PJ, Douglas N, Troop M, Greenough TC, Weber J, et al.
Sequence variations in human immunodeficiency virus type 1 Nef are associated
with different stages of disease. J Virol. 1999;73(7):5497-508.
240. Casartelli N, Di Matteo G, Argentini C, Cancrini C, Bernardi S, Castelli G, et al.
Structural defects and variations in the HIV-1 nef gene from rapid, slow and non-
progressor children. AIDS. 2003;17(9):1291-301.
63
241. Shugars DC, Smith MS, Glueck DH, Nantermet PV, Seillier-Moiseiwitsch F,
Swanstrom R. Analysis of human immunodeficiency virus type 1 nef gene
sequences present in vivo. J Virol. 1993;67(8):4639-50.
242. Verma S, Ronsard L, Kapoor R, Banerjea AC. Genetic characterization of natural
variants of Vpu from HIV-1 infected individuals from Northern India and their
impact on virus release and cell death. PLoS One. 2013;8(3):e59283.
243. Zhang L, Huang Y, Yuan H, Tuttleton S, Ho DD. Genetic characterization of vif,
vpr, and vpu sequences from long-term survivors of human immunodeficiency
virus type 1 infection. Virology. 1997;228(2):340-9.
244. Guy B, Kieny MP, Riviere Y, Le Peuch C, Dott K, Girard M, et al. HIV F/3' orf
encodes a phosphorylated GTP-binding protein resembling an oncogene product.
Nature. 1987;330(6145):266-9.
245. Franchini G, Robert-Guroff M, Ghrayeb J, Chang NT, Wong-Staal F. Cytoplasmic
localization of the HTLV-III 3' orf protein in cultured T cells. Virology.
1986;155(2):593-9.
246. Kaminchik J, Margalit R, Yaish S, Drummer H, Amit B, Sarver N, et al. Cellular
distribution of HIV type 1 Nef protein: identification of domains in Nef required
for association with membrane and detergent-insoluble cellular matrix. AIDS Res
Hum Retroviruses. 1994;10(8):1003-10.
247. Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y. Structural basis of evasion
of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol. 2012;19(7):701-
6.
248. Iijima S, Lee YJ, Ode H, Arold ST, Kimura N, Yokoyama M, et al. A noncanonical
mu-1A-binding motif in the N terminus of HIV-1 Nef determines its ability to
downregulate major histocompatibility complex class I in T lymphocytes. J Virol.
2012;86(7):3944-51.
249. Ren X, Park SY, Bonifacino JS, Hurley JH. How HIV-1 Nef hijacks the AP-2
clathrin adaptor to downregulate CD4. Elife. 2014;3:e01754.
250. Janvier K, Craig H, Le Gall S, Benarous R, Guatelli J, Schwartz O, et al. Nef-
induced CD4 downregulation: a diacidic sequence in human immunodeficiency
virus type 1 Nef does not function as a protein sorting motif through direct binding
to beta-COP. J Virol. 2001;75(8):3971-6.
251. Schaefer MR, Wonderlich ER, Roeth JF, Leonard JA, Collins KL. HIV-1 Nef
targets MHC-I and CD4 for degradation via a final common beta-COP-dependent
pathway in T cells. PLoS Pathog. 2008;4(8):e1000131.
252. Piguet V, Wan L, Borel C, Mangasarian A, Demaurex N, Thomas G, et al. HIV-1
Nef protein binds to the cellular protein PACS-1 to downregulate class I major
histocompatibility complexes. Nat Cell Biol. 2000;2(3):163-7.
253. Geyer M, Yu H, Mandic R, Linnemann T, Zheng YH, Fackler OT, et al. Subunit H
of the V-ATPase binds to the medium chain of adaptor protein complex 2 and
connects Nef to the endocytic machinery. J Biol Chem. 2002;277(32):28521-9.
64
254. Lu X, Yu H, Liu SH, Brodsky FM, Peterlin BM. Interactions between HIV1 Nef
and vacuolar ATPase facilitate the internalization of CD4. Immunity.
1998;8(5):647-56.
255. Grzesiek S, Stahl SJ, Wingfield PT, Bax A. The CD4 determinant for
downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding
surface by NMR. Biochemistry. 1996;35(32):10256-61.
256. Lee CH, Leung B, Lemmon MA, Zheng J, Cowburn D, Kuriyan J, et al. A single
amino acid in the SH3 domain of Hck determines its high affinity and specificity
in binding to HIV-1 Nef protein. EMBO J. 1995;14(20):5006-15.
257. Arold S, Franken P, Strub MP, Hoh F, Benichou S, Benarous R, et al. The crystal
structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role
for this complex in altered T cell receptor signaling. Structure. 1997;5(10):1361-
72.
258. Lee CH, Saksela K, Mirza UA, Chait BT, Kuriyan J. Crystal structure of the
conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell.
1996;85(6):931-42.
259. Jung J, Byeon IJ, Ahn J, Gronenborn AM. Structure, dynamics, and Hck interaction
of full-length HIV-1 Nef. Proteins. 2011;79(5):1609-22.
260. Alvarado JJ, Tarafdar S, Yeh JI, Smithgall TE. Interaction with the Src homology
(SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for
kinase activation and effector recruitment. J Biol Chem. 2014;289(41):28539-53.
261. Agopian K, Wei BL, Garcia JV, Gabuzda D. A hydrophobic binding surface on the
human immunodeficiency virus type 1 Nef core is critical for association with p21-
activated kinase 2. J Virol. 2006;80(6):3050-61.
262. Van Nuffel A, Arien KK, Stove V, Schindler M, O'Neill E, Schmokel J, et al.
Primate lentiviral Nef proteins deregulate T-cell development by multiple
mechanisms. Retrovirology. 2013;10:137.
263. Arold S, Hoh F, Domergue S, Birck C, Delsuc MA, Jullien M, et al.
Characterization and molecular basis of the oligomeric structure of HIV-1 nef
protein. Protein Sci. 2000;9(6):1137-48.
264. Vigan R, Neil SJ. Determinants of tetherin antagonism in the transmembrane
domain of the human immunodeficiency virus type 1 Vpu protein. J Virol.
2010;84(24):12958-70.
265. Skasko M, Wang Y, Tian Y, Tokarev A, Munguia J, Ruiz A, et al. HIV-1 Vpu
protein antagonizes innate restriction factor BST-2 via lipid-embedded helix-helix
interactions. J Biol Chem. 2012;287(1):58-67.
266. Kueck T, Foster TL, Weinelt J, Sumner JC, Pickering S, Neil SJ. Serine
Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction
with Clathrin Adaptors. PLoS Pathog. 2015;11(8):e1005141.
65
267. Margottin F, Bour SP, Durand H, Selig L, Benichou S, Richard V, et al. A novel
human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to
the ER degradation pathway through an F-box motif. Mol Cell. 1998;1(4):565-74.
268. Park SH, Mrse AA, Nevzorov AA, Mesleh MF, Oblatt-Montal M, Montal M, et al.
Three-dimensional structure of the channel-forming trans-membrane domain of
virus protein "u" (Vpu) from HIV-1. J Mol Biol. 2003;333(2):409-24.
269. Bonifacino JS, Glick BS. The mechanisms of vesicle budding and fusion. Cell.
2004;116(2):153-66.
270. Bonifacino JS. Vesicular transport earns a Nobel. Trends Cell Biol. 2014;24(1):3-
5.
271. Davey NE, Trave G, Gibson TJ. How viruses hijack cell regulation. Trends
Biochem Sci. 2011;36(3):159-69.
272. Park SY, Guo X. Adaptor protein complexes and intracellular transport. Biosci Rep.
2014;34(4).
273. Noviello CM, Benichou S, Guatelli JC. Cooperative binding of the class I major
histocompatibility complex cytoplasmic domain and human immunodeficiency
virus type 1 Nef to the endosomal AP-1 complex via its mu subunit. J Virol.
2008;82(3):1249-58.
274. Greenberg ME, Iafrate AJ, Skowronski J. The SH3 domain-binding surface and an
acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. EMBO
J. 1998;17(10):2777-89.
275. Chaudhuri R, Mattera R, Lindwasser OW, Robinson MS, Bonifacino JS. A Basic
Patch on alpha-Adaptin Is Required for Binding of Human Immunodeficiency
Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex. J Virol.
2009;83(6):2518-30.
276. Mangasarian A, Piguet V, Wang JK, Chen YL, Trono D. Nef-induced CD4 and
major histocompatibility complex class I (MHC-I) down-regulation are governed
by distinct determinants: N-terminal alpha helix and proline repeat of Nef
selectively regulate MHC-I trafficking. J Virol. 1999;73(3):1964-73.
277. Greenberg M, DeTulleo L, Rapoport I, Skowronski J, Kirchhausen T. A dileucine
motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for
downregulation of CD4. Curr Biol. 1998;8(22):1239-42.
278. Aiken C, Krause L, Chen YL, Trono D. Mutational analysis of HIV-1 Nef:
identification of two mutants that are temperature-sensitive for CD4
downregulation. Virology. 1996;217(1):293-300.
279. Stevens TH, Forgac M. Structure, function and regulation of the vacuolar (H+)-
ATPase. Annu Rev Cell Dev Biol. 1997;13:779-808.
280. Stove V, Van de Walle I, Naessens E, Coene E, Stove C, Plum J, et al. Human
immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor
CD8alphabeta. J Virol. 2005;79(17):11422-33.
66
281. Heigele A, Kmiec D, Regensburger K, Langer S, Peiffer L, Sturzel CM, et al. The
Potency of Nef-Mediated SERINC5 Antagonism Correlates with the Prevalence of
Primate Lentiviruses in the Wild. Cell Host Microbe. 2016;20(3):381-91.
282. Ren X, Farias GG, Canagarajah BJ, Bonifacino JS, Hurley JH. Structural basis for
recruitment and activation of the AP-1 clathrin adaptor complex by Arf1. Cell.
2013;152(4):755-67.
283. Wonderlich ER, Leonard JA, Kulpa DA, Leopold KE, Norman JM, Collins KL.
ADP ribosylation factor 1 activity is required to recruit AP-1 to the major
histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I
trafficking in HIV-1-infected primary T cells. J Virol. 2011;85(23):12216-26.
284. Shen QT, Ren X, Zhang R, Lee IH, Hurley JH. HIV-1 Nef hijacks clathrin coats by
stabilizing AP-1:Arf1 polygons. Science. 2015;350(6259):aac5137.
285. Serafini T, Orci L, Amherdt M, Brunner M, Kahn RA, Rothman JE. ADP-
ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a
novel role for a GTP-binding protein. Cell. 1991;67(2):239-53.
286. Donaldson JG, Cassel D, Kahn RA, Klausner RD. ADP-ribosylation factor, a small
GTP-binding protein, is required for binding of the coatomer protein beta-COP to
Golgi membranes. Proc Natl Acad Sci U S A. 1992;89(14):6408-12.
287. Piguet V, Gu F, Foti M, Demaurex N, Gruenberg J, Carpentier JL, et al. Nef-
induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal
targeting signal through the binding of beta-COP in endosomes. Cell.
1999;97(1):63-73.
288. Thomas G, Aslan JE, Thomas L, Shinde P, Shinde U, Simmen T. Caught in the act
- protein adaptation and the expanding roles of the PACS proteins in tissue
homeostasis and disease. J Cell Sci. 2017;130(11):1865-76.
289. Crump CM, Xiang Y, Thomas L, Gu F, Austin C, Tooze SA, et al. PACS-1 binding
to adaptors is required for acidic cluster motif-mediated protein traffic. EMBO J.
2001;20(9):2191-201.
290. Kottgen M, Benzing T, Simmen T, Tauber R, Buchholz B, Feliciangeli S, et al.
Trafficking of TRPP2 by PACS proteins represents a novel mechanism of ion
channel regulation. EMBO J. 2005;24(4):705-16.
291. Pawlak EN, Dikeakos JD. HIV-1 Nef: a master manipulator of the membrane
trafficking machinery mediating immune evasion. Biochim Biophys Acta.
2015;1850(4):733-41.
292. Piper RC, Dikic I, Lukacs GL. Ubiquitin-dependent sorting in endocytosis. Cold
Spring Harb Perspect Biol. 2014;6(1).
293. Dowlatshahi DP, Sandrin V, Vivona S, Shaler TA, Kaiser SE, Melandri F, et al.
ALIX is a Lys63-specific polyubiquitin binding protein that functions in retrovirus
budding. Dev Cell. 2012;23(6):1247-54.
294. Amorim NA, da Silva EM, de Castro RO, da Silva-Januario ME, Mendonca LM,
Bonifacino JS, et al. Interaction of HIV-1 Nef protein with the host protein Alix
67
promotes lysosomal targeting of CD4 receptor. J Biol Chem. 2014;289(40):27744-
56.
295. daSilva LL, Sougrat R, Burgos PV, Janvier K, Mattera R, Bonifacino JS. Human
immunodeficiency virus type 1 Nef protein targets CD4 to the multivesicular body
pathway. J Virol. 2009;83(13):6578-90.
296. Chandrasekaran P, Buckley M, Moore V, Wang LQ, Kehrl JH, Venkatesan S. HIV-
1 Nef impairs heterotrimeric G-protein signaling by targeting Galpha(i2) for
degradation through ubiquitination. J Biol Chem. 2012;287(49):41481-98.
297. Jin YJ, Cai CY, Zhang X, Burakoff SJ. Lysine 144, a ubiquitin attachment site in
HIV-1 Nef, is required for Nef-mediated CD4 down-regulation. J Immunol.
2008;180(12):7878-86.
298. Cai CY, Zhang X, Sinko PJ, Burakoff SJ, Jin YJ. Two sorting motifs, a
ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian
immunodeficiency virus Nef-mediated receptor endocytosis. J Immunol.
2011;186(10):5807-14.
299. Trible RP, Emert-Sedlak L, Smithgall TE. HIV-1 Nef selectively activates Src
family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction. J Biol
Chem. 2006;281(37):27029-38.
300. Saksela K, Permi P. SH3 domain ligand binding: What's the consensus and where's
the specificity? FEBS Lett. 2012;586(17):2609-14.
301. Dikeakos JD, Atkins KM, Thomas L, Emert-Sedlak L, Byeon IJL, Jung JW, et al.
Small Molecule Inhibition of HIV-1-Induced MHC-I Down-Regulation Identifies
a Temporally Regulated Switch in Nef Action. Mol Biol Cell. 2010;21(19):3279-
92.
302. Chutiwitoonchai N, Hiyoshi M, Mwimanzi P, Ueno T, Adachi A, Ode H, et al. The
identification of a small molecule compound that reduces HIV-1 Nef-mediated
viral infectivity enhancement. PLoS One. 2011;6(11):e27696.
303. Biggs TE, Cooke SJ, Barton CH, Harris MP, Saksela K, Mann DA. Induction of
activator protein 1 (AP-1) in macrophages by human immunodeficiency virus type-
1 NEF is a cell-type-specific response that requires both hck and MAPK signaling
events. J Mol Biol. 1999;290(1):21-35.
304. Schindler M, Wurfl S, Benaroch P, Greenough TC, Daniels R, Easterbrook P, et al.
Down-modulation of mature major histocompatibility complex class II and up-
regulation of invariant chain cell surface expression are well-conserved functions
of human and simian immunodeficiency virus nef alleles. J Virol.
2003;77(19):10548-56.
305. Galaski J, Ahmad F, Tibroni N, Pujol FM, Muller B, Schmidt RE, et al. Cell Surface
Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef
Alleles. J Acquir Immune Defic Syndr. 2016;72(1):1-10.
68
306. Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ, Saksela K. SH3-Domain
binding function of HIV-1 Nef is required for association with a PAK-related
kinase. Virology. 1998;250(2):273-82.
307. Wiskerchen M, ChengMayer C. HIV-1 Nef association with cellular serine kinase
correlates with enhanced virion infectivity and efficient proviral DNA synthesis.
Virology. 1996;224(1):292-301.
308. Sawai ET, Baur A, Struble H, Peterlin BM, Levy JA, Cheng-Mayer C. Human
immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T
lymphocytes. Proc Natl Acad Sci U S A. 1994;91(4):1539-43.
309. Nunn MF, Marsh JW. Human immunodeficiency virus type 1 Nef associates with
a member of the p21-activated kinase family. J Virol. 1996;70(9):6157-61.
310. Renkema GH, Manninen A, Mann DA, Harris M, Saksela K. Identification of the
Nef-associated kinase as p21-activated kinase 2. Curr Biol. 1999;9(23):1407-10.
311. Arora VK, Molina RP, Foster JL, Blakemore JL, Chernoff J, Fredericksen BL, et
al. Lentivirus Nef specifically activates Pak2. J Virol. 2000;74(23):11081-7.
312. Rauch S, Pulkkinen K, Saksela K, Fackler OT. Human immunodeficiency virus
type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface
into plasma membrane microdomains for association with p21-activated kinase 2
activity. J Virol. 2008;82(6):2918-29.
313. Stolp B, Abraham L, Rudolph JM, Fackler OT. Lentiviral Nef proteins utilize
PAK2-mediated deregulation of cofilin as a general strategy to interfere with actin
remodeling. J Virol. 2010;84(8):3935-48.
314. Lee JH, Wittki S, Brau T, Dreyer FS, Kratzel K, Dindorf J, et al. HIV Nef, paxillin,
and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases. Mol
Cell. 2013;49(4):668-79.
315. Kienzle N, Freund J, Kalbitzer HR, Mueller-Lantzsch N. Oligomerization of the
Nef protein from human immunodeficiency virus (HIV) type 1. Eur J Biochem.
1993;214(2):451-7.
316. Poe JA, Smithgall TE. HIV-1 Nef dimerization is required for Nef-mediated
receptor downregulation and viral replication. J Mol Biol. 2009;394(2):329-42.
317. Ye H, Choi HJ, Poe J, Smithgall TE. Oligomerization is required for HIV-1 Nef-
induced activation of the Src family protein-tyrosine kinase, Hck. Biochemistry.
2004;43(50):15775-84.
318. Poe JA, Vollmer L, Vogt A, Smithgall TE. Development and validation of a high-
content bimolecular fluorescence complementation assay for small-molecule
inhibitors of HIV-1 Nef dimerization. J Biomol Screen. 2014;19(4):556-65.
319. Liu LX, Heveker N, Fackler OT, Arold S, Le Gall S, Janvier K, et al. Mutation of
a conserved residue (D123) required for oligomerization of human
immunodeficiency virus type 1 Nef protein abolishes interaction with human
thioesterase and results in impairment of Nef biological functions. J Virol.
2000;74(11):5310-9.
69
320. Wu M, Alvarado JJ, Augelli-Szafran CE, Ptak RG, Smithgall TE. A single beta-
octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of
partner protein binding. PLoS One. 2018;13(2):e0192512.
321. Kwak YT, Raney A, Kuo LS, Denial SJ, Temple BR, Garcia JV, et al. Self-
association of the Lentivirus protein, Nef. Retrovirology. 2010;7:77.
322. Emert-Sedlak LA, Narute P, Shu ST, Poe JA, Shi H, Yanamala N, et al. Effector
kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists
with antiretroviral activity. Chem Biol. 2013;20(1):82-91.
323. Nieva JL, Madan V, Carrasco L. Viroporins: structure and biological functions. Nat
Rev Microbiol. 2012;10(8):563-74.
324. Cordes FS, Tustian AD, Sansom MS, Watts A, Fischer WB. Bundles consisting of
extended transmembrane segments of Vpu from HIV-1: computer simulations and
conductance measurements. Biochemistry. 2002;41(23):7359-65.
325. Lopez CF, Montal M, Blasie JK, Klein ML, Moore PB. Molecular dynamics
investigation of membrane-bound bundles of the channel-forming transmembrane
domain of viral protein U from the human immunodeficiency virus HIV-1. Biophys
J. 2002;83(3):1259-67.
326. Hussain A, Das SR, Tanwar C, Jameel S. Oligomerization of the human
immunodeficiency virus type 1 (HIV-1) Vpu protein--a genetic, biochemical and
biophysical analysis. Virol J. 2007;4:81.
327. Schubert U, Ferrer-Montiel AV, Oblatt-Montal M, Henklein P, Strebel K, Montal
M. Identification of an ion channel activity of the Vpu transmembrane domain and
its involvement in the regulation of virus release from HIV-1-infected cells. FEBS
Lett. 1996;398(1):12-8.
328. Coady MJ, Daniel NG, Tiganos E, Allain B, Friborg J, Lapointe JY, et al. Effects
of Vpu expression on Xenopus oocyte membrane conductance. Virology.
1998;244(1):39-49.
329. Hsu K, Han J, Shinlapawittayatorn K, Deschenes I, Marban E. Membrane potential
depolarization as a triggering mechanism for Vpu-mediated HIV-1 release.
Biophys J. 2010;99(6):1718-25.
330. Magadan JG, Bonifacino JS. Transmembrane domain determinants of CD4
Downregulation by HIV-1 Vpu. J Virol. 2012;86(2):757-72.
331. Bolduan S, Votteler J, Lodermeyer V, Greiner T, Koppensteiner H, Schindler M,
et al. Ion channel activity of HIV-1 Vpu is dispensable for counteraction of CD317.
Virology. 2011;416(1-2):75-85.
332. Willey RL, Buckler-White A, Strebel K. Sequences present in the cytoplasmic
domain of CD4 are necessary and sufficient to confer sensitivity to the human
immunodeficiency virus type 1 Vpu protein. J Virol. 1994;68(2):1207-12.
333. Margottin F, Benichou S, Durand H, Richard V, Liu LX, Gomas E, et al. Interaction
between the cytoplasmic domains of HIV-1 Vpu and CD4: role of Vpu residues
70
involved in CD4 interaction and in vitro CD4 degradation. Virology.
1996;223(2):381-6.
334. Chen MY, Maldarelli F, Karczewski MK, Willey RL, Strebel K. Human
immunodeficiency virus type 1 Vpu protein induces degradation of CD4 in vitro:
the cytoplasmic domain of CD4 contributes to Vpu sensitivity. J Virol.
1993;67(7):3877-84.
335. Bour S, Schubert U, Strebel K. The human immunodeficiency virus type 1 Vpu
protein specifically binds to the cytoplasmic domain of CD4: implications for the
mechanism of degradation. J Virol. 1995;69(3):1510-20.
336. Tiganos E, Yao XJ, Friborg J, Daniel N, Cohen EA. Putative alpha-helical
structures in the human immunodeficiency virus type 1 Vpu protein and CD4 are
involved in binding and degradation of the CD4 molecule. J Virol.
1997;71(6):4452-60.
337. Magadan JG, Perez-Victoria FJ, Sougrat R, Ye Y, Strebel K, Bonifacino JS.
Multilayered mechanism of CD4 downregulation by HIV-1 Vpu involving distinct
ER retention and ERAD targeting steps. PLoS Pathog. 2010;6(4):e1000869.
338. Tiganos E, Friborg J, Allain B, Daniel NG, Yao XJ, Cohen EA. Structural and
functional analysis of the membrane-spanning domain of the human
immunodeficiency virus type 1 Vpu protein. Virology. 1998;251(1):96-107.
339. Buonocore L, Turi TG, Crise B, Rose JK. Stimulation of heterologous protein
degradation by the Vpu protein of HIV-1 requires the transmembrane and
cytoplasmic domains of CD4. Virology. 1994;204(1):482-6.
340. Jia X, Weber E, Tokarev A, Lewinski M, Rizk M, Suarez M, et al. Structural basis
of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor
protein complex 1. Elife. 2014;3:e02362.
341. Kueck T, Neil SJ. A cytoplasmic tail determinant in HIV-1 Vpu mediates targeting
of tetherin for endosomal degradation and counteracts interferon-induced
restriction. PLoS Pathog. 2012;8(3):e1002609.
342. Pickering S, Hue S, Kim EY, Reddy S, Wolinsky SM, Neil SJ. Preservation of
tetherin and CD4 counter-activities in circulating Vpu alleles despite extensive
sequence variation within HIV-1 infected individuals. PLoS Pathog.
2014;10(1):e1003895.
343. Schubert U, Schneider T, Henklein P, Hoffmann K, Berthold E, Hauser H, et al.
Human-immunodeficiency-virus-type-1-encoded Vpu protein is phosphorylated by
casein kinase II. Eur J Biochem. 1992;204(2):875-83.
344. Madjo U, Leymarie O, Fremont S, Kuster A, Nehlich M, Gallois-Montbrun S, et
al. LC3C Contributes to Vpu-Mediated Antagonism of BST2/Tetherin Restriction
on HIV-1 Release through a Non-canonical Autophagy Pathway. Cell Rep.
2016;17(9):2221-33.
345. Sanjuan MA, Milasta S, Green DR. Toll-like receptor signaling in the lysosomal
pathways. Immunol Rev. 2009;227(1):203-20.
71
346. Mangeat B, Gers-Huber G, Lehmann M, Zufferey M, Luban J, Piguet V. HIV-1
Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its
beta-TrCP2-dependent degradation. PLoS Pathog. 2009;5(9):e1000574.
347. Lee EK, Diehl JA. SCFs in the new millennium. Oncogene. 2014;33(16):2011-8.
348. Binette J, Dube M, Mercier J, Halawani D, Latterich M, Cohen EA. Requirements
for the selective degradation of CD4 receptor molecules by the human
immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.
Retrovirology. 2007;4:75.
349. Schubert U, Strebel K. Differential activities of the human immunodeficiency virus
type 1-encoded Vpu protein are regulated by phosphorylation and occur in different
cellular compartments. J Virol. 1994;68(4):2260-71.
350. Paul M, Jabbar MA. Phosphorylation of both phosphoacceptor sites in the HIV-1
Vpu cytoplasmic domain is essential for Vpu-mediated ER degradation of CD4.
Virology. 1997;232(1):207-16.
351. Tokarev AA, Munguia J, Guatelli JC. Serine-threonine ubiquitination mediates
downregulation of BST-2/tetherin and relief of restricted virion release by HIV-1
Vpu. J Virol. 2011;85(1):51-63.
352. Janvier K, Pelchen-Matthews A, Renaud JB, Caillet M, Marsh M, Berlioz-Torrent
C. The ESCRT-0 component HRS is required for HIV-1 Vpu-mediated BST-
2/tetherin down-regulation. PLoS Pathog. 2011;7(2):e1001265.
353. Mitchell RS, Katsura C, Skasko MA, Fitzpatrick K, Lau D, Ruiz A, et al. Vpu
antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-
lysosomal trafficking. PLoS Pathog. 2009;5(5):e1000450.
354. Roy N, Pacini G, Berlioz-Torrent C, Janvier K. Characterization of E3 ligases
involved in lysosomal sorting of the HIV-1 restriction factor BST2. J Cell Sci.
2017;130(9):1596-611.
355. Kanarek N, Ben-Neriah Y. Regulation of NF-kappaB by ubiquitination and
degradation of the IkappaBs. Immunol Rev. 2012;246(1):77-94.
356. Akari H, Bour S, Kao S, Adachi A, Strebel K. The human immunodeficiency virus
type 1 accessory protein Vpu induces apoptosis by suppressing the nuclear factor
kappaB-dependent expression of antiapoptotic factors. J Exp Med.
2001;194(9):1299-311.
357. Xia Y, Padre RC, De Mendoza TH, Bottero V, Tergaonkar VB, Verma IM.
Phosphorylation of p53 by IkappaB kinase 2 promotes its degradation by beta-
TrCP. Proc Natl Acad Sci U S A. 2009;106(8):2629-34.
358. Dirk BS, Jacob RA, Johnson AL, Pawlak EN, Cavanagh PC, Van Nynatten L, et
al. Viral bimolecular fluorescence complementation: a novel tool to study
intracellular vesicular trafficking pathways. PLoS One. 2015;10(4):e0125619.
359. Kuang XT, Li X, Anmole G, Mwimanzi P, Shahid A, Le AQ, et al. Impaired Nef
function is associated with early control of HIV-1 viremia. J Virol.
2014;88(17):10200-13.
72
360. Gupta P, Husain M, Hans C, Ram H, Verma SS, Misbah M, et al. Sequence
heterogeneity in human immunodeficiency virus type 1 nef in patients presenting
with rapid progression and delayed progression to AIDS. Arch Virol.
2014;159(9):2303-20.
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Chapter 2
2 The downregulation of CD28 by HIV-1 Nef and Vpu
2.1 Introduction
Adaptive immune responses are primarily driven by B and T lymphocytes, which facilitate
humoral and cell-mediated immunity (reviewed in [1]). Although a simplification, thymus
derived lymphocytes, or T cells, can be classified as CD8+ CTLs or CD4+ T helper cells,
and these cells play key roles during anti-viral responses. Naïve T cells require signaling
from APCs to become competent effector cells. According to the two-signal model of T
cell activation, APCs provide two signals to enable these cells to become productive
effector cells. The first signal is established during MHC-restricted binding of the TCR:
antigen complex, while signal 2 consists of co-stimulatory signaling (reviewed in [2, 3]).
A lack of co-stimulatory signaling results in cells becoming unresponsive or anergic. Key
co-stimulatory signaling is provided by binding of CD28, a transmembrane receptor
expressed on the surface of T cells, to B7.1/B7.2 on the surface of APCs. Indeed, CD28
receptor ligation initiates downstream signaling, which in conjunction with TCR signaling,
results in cell activation and proliferation essential for mounting an immune response [4,
5].
The importance of the TCR and co-stimulatory signaling to the development of an effective
immune response makes them prime targets of intracellular pathogens. Indeed, many
viruses have evolved the capabilities to intricately modulate these T cell activation cues to
optimize their replication and persistence. For instance, lymphotropic viruses, including
HIV-1, HTLV, and Epstein-Barr Virus inhibit T cell activation by downregulating
components of the TCR and the TCR-associated kinases essential for TCR signaling [6-
11]. Moreover, viruses inhibit signaling downstream of co-stimulatory receptors, or alter
the levels of cell surface co-stimulatory or inhibitory molecules [12-15]. Ultimately, virus-
induced changes in T cell activation can result in immune evasion, enhanced viral
replication, and increased viral persistence. The importance of viral alterations to immune
cell activation are evidenced by the specific HIV-1 proteins that play key roles in T cell
activation.
74
HIV-1 encodes four accessory proteins that lack any known enzymatic or structural
functions: Vif, Vpr, Nef and Vpu (reviewed in [16-18]). Collectively, these viral proteins
play critical roles in increasing infectivity, persistence and pathogenesis. While Nef and
Vpu are arguably the most extensively studied accessory proteins, their roles in T cell
activation are not fully understood. The ability of Nef and Vpu to downregulate various
host cell receptors is a key function mediating their effects. Indeed, both Nef and Vpu
downregulated multiple receptors in a screen to identify cell surface proteins that are
altered by these viral proteins [19]. Specifically, Nef and Vpu facilitate degradation or
intracellular sequestration of host receptors, including restriction factors and immune cell
receptors, to thwart their cell surface expression and ultimately, their function [16]. By
hijacking membrane trafficking proteins, such as the adaptor proteins AP-1 or AP-2, Nef
and Vpu connect multiple cellular receptors to additional cellular trafficking machinery
which alters the receptors’ subcellular localization. For example, Nef hijacks AP-1 to
facilitate the endocytosis and sequestration of MHC-I molecules [17, 20-22], limiting
recognition of infected cells by the immune system [23]. In parallel, the ability of Nef to
hijack AP-2 results in the endocytosis and degradation of CD4, which limits superinfection
and ADCC [24-31]. Similarly, Vpu hijacks BST-2 trafficking by associating with adaptor
proteins to facilitate its sequestration and degradation [32-36]. Vpu also enables the
degradation of CD4 by targeting newly synthesized CD4 to the ERAD pathway [37].
Interestingly, Nef expression leads to endocytosis of CD28 from the cell surface, in a
manner dependent on both AP-1 and AP-2 [38, 39]. However, the fate of CD28 after Nef-
mediated endocytosis remains poorly understood and the effects of Vpu on CD28 are
unexplored.
In this chapter, we report that Nef and Vpu decrease cell surface and total cellular CD28
levels in infected CD4+ T cells. Moreover, these decreases in total CD28 are blocked by
inhibiting the cellular degradation machinery, consistent with Nef or Vpu facilitating
trafficking of CD28 to an acidic lysosomal compartment. Additionally, a mutant Nef
protein associated with impaired binding to the vacuolar ATPase was compromised in its
ability to reduce cell surface and total CD28 levels, while a non-phosphorylatable Vpu
protein unable to recruit protein degradation machinery was impaired in its ability to reduce
cell surface and total CD28 levels. Moreover, we sought to determine if knock-downs of
75
specific host cell trafficking machinery impaired the observed downregulation of CD28.
Finally, we found that the ability of Vpu to downregulate CD28 is not limited to the lab
adapted HIV-1 strain NL4.3 Vpu, and infection of cells with viruses encoding Nef or Vpu
have differential effects on activation upon stimulation of CD3 and CD28.
2.2 Results
2.2.1 The HIV-1 accessory proteins Nef and Vpu downregulate cell surface and total
CD28 protein levels
To investigate the effects of the HIV-1 accessory proteins Nef and Vpu on endogenous
CD28 levels, CD4+ Sup-T1 T cells were infected with Gag-Pol truncated, vesicular
stomatitis virus envelope glycoprotein (VSV-G) pseudotyped and eGFP expressing HIV-
1 NL4.3 viruses encoding both Nef and Vpu (NL4.3), Vpu alone (dNef), Nef alone (dVpu),
or neither accessory protein (dVpu dNef). Infected Sup-T1 cells were analyzed by flow
cytometry after staining for cell surface CD28 (Figure 2.1). Upon gating on live (Zombie
RedTM-), infected (GFP+) single cells (Figure 2.2), significantly greater cell surface levels
of CD28 were present on cells infected with viruses lacking Nef (dNef), Vpu (dVpu) or
both Nef and Vpu (dVpu dNef), compared to cells infected with virus encoding both Nef
and Vpu (NL4.3; Figure 2.1A, B, C). Accordingly, Western blot analysis confirmed the
presence or absence of Nef and Vpu in infected Sup-T1 cells (Figure 2.1D). Moreover, we
examined the ability of Nef and Vpu expressed from replication competent NL4.3 to
downregulate cell surface CD28 in infected CD4+ T cells purified from peripheral blood
mononuclear cells (PBMCs) (Figure 2.1E, F; Figure 2.3). In line with our observations in
Sup-T1 cells, in the absence of Nef (dNef), Vpu (dVpu), or both Nef and Vpu (dVpu dNef)
significantly greater cell surface levels of CD28 were present when compared to cells
infected with NL4.3 (Figure 2.1E), indicating that Nef and Vpu independently
downregulate cell surface CD28 in both primary CD4+ T cells and the immortal CD4+ T
cell line, Sup-T1 cells.
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Figure 2.1 HIV-1 Nef and Vpu downregulate cell surface CD28 protein levels
CD4+ Sup-T1 and primary CD4+ T cells were infected with VSV-G pseudotyped or
replication competent NL4.3. Viruses encoded Nef and Vpu (NL4.3, red), lacked Nef (dNef,
77
blue) or Vpu (dVpu, orange), or lacked both Nef and Vpu (dVpu dNef, green). Infected cells
were stained for CD28 and analyzed by flow cytometry. Live infected Sup-T1 cells were
analyzed by gating on Zombie RedTM- and GFP+ cells and infected primary CD4+ T cells
were analyzed by gating on p24+ cells. (A) Representative dot plots illustrating cell surface
CD28 (APC) and infection (GFP+) of live (Zombie RedTM-) Sup-T1 cells. (B)
Representative histograms illustrating cell surface levels of CD28 or the appropriate isotype
control on Sup-T1 cells after gating on live (Zombie RedTM-) and infected (GFP+) cells.
CD28 (APC) geometric mean fluorescence intensities (MFI) are indicated. (C) Summary of
the relative mean (+/-SE) cell surface CD28 levels on infected (GFP+) Sup-T1 cells based
on MFIs (n=17). (D) Western blot illustrating expression of Nef and Vpu in infected Sup-
T1 cells. (E) Representative histograms illustrating cell surface levels of CD28 or the
appropriate isotype control on uninfected (UI) or infected (p24+) CD4+ PBMCs. MFIs are
indicated. (F) Summary of the relative mean (+/-SE) cell surface CD28 levels on infected
CD4+ T cells based on MFIs obtained from infection of CD4+ T cells from two healthy
donors (n ≥3). (UI: uninfected; SE: standard error; Mr: molecular weight; GAPDH:
glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; SE: standard
error; *p≤ 0.05; **p≤ 0.01; ****p≤ 0.0001).
78
Figure 2.2 Gating of live infected Sup-T1 cells infected with Gag-Pol truncated
VSV-G pseudotyped NL4.3
To examine live infected cells, dead cells were excluded by gating on Zombie RedTM-
cells, doublets were excluded and subsequently infected (GFP+) cells were gated on. In
a representative experiment, 80.3% of cells were live (Zombie RedTM-) and 82.1% of live
single cells were infected (GFP+). Gates were set based on FMO (fluorescence minus
one) controls stained for all fluorophores except that which is being gated on.
79
Figure 2.3 Analysis of primary CD4+ T cells infected with replication competent
virus
Primary CD4+ T cells were purified and infected with replication competent NL4.3
viruses and stained for cell surface CD28 and intracellular p24. (A) To examine the cell
surface CD28 levels on infected cells, single cells were gated on followed by gating on
the p24 (PE) and CD28 (APC) high populations. In a representative experiment, 5.22%
of cells were p24 high. (B) Representative dot plots illustrating p24 (PE) and CD28
(APC) on cells that were either uninfected and unstained or stained with the appropriate
APC isotype control or infected and stained with the appropriate PE isotype control or
both anti-CD28 (APC) and anti-p24 (PE).
80
Given that HIV-1 Nef and Vpu are known to decrease cell surface levels of various
receptors by mediating their degradation, we next sought to determine if these viral proteins
alter total CD28 protein levels. To examine total CD28 protein levels and due to the
unavailability of an antibody capable of detecting endogenous CD28 on Western blots, we
engineered an expression vector to facilitate overexpression of CD28 in cells. This vector
was designed to optimize protein expression by fusing the CD8 α signal peptide to the
CD28 protein coding sequence [40], as has been done previously to overexpress other cell
receptors [41, 42]. Moreover, this vector contains an epitope (FLAG) tag to enable
detection (Figure 2.4). We subsequently examined the effects of Nef on total CD28-FLAG
protein levels, as Nef had the largest effect on cell surface CD28, as measured by flow
cytometry (Figure 2.1C). Thus, HeLa cells were co-transfected with expression vectors
encoding CD28-FLAG and Nef-eGFP or eGFP alone and total protein was examined by
Western blot. A decrease in CD28-FLAG levels in the presence of Nef was observed
(Figure 2.5). To further investigate Nef-mediated decreases in total CD28 protein levels in
T cells, CD4+ Sup-T1 cells were transduced with VSV-G pseudotyped HIV-1 NL4.3
engineered to encode epitope tagged CD28 and Nef (F2A CD28-FLAG Nef) or no Nef
(F2A CD28-FLAG dNef; Figure 2.6). Forty-eight hours post transduction cells were lysed
and total cellular CD28-FLAG was visualized by Western blot (Figure 2.7). As in
transfected HeLa cells, decreases in CD28-FLAG in the presence of Nef were observed.
Notably, Nef-mediated decreases in CD28-FLAG were observed inconsistently, which we
hypothesize may be due to the overexpression of CD28-FLAG above level of endogenous
CD28 protein, thus limiting our ability to detect viral protein mediated decreases.
81
Figure 2.4 Schematic of CD28-FLAG construct utilized to overexpress CD28 in
cells
The CD8α signal peptide (blue) was cloned in frame with the coding sequence for the
CD28 protein (residues 19-220 of CD28 protein), including the CD28 extracellular
domain (orange; residues 153-179 of CD28 protein), transmembrane domain (red;
residues 153-179 of CD28 protein) and cytoplasmic tail (green; residues 180-220 of
CD28 protein) and was epitope (purple; DYKDDDDK) tagged on the C-terminus.
Numbers denote residues in the CD28-FLAG construct.
82
Figure 2.5 Decreased CD28-FLAG protein levels are detected in the presence of
Nef in HeLa cells
HeLa cells were transfected with plasmids encoding CD28-FLAG and Nef-eGFP or
eGFP. Forty-eight hours post transfection, cells were lysed and lysate analyzed for the
presence of CD28-FLAG (top), Nef-eGFP or eGFP (middle) and a loading control (actin,
bottom) via Western blot. Indicated are relative intensities of the band corresponding to
CD28-FLAG as determined using LI-COR Image Studio software (top). (eGFP:
enhanced green fluorescent protein; Mr: molecular weight; NT: non-transfected)
83
Figure 2.6 HIV-1 lentiviral vector utilized to express epitope tagged CD28 in the
presence or absence of Nef
Illustration of a linear map of the lentiviral vector utilized to overexpress epitope tagged
CD28 in T cells. The vector was modified from previously described lentiviral vectors
produced from modification of the HIV-1 laboratory strain NL4.3 [43]. The HIV-1 gag
and pol genes are truncated to encode the foot-and-mouth disease virus 2A peptide (F2A)
and the CD8α signal peptide fused to a FLAG-tagged CD28-encoding gene. The
remaining pUC vector backbone is not shown.
84
Figure 2.7 CD28-FLAG protein levels are reduced in the presence of Nef in CD4+
Sup-T1 T cells
Sup-T1 T cells were transduced with a VSV-G pseudotyped lentivirus encoding CD28-
FLAG and Nef (F2A CD28-FLAG Nef) or CD28-FLAG and no Nef (F2A CD28-FLAG
dNef). Forty-eight hours post transduction cells were lysed and proteins were analyzed
by Western blot. Shown is CD28-FLAG (top), Nef (middle) and actin (bottom). (Mr:
molecular weight)
85
Due to the inconsistent nature of our results obtained when using overexpressed epitope
tagged CD28, we next sought to examine the effects of Nef and Vpu on endogenous CD28
total protein levels within CD4+ T cells, a more biologically relevant system. Briefly, Sup-
T1 cells or whole peripheral blood mononuclear cells (PBMCs) were infected with VSV-
G pseudotyped HIV-1 NL4.3 viruses, as in Figure 2.1. Infected cells were stained for total
CD28 levels by permeabilizing the cells prior to staining for CD28 and cells were
subsequently analyzed by flow cytometry. Levels of CD28 were analyzed in Sup-T1 cells
after gating on single live (Zombie NIRTM-) infected (GFP+) cells (Figure 2.2), while
PBMCs were gated on CD4+ and infected (GFP+) cells. As with cell surface CD28, total
CD28 levels were significantly higher in cells infected with viruses lacking Nef (dNef) or
Vpu (dVpu), relative to cells infected with viruses encoding both viral proteins (NL4.3), in
both Sup-T1 and primary CD4+ cells (Figure 2.8). Moreover, the highest total CD28 levels
were observed in Sup-T1 cells infected with virus lacking functional nef and vpu genes
(dVpu dNef; Figure 2.8C).
86
Figure 2.8 HIV-1 Nef and Vpu downregulate total CD28 protein levels
CD4+ Sup-T1 cells or peripheral blood mononuclear cells were infected with VSV-G
pseudotyped NL4.3. Viruses encoded Nef and Vpu (NL4.3, red), lacked Nef (dNef,
blue), lacked Vpu (dVpu, orange) or lacked both Nef and Vpu (dNef dVpu, green).
Forty-eight hours post-infection, cells were permeabilized, stained for CD28 and
analyzed by flow cytometry. (A) Representative dot plots illustrating total CD28 (APC)
87
and infection (GFP) levels of live (Zombie RedTM-) Sup-T1 cells. (B) Representative
histograms illustrating total levels of CD28 (APC) after gating on live (Zombie RedTM-)
infected (GFP+) cells. Geometric mean fluorescence intensities (MFI) are indicated. (C)
Summary of mean (+/-SE) relative total CD28 based on MFI (n=12). (D) Representative
dot plots illustrating purified CD4+ T cells infection (GFP) and total CD28 (APC) levels.
(E) Summary of mean (+/-SE) total CD28 levels on infected (GFP+) CD4+ T cells (n=5).
Data were obtained from infection of cells obtained from two healthy donors. (UI:
uninfected; SE: standard error; *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001; GFP:
green fluorescent protein).
88
To alternatively confirm that the HIV-1 accessory proteins Nef and Vpu alter endogenous
CD28, we infected Sup-T1 cells with VSV-G pseudotyped NL4.3 viruses encoding or
lacking the viral proteins and examined CD28 localization using widefield microscopy
(Figure 2.9). Specifically, due to the observed decreases in total CD28 protein levels
(Figure 2.5; Figure 2.7; Figure 2.8), we stained cells for endogenous CD28 and lysosomal-
associated membrane protein 1 (LAMP-1), a marker of the degradative lysosomal
compartment. Interestingly, we observed that in the presence of Nef and Vpu, CD28
localized away from the cell surface (NL4.3; Figure 2.9A), inconsistent with the plasma
membrane localization observed in uninfected cells (UI; Figure 2.9A). Moreover, upon
elimination of the viral proteins we observed a rescue in CD28’s localization to the cell
surface (dVpu dNef; Figure 2.9A). Upon quantification of CD28: LAMP-1 co-localization,
we found that in the presence of both viral proteins (NL4.3) the co-localization of CD28
with LAMP-1 was significantly greater than in the absence of either (dNef, dVpu) or both
(dVpu dNef) viral proteins (Figure 2.9B). This microscopy analysis confirms that Nef and
Vpu downregulate endogenous CD28 and suggests this downregulation is mediated by
transport of CD28 to the degradative lysosome.
90
CD4+ Sup-T1 cells were infected with VSV-G pseudotyped NL4.3 viruses encoding or
lacking Nef and/or Vpu. Infected cells were stained for CD28 and the lysosomal marker
LAMP-1 and visualized by widefield microscopy. (A) Shown are representative infected
(GFP+) cells (left) and a graphical representation of CD28 (blue) and LAMP-1 (red)
fluorescence intensity relative to the maximum along the illustrated line (right). The scale
bar indicates 10 m and the vertical lines labelled PM indicate where the illustrated line
meets the plasma membrane (PM). Insets in the merged panel illustrate the GFP channel.
(B) The percentage overlap between CD28 and LAMP-1 is calculated based on the mean
(+/- SD) Manders’ overlap coefficients from at least 30 cells and 3 independent
experiments. (UI: uninfected; LAMP-1: Lysosomal-associated membrane protein 1; SD:
standard deviation; ****p≤ 0.0001).
91
2.2.2 Nef and Vpu-mediated effects on CD28 are dependent on the cellular
degradation machinery
Cellular protein levels can be reduced via degradation by acidic hydrolases within acidified
endosomal compartments [44]. Since we observed the co-localization of CD28 and LAMP-
1 in infected cells (Figure 2.9), we sought to determine if Nef- and Vpu-mediated
reductions in total CD28 could be blocked by inhibition of endosomal acidification. To test
this, we treated infected Sup-T1 cells with the base ammonium chloride to increase intra-
vesicular pH [45]. We specifically measured total CD28 within Sup-T1 cells infected with
pseudotyped NL4.3 and treated with ammonium chloride prior to staining. Interestingly,
upon ammonium chloride treatment, total CD28 detected by flow cytometry increased
relative to untreated cells (Figure 2.10). Moreover, the fold increase, or recovery, of total
CD28 protein levels upon ammonium chloride treatment was significantly lower when
cells were infected with viruses lacking either (dNef, dVpu) or both Nef and Vpu (dVpu
dNef), compared to Nef- and Vpu-encoding virus (NL4.3; Figure 2.10A). As a control, we
measured total CD4 within infected cells treated or not with ammonium chloride. Similar
to CD28, the recovery of CD4 levels upon ammonium chloride treatment was significantly
less in cells infected with virus lacking one or both of the accessory proteins (dNef, dVpu,
dVpu dNef; Figure 2.11). Therefore, in the presence of the viral accessory proteins Nef and
Vpu, a decrease in total CD28 occurs and this effect is most likely mitigated by inhibition
of endosomal acidification.
To further confirm these findings, we used widefield microscopy to visualize CD28 within
Sup-T1 cells infected with VSV-G pseudotyped NL4.3. Specifically, we examined the
subcellular localization of CD28 within infected cells treated with the general endosome
acidification inhibitor ammonium chloride or treated with Bafilomycin A1 or
Concanamycin A, specific inhibitors of the endosome acidifying proton pump, the vacuolar
ATPase [46, 47]. We observed that upon treatment with ammonium chloride, Bafilomycin
A1 or Concanamycin A, the distribution of CD28 within VSV-G pseudotyped NL4.3
infected cells was altered relative to vehicle (DMSO) treated cells (Figure 2.10). Indeed,
inhibitor treatment resulted in the increased localization of CD28 to the cell surface when
compared to cells treated with vehicle. Moreover, quantification of the co-localization of
CD28 and LAMP-1 in inhibitor treated cells indicated that upon inhibitor treatment CD28
92
co-localizes significantly less with the lysosomal marker LAMP-1 (Figure 2.10C). These
results suggest that the ability of Nef and Vpu to downregulate CD28 is dependent on
lysosomal acidification.
93
Figure 2.10 Inhibition of the degradation machinery increases CD28 protein levels
in infected cells
94
CD4+ Sup-T1 cells were infected with VSV-G pseudotyped NL4.3 encoding or lacking
Nef and/or Vpu. Infected cells were analyzed 48 hours post-infection after treatment for
24 hours with 40 mM ammonium chloride or treatment for five hours with 100 nM
Bafilomycin A1 or 100 nM Concanamycin. Cells were stained for CD28 and the
lysosomal marker LAMP-1 and analyzed by widefield microscopy or stained for CD28
and analyzed by flow cytometry. (A) Representative histograms illustrating total CD28
levels of infected cells treated with complete RPMI with (blue) or without (red) 40 mM
ammonium chloride. Geometric mean fluorescence intensities (MFIs) are indicated. MFI
of infected cells were determined after gating on live (Zombie RedTM-), infected (GFP+)
cells and the relative fold increase (+/-SE) in total CD28 upon ammonium chloride (n≥5)
treatment is illustrated (right). (B) Representative infected (GFP+) Sup-T1 cells
subjected to treatment with vehicle (DMSO), ammonium chloride (NH4Cl),
Concanamycin A (ConA) or Bafilomycin A1 (BafA) and visualized by widefield
microscopy (left). The scale bar indicates 10 m and insets in merged panel illustrate
GFP channel. A graphical representation of CD28 and LAMP-1 fluorescence intensity
relative to max along the illustrated line is shown (right). The vertical lines labelled PM
indicate where the illustrated line meets the plasma membrane (PM). (C) The percentage
of overlap between CD28 and LAMP-1 is based on the mean (+/-SD) Manders’ overlap
coefficient measured on at least 30 cells from 3 independent experiments (UI:
uninfected; SD: standard deviation; LAMP-1: Lysosomal-associated membrane protein;
****p≤ 0.0001).
95
Figure 2.11 Ammonium chloride treatment increases total CD4 levels in infected
cells
CD4+ Sup-T1 cells were infected with Gag-Pol truncated VSV-G pseudotyped NL4.3
encoding or lacking Nef and/or Vpu. Infected cells were treated with 40 mM ammonium
chloride for twenty-four hours prior to staining for CD4 and analyzed by flow cytometry.
(A) Representative histograms illustrating CD4 (APC) levels on live infected cells. Mean
geometric fluorescence intensities (MFIs) are indicated. (B) MFIs of infected cells were
determined after gating on live, infected (Zombie RedTM- and GFP+) cells and the relative
fold increase (+/-SE) in total CD4 (n≥5) upon ammonium chloride treatment is
illustrated. (SE: standard error; ****p≤ 0.0001).
96
2.2.3 Motifs ascribed to specific Nef: host cell protein interactions are critical for
Nef-mediated CD28 downregulation
To further elucidate the mechanisms used by Nef to downregulate CD28, we sought to
determine if this function is dependent on motifs within Nef that have been previously
implicated in interacting with membrane trafficking regulators that act to downregulate
other cell surface receptors. Accordingly, Sup-T1 cells were infected with isogenic Gag-
Pol truncated VSV-G pseudotyped NL4.3 viruses that only differed at the following Nef
motifs: the myristoylation site (G2; [48]), the methionine residue critical for Nef: AP-1:
MHC-I complex formation (M20; [21]), the dileucine motif implicated in adaptor protein-
complex formation (LL165; [49]) and the diacidic motif implicated in vacuolar ATPase
binding (DD175; [50]). Specifically, Sup-T1 cells were infected with pseudotyped NL4.3
encoding a non-functional Vpu protein and the various Nef mutants. Subsequently, live
and infected single cells were analyzed (Figure 2.2). In addition, cells were infected with
pseudotyped NL4.3 encoding a functional Vpu protein and the various Nef mutants (Figure
2.12; Figure 2.13). As expected, cells infected with virus encoding Nef G2A [48], which
disrupts most known Nef functions, exhibited significantly higher cell surface levels of
CD28 compared to cells infected with virus encoding wild-type Nef (Figure 2.13; Figure
2.14A, B). Interestingly, cells infected with viruses encoding the LL165AA and DD175GA
Nef mutations also showed significantly higher cell surface levels of CD28, suggesting that
the LL165 and DD175 motifs are critical for cell surface CD28 downregulation (Figure
2.13A; Figure 2.14A, B). Conversely, mutation of M20 (M20A), a residue essential for the
downregulation of MHC-I via the interaction with AP-1 [21, 51], did not affect CD28 cell
surface downregulation (Figure 2.13A; Figure 2.14A, B). Importantly, the dileucine
(LL165AA) and diacidic (DD175GA) motif mutations did not inhibit other Nef functions, as
these mutated Nef proteins retained the ability to downregulate cell surface MHC-I (Figure
2.13C), demonstrating that the dileucine and diacidic motifs function in downregulating
CD28.
In parallel, Sup-T1 cells were infected with the isogenic Gag-Pol truncated VSV-G
pseudotyped NL4.3 viruses that only differed at various Nef motifs and total CD28 levels
were measured by flow cytometry. The observed decrease in total CD28 in the presence of
Nef (Figure 2.14C) was abolished by mutation of the myristolation site (G2A) and the
97
diacidic motif (DD175GA), as cells infected with viruses encoding these mutated Nef
proteins exhibited significantly higher total levels of CD28 compared to those infected with
virus encoding wild-type Nef (Figure 2.13; Figure 2.14C). However, unlike cell surface
CD28, the total CD28 levels did not differ significantly between cells infected with virus
encoding wild-type Nef versus Nef LL165AA in cells infected with viruses also encoding
Vpu (Figure 2.13B). Contrastingly, in cells infected with virus lacking Vpu and encoding
the Nef LL165AA mutation, we observed significantly greater levels of cell surface CD28
(Figure 2.14C). Finally, expression of the mutated Nef proteins was confirmed by Western
blot (Figure 2.14). Taken together, these findings implicate the Nef diacidic motif (DD175)
in the downregulation of total and cell surface CD28.
98
Figure 2.12 Gating of Sup-T1 cells infected with VSV-G pseudotyped NL4.3
encoding various Nef mutants
To examine the population of interest, cells were gated on, followed by gating on
infected (GFP+) cells. In a representative experiment 97.9% of cells were infected
(GFP+).
99
Figure 2.13 Nef: host protein interaction motifs are critical for Nef-mediated
CD28 downregulation in the presence of Vpu
Infected CD4+ Sup-T1 cells were stained for CD28 or MHC-I and analyzed by flow
cytometry. Cells infected with VSV-G pseudotyped wild-type NL4.3 (NL4.3, red) or
NL4.3 lacking Nef (dNef, blue) were used as controls. (A) Mean (+/- SE) relative cell
surface CD28 of cells infected with NL4.3 encoding various mutations in the nef gene
100
(n ≥5). (B) Mean (+/-SE) relative cell surface MHC-I on cells infected with NL4.3
encoding various nef mutations (n≥4). (C) Relative mean (+/- SE) total CD28 within live
cells infected with NL4.3 encoding various nef mutations (n≥6). (SE: standard error;
*p≤ 0.05; **p≤ 0.01; ****p≤ 0.0001)
101
Figure 2.14 Nef: host cell protein interaction motifs are critical for Nef-mediated
CD28 downregulation
Infected CD4+ Sup-T1 cells were stained for CD28 and analyzed by flow cytometry.
Cells infected with VSV-G pseudotyped NL4.3 lacking Vpu (dVpu, orange) or Vpu and
Nef (dVpu dNef, green) were used as controls. (A) Representative dot plots illustrating
CD28 (APC) and infection (GFP) in live (Zombie RedTM-) cells. (B) Mean (+/-SE)
relative cell surface CD28 MFIs of cells infected with NL4.3 lacking Vpu and encoding
the indicated mutations in nef (n ≥4). (C) Relative mean (+/-SE) total CD28 within live
cells infected with NL4.3 encoding various Nef mutations (n≥5). (D) Western blot
illustrating expression of the mutated Nef proteins. (Mr: molecular weight; UI:
uninfected; SE: standard error; *p≤ 0.05; ***p≤ 0.001; ****p≤ 0.0001)
102
2.2.4 Motifs in Vpu implicated in interactions with specific host cellular proteins are
critical for CD28 downregulation
Next, we sought to gain insight into the mechanism used by Vpu to downregulate CD28
by examining the ability of mutant Vpu proteins to modulate cell surface and total CD28
levels. Mutated motifs within the NL4.3 vpu gene which have been previously implicated
in the ability of Vpu to downregulate CD4 [52-54] or the restriction factor BST-2 [55-57]
were inserted into a NL4.3 viral backbone encoding eGFP, but lacking a functional nef
gene (pNL4.3 dG/P eGFP dNef). Sup-T1 cells were infected with these VSV-G
pseudotyped isogenic NL4.3 viruses, which only differ in the specific vpu gene mutations.
Relative to cells infected with virus encoding wild-type Vpu (dNef), significantly greater
levels of cell surface CD28 were present on cells infected with viruses encoding Vpu
mutations at the adaptor protein interaction interface (LV64AA Vpu dNef) and serine
residues (S52/56N Vpu dNef) which are phosphorylated by casein kinase II to facilitate
recruitment of E3 ubiquitin ligase complex components (Figure 2.15; [37]). In contrast,
relative to cells infected with virus encoding wild-type Vpu (dNef), cell surface CD28
levels were not significantly different on cells infected with virus encoding a mutation
within a Vpu transmembrane domain residue implicated in downregulation of CD4 and
BST-2 (W22L Vpu dNef [53, 56]; Figure 2.15A, B). The functionality of the mutant Vpu
proteins was tested by examining the effects of the mutations on Vpu-mediated CD4
downregulation in Sup-T1 cells (Figure 2.16). Indeed, upon infection with viruses
encoding the W22L, S52/56N or LV64AA mutations, significantly greater cell surface levels
of CD4 were present relative to cells infected with virus encoding wild-type Vpu (dNef;
Figure 2.16A), suggesting that the W22 motif is critical for CD4, but not cell surface CD28
downregulation.
In parallel, viruses encoding mutated Vpu proteins were also tested for their effects on the
downregulation of total CD28. Total CD28 levels were not significantly different when
comparing cells infected with virus encoding wild-type Vpu or the W22L or LV64AA
mutations (Figure 2.15C). In contrast, there was significantly greater total CD28 in the
absence of Vpu (dVpu dNef) and in the presence of Vpu encoding the S52/56N mutation
(S52/56N dNef) relative to virus encoding wild-type Vpu (dNef; Figure 2.15C). As an
additional control, we demonstrated that cells infected with virus encoding the W22L,
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S52/56N or LV64AA mutations exhibited significantly greater total levels of CD4 relative to
cells infected with virus encoding wild-type Vpu (Figure 2.16B). Expression of all mutated
Vpu proteins was confirmed by Western blot (Figure 2.15D). As with our findings for cell
surface CD28 downregulation, this suggests that the molecular determinants of CD28
downregulation by Vpu are distinct from those utilized to downregulate CD4.
105
Figure 2.15 Motifs in Vpu are critical for downregulation of CD28
Infected CD4+ Sup-T1 cells were stained for CD28 and analyzed by flow cytometry.
Mean geometric fluorescence intensities (MFI) of cells were determined after gating on
live and infected (Zombie RedTM- and GFP+) cells. Cells infected with VSV-G
pseudotyped NL4.3 lacking Nef (dNef, blue) and both Nef and Vpu (dNef dVpu, green)
were used as controls. (A) Representative histograms illustrating cell surface CD28 on
live (Zombie RedTM-) infected (GFP+) cells. MFIs are indicated. (B) Mean (+/-SE)
relative cell surface CD28 on cells infected with viruses encoding mutations in vpu (n
≥9). (C) Relative mean (+/-SE) total CD28 within cells infected with viruses encoding
the indicated vpu mutations (n≥7). (D) Western blot illustrating expression of mutated
Vpu proteins. (Mr: molecular weight; UI: uninfected; SE: standard error; *p≤ 0.05;
**p≤ 0.01; ****p≤ 0.0001)
106
Figure 2.16 Specific motifs in Vpu are critical for downregulation of CD4
Infected CD4+ Sup-T1 cells were stained for CD4 and analyzed by flow cytometry. Mean
geometric fluorescence intensities of cells (MFI) were determined after gating on live
and infected (Zombie RedTM- and GFP+) cells. Cells infected with VSV-G pseudotyped
NL4.3 lacking Nef (dNef, blue) and both Nef and Vpu (dNef dVpu, green) were used as
controls. (A) Mean (+/-SE) relative cell surface CD4 on cells infected with NL4.3
encoding various mutations in vpu (n≥4). (B) Relative mean (+/- SE) total CD4 within
cells infected with NL4.3 encoding various Vpu mutations (n≥5). (SE: standard error;
*p≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001)
107
2.2.5 Implicating host cell proteins in Nef- and Vpu-mediated CD28 downregulation
We next sought to utilize the information gained through our mutational analysis to test for
the requirements of putative host cell proteins in Nef- or Vpu-mediated downregulation of
CD28. Specifically, the Nef LL165 and DD175 motifs, which were critical for CD28
downregulation (Figure 2.13; Figure 2.14), and the Vpu LV64 motif, which was critical for
cell surface CD28 downregulation (Figure 2.15), have been implicated in binding to the
membrane trafficking adaptor protein AP-2 [24, 50, 57]. Thus, we sought to test for the
requirement of AP-2 in CD28 downregulation. To test this, CD4+ Sup-T1 T cells were
infected with lentiviral vectors encoding eGFP and shRNAs targeting the AP-2 α and μ
subunits. The next day, these cells were infected with VSV-G pseudotyped NL4.3 encoding
mStrawberry and Nef, Vpu, or Nef and Vpu. Forty-eight hours later cell surface CD28 was
measured by flow cytometry. Live (Zombie NIRTM-) cells transduced with both the shRNA
encoding viruses (eGFP+) and viral protein encoding viruses (mStrawberry+) were
analyzed for cell surface CD28 levels by gating on double positive cells (eGFP+
mStrawberry+), and the relative downregulation mediated by Nef or Vpu was determined.
Compared to cells transduced with virus encoding a non-specific (NS) shRNA, no
significant effect of AP-2 α and μ knock-down on Nef or Vpu mediated CD28
downregulation was observed (Figure 2.17A). Knock-down of the AP-2 subunits was
confirmed using Western blotting (Figure 2.17 B, C). These findings suggest that Nef- and
Vpu-mediated decreases in cell surface levels of CD28 may be AP-2 independent, or
alternatively may demonstrate that cells can compensate for the loss of the AP-2 α and μ
subunits.
108
Figure 2.17 Downregulation of cell surface CD28 by Nef and Vpu in the presence
of shRNA targeting AP-2
Sup-T1 T cells were infected with lentiviruses encoding eGFP and non-specific (NS) or
AP-2 α and μ subunit targeting shRNA. Twenty-four hours post transduction cells were
infected with VSV-G pseudotyped NL4.3 encoding mStrawberry and encoding or
lacking Nef and Vpu. Forty-eight hours later, cells were stained for cell surface CD28
and analyzed by flow cytometry. Cell surface CD28 levels on live (Zombie NIRTM-),
dual infected (eGFP+ mStrawberry+) cells was determined. (A) Mean (+/-SD) relative
levels of CD28 downregulation mediated by the various viral proteins in the presence or
absence of Nef and/or Vpu (n=8). Downregulation was calculated by comparing cell
surface levels of CD28 on cells expressing vs. lacking the viral protein of interest, and
downregulation in the presence of the AP-2 α- and μ-targeting shRNAs was normalized
to downregulation in the presence of non-specific (NS) control shRNA (set to 1). (B)
Western blot illustrating AP-2 μ protein levels in cells transduced with virus encoding
the knock-down (KD) shRNA or non-specific shRNA (NS). (C) Western blot illustrating
AP-2 α protein levels in cells transduced with virus encoding the knock-down (KD)
shRNA or non-specific shRNA (NS). (SD: standard deviation; AP-2: adaptor protein 2;
NS: non-specific; KD: knock-down; GFP: green fluorescent protein; GAPDH:
glyceraldehyde 3-phosphate dehydrogenase)
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In order to further confirm that knock-down of AP-2 does not affect Nef-mediated CD28
downregulation, we tested for the requirement of the membrane trafficking regulator
sorting nexin 9 (SNX9), which functions in membrane trafficking by binding to AP-2 [58].
Moreover, in uninfected cells CD28 is removed from the cell surface in a SNX9 dependent
manner [59]. To test for the requirement of SNX9 in Nef-mediated cell surface CD28
downregulation, CD4+ Sup-T1 cells were transduced with VSV-G pseudotyped NL4.3
which was modified to express a previously described non-functional dominant negative
SNX9 protein lacking the PX and BAR domains [58], as well as Nef-eGFP or a non-
functional mutant Nef G2A-eGFP. In parallel, cells were infected with viruses encoding
wild-type SNX9 and Nef-eGFP or Nef G2A-eGFP. These vectors also lacked a functional
vpu gene to ensure only Nef-mediated CD28 downregulation could occur. Forty-eight
hours post transduction, cells were analyzed for cell surface CD28 levels using flow
cytometry. The downregulation of CD28 in live, transduced (Zombie RedTM- and GFP+)
cells was calculated based on mean fluorescence intensity of cells expressing Nef-eGFP
versus Nef G2A-eGFP. Subsequently, cell surface CD28 downregulation was compared
between cells transduced with virus encoding wild-type SNX9 (F2A SNX9 (WT)) or
dominant negative SNX9 (F2A SNX9 (DN); Figure 2.18A). Expression of the SNX9 and
Nef proteins of interest within transduced cells was confirmed by Western blotting (Figure
2.18B). No difference in Nef-mediated downregulation of cell surface CD28 was seen
between cells expressing wild-type versus dominant negative SNX9 (Figure 2.18A),
indicating that SNX9 may be dispensable for cell surface downregulation of CD28 by Nef.
Alternatively, cells may compensate for the disruption in SNX9-mediated trafficking by
utilizing alternate trafficking pathways.
110
Figure 2.18 Nef-mediated cell surface downregulation of CD28 in the presence of
overexpressed wild-type or dominant negative non-functional SNX9
Sup-T1 T cells were infected with VSV-G pseudotyped lentiviruses encoding wild-type
or dominant negative SNX9 and Nef-eGFP or a non-functional Nef mutant (Nef G2A-
eGFP). Forty-eight hours later, cells were stained for cell surface CD28 and analyzed by
flow cytometry. (A) Mean (+/-SD) relative levels of CD28 downregulation mediated by
Nef in live (Zombie RedTM-) and infected (eGFP+) cells. (n=8) (B) Western blot
illustrating expression of wild type or dominant negative SNX9-FLAG and Nef-eGFP
or Nef G2A-eGFP within transduced cells. (SD: standard deviation; SNX9: sorting nexin
9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; eGFP: enhanced green
fluorescent protein; WT: wild-type; DN: dominant negative; UI: uninfected)
111
In addition to testing the requirement of SNX9 for the downregulation of cell surface
CD28, we also sought to determine if SNX9 is required for Nef-mediated decreases in total
CD28 protein levels. To test this, HeLa cell lines stably expressing SNX9-targeting or non-
specific shRNA were generated. These cells were subsequently transfected with expression
vectors encoding epitope tagged CD28 and Nef, and total protein was examined using
Western blotting (Figure 2.19). Decreased CD28-FLAG was observed in cells expressing
Nef and a non-specific shRNA, while this Nef-mediated decrease in CD28-FLAG was
absent in cells expressing shRNA targeting SNX9 (Figure 2.19), indicating SNX9 may be
required for Nef-mediated reductions in total CD28 protein levels. To test this in T cells,
CD4+ Sup-T1 cells were infected with VSV-G pseudotyped lentiviruses encoding SNX9-
targeting or non-specific shRNA. The next day, infected cells were transduced with a
modified NL4.3 virus, as in Figure 2.7, that encodes CD28-FLAG and Nef (F2A CD28-
FLAG Nef) or no Nef (F2A CD28-FLAG dNef; Figure 2.6). Forty-eight hours post
transduction, cells were lysed and total CD28-FLAG was visualized by Western blot
(Figure 2.20). In cells transduced with virus expressing a non-specific shRNA (NS), there
was a reduction in CD28-FLAG detected in cells expressing Nef, compared to those
lacking Nef. However, in cells transduced with lentivirus encoding a SNX9-targeting
shRNA there was no difference in CD28-FLAG levels in the presence or absence of Nef
(Figure 2.20). Overall, these findings suggest that the AP-2-binding membrane trafficking
regulator SNX9 may be required for the ability of Nef to decrease total CD28 protein levels.
112
Figure 2.19 HeLa cells with decreased SNX9 expression exhibit reductions in Nef-
mediated decreases in total CD28-FLAG
HeLa cell lines were established that stably express shRNA targeting SNX9 (clone D1)
or non-specific shRNA (clone NS1). These cells were transfected with vectors encoding
CD28-FLAG and Nef-eGFP or eGFP alone. Forty-eight hours post transfection cells
were lysed and proteins analyzed by Western blot. (A) Western blot illustrating CD28-
FLAG and Nef-eGFP or eGFP expression in cells expressing SNX9-targeting (anti-
SNX9; left) or non-specific (right) shRNA. (B) Western blot illustrating SNX9 protein
levels in the stable cell lines utilized. Clone D1 was transduced with a virus encoding
SNX9-targeting shRNA, clone NS1 was transduced with virus encoding a non-specific
shRNA. (eGFP: enhanced green fluorescent protein; Mr: molecular weight; SNX9:
sorting nexin 9)
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Figure 2.20 SNX9-targeting shRNA reduces Nef-mediated decreases in total CD28
in Sup-T1 cells
CD4+ Sup-T1 T cells were transduced with lentiviruses encoding shRNA that targets
SNX9 or non-specific shRNA. The next day, cells were transduced with lentiviruses
encoding CD28-FLAG and Nef (F2A CD28-FLAG Nef) or no Nef (F2A CD28-FLAG
dNef). Forty-eight hours post infection cells were lysed and analyzed by Western blot.
Shown are Western blots illustrating CD28-FLAG (top) protein levels in cells transduced
with viruses encoding SNX9-targeting (left) or non-specific-targeting (right) shRNAs
and Nef (middle) and actin (bottom) protein levels in these cells. (Mr: molecular weight)
114
2.2.6 Patient-derived Vpu proteins mediate CD28 downregulation
Our findings suggest that HIV-1 NL4.3 can downregulate CD28 by both Nef and Vpu.
However, as HIV-1 is genetically diverse, functions observed in the laboratory strain
NL4.3 may not necessarily play a prominent role in the epidemic at large. We therefore
wanted to determine if Vpu-mediated CD28 downregulation is a conserved function. Thus,
we tested the ability of multiple group M Vpu proteins to downregulate cell surface and
total CD28. To test this, the NL4.3 vpu gene from a gag-pol truncated NL4.3 virus lacking
Nef (NL4.3 dG/P eGFP dNef) was replaced with vpu genes from a HIV-1 subtype B
reference strain (B.US.86.JRFL), a subtype C reference strain (C.BR.92.92BR025; [60]),
or a gene encoding a subtype C consensus protein (CC). Sup-T1 cells were infected with
these VSV-G pseudotyped isogenic viruses which differed only in the encoded Vpu
proteins, and cell surface and total CD28 levels were measured by flow cytometry. Cells
infected with virus encoding a subtype C consensus protein or a subtype B reference strain
(B.US.86.JRFL) Vpu protein did not significantly differ in cell surface levels of CD28
relative to cells infected with virus encoding NL4.3 Vpu (Figure 2.21A, B), suggesting that
these additional Vpu proteins were capable of downregulating cell surface CD28. In
contrast, cells infected with virus encoding a subtype C reference strain
(C.BR.92.92BR025) vpu gene had significantly greater cell surface levels of CD28, as with
virus lacking a functional vpu gene (dVpu dNef; Figure 2.21A, B). Moreover, staining for
total cellular CD28 revealed that cells infected with virus encoding the B.US.86.JRFL and
consensuses C vpu genes did not differ in total cellular CD28 levels relative to cells infected
with virus encoding NL4.3 vpu, while the C.BR.92.92BR025 Vpu protein did not
downregulate total CD28 (Figure 2.21C). To confirm that these findings were not unique
to the Sup-T1 cell line, PBMCs were infected with the same pseudotyped NL4.3 viruses
(Figure 2.21A) and cell surface CD28 levels on infected (GFP+) CD4+ cells was measured
(Figure 2.1E, F, G; Figure 2.22). A similar trend to that observed in Sup-T1 cells was seen
whereby the levels of CD28 were greater on cells infected with virus lacking the vpu gene
than in cells infected with virus encoding the B.US.86.JRFL vpu gene. However, unlike in
Sup-T1 cells, significantly more cell surface CD28 is present on cells infected with virus
encoding CC Vpu than NL4.3 Vpu. Moreover, expression of these proteins in a
heterologous system revealed equivalent protein levels for all Vpus except for Vpu from
115
C.BR.92.92BR025, which is weakly expressed (Figure 2.21D). Overall, this suggests that
Vpu-mediated cell surface and total CD28 downregulation is not limited to the laboratory
adapted HIV-1 strain NL4.3.
116
Figure 2.21 Non-NL4.3 Vpu proteins downregulate cell surface CD28
CD4+ Sup-T1 and peripheral blood mononuclear cells (PBMCs) were infected with
VSV-G pseudotyped NL4.3 viruses lacking Nef and encoding wild-type Vpu (dNef,
117
blue), no Vpu (dNef dVpu, green) or three non-NL4.3 Vpu proteins (grey). Cells were
analyzed by flow cytometry and mean geometric fluorescence intensities (MFI) of
infected (GFP+) cells were determined. (A) Representative histograms illustrating cell
surface CD28 on live infected Sup-T1 cells. MFIs are indicated. (B) Mean (+/-SE)
relative cell surface CD28 on Sup-T1 cells infected with NL4.3 lacking a functional Nef
protein (dNef), but encoding various Vpu proteins (n=5). (C) Mean (+/-SE) relative total
cellular CD28 of Sup-T1 cells infected with NL4.3 encoding various Vpu proteins (n≥8).
(D) Western blot illustrating expression of eGFP-tagged versions of the analyzed Vpu
proteins from transfected HEK293T cells. (E) Representative dot plots illustrating GFP
and cell surface CD28 (APC) on CD4+ cells. (F) Representative histograms illustrating
cell surface CD28 on either uninfected (isotype control, uninfected) or CD4+ infected
(GFP+) cells. MFIs are indicated. (G) Mean (+/-SE) relative cell surface CD28 on CD4+
cells infected with viruses encoding various vpu genes (n=8). Data were obtained from
infection of PBMCs from two healthy donors. (NT: non-transfected; Mr: molecular
weight; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent
protein; SE: standard error; UI: uninfected; *p≤ 0.05; **p≤0.01; ****p≤ 0.0001).
118
Figure 2.22 Gating of CD4+ peripheral blood mononuclear cells infected with
VSV-G pseudotyped NL4.3
To examine the population of interest, lymphocytes were gated on, followed by gating
on CD4+ (APC-Cy7) and infected (GFP+) positive cells. In a representative experiment
35.9% of single lymphocytes were CD4+ and 1.3% of these were infected (GFP+). Gates
were set based on isotype stained (APC-Cy7) and uninfected controls.
119
2.2.7 Nef and Vpu differentially alter responsiveness to CD28-mediated stimulation
Given that HIV-1 decreases cell surface levels of CD28 (Figure 2.1), a key immune cell
stimulatory receptor, we postulated that decreased cell surface levels of CD28 on infected
cells leads to a decreased ability of cells to become activated by CD28/CD3 stimulation.
Thus, we wished to test what effects Nef and Vpu may have on activation in cells
stimulated by CD28/CD3. We therefore infected primary CD4+ T cells with VSV-G
pseudotyped NL4.3 encoding both Nef and Vpu (NL4.3), Nef alone (dVpu), Vpu alone
(dNef) or lacking both Nef and Vpu (dVpu dNef). Infected cells were stimulated with
soluble anti-CD28 and plate bound anti-CD3, and activation was determined by measuring
intracytoplasmic production of IL-2, an event that occurs downstream from CD28
activation (Figure 2.23; [61]). Upon infection with virus encoding Vpu alone (dNef), the
percentage of infected cells that produced IL-2 after stimulation was significantly lower
than in cells infected with virus encoding Nef and Vpu (NL4.3; Figure 2.23B), suggesting
that the presence of Nef leads to increased activation, consistent with Nef’s previously
reported role in T-cell activation upon anti-CD3 and anti-CD28 treatment of Jurkat cells
[62]. In contrast, in primary cells infected with a virus lacking Vpu (dVpu), the proportion
of IL-2 positive infected cells were significantly increased, suggesting that Vpu decreases
the anti-CD3/CD28 dependent cell activation. Overall, this suggests that the Nef and Vpu,
which influence cell surface and total CD28 levels, differentially regulate the
responsiveness to CD28 stimulation.
121
Purified CD4+ T cells were infected with VSV-G pseudotyped NL4.3 encoding Nef and
Vpu (NL4.3, red), lacking Nef (dNef, blue) or Vpu (dVpu, orange), or lacking both Nef
and Vpu (dVpu dNef, green). Twenty-four hours post-infection cells were activated with
anti-CD3/anti-CD28 for 24 hours. Cells were then incubated with Brefeldin A for 12
hours and stained for intracellular IL-2 prior to analysis by flow cytometry. (A)
Representative dot plots illustrating intracellular IL-2 (APC) and infection (eGFP)
levels. Quadrants were selected based on the IL-2 isotype and uninfected controls. The
percentage of infected cells that are IL-2 positive is indicated (red). The percentage of
cells making up the uninfected and IL-2 negative population (Q4) are as follows:
uninfected unstimulated: 99.8%, isotype control: 99.7%, uninfected stimulated: 92.8%,
NL4.3: 91.3, dNef: 95.5%, dVpu: 90.1, dVpu dNef: 89.3%. (B) Mean (+/-SE) percentage
of infected cells that are IL-2 positive (n≥6). The means were obtained by analysis of
infected cells from two healthy donors. (SE: standard error; *p≤ 0.05)
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2.3 Discussion
In this chapter, we describe the ability of both HIV-1 Nef and Vpu to decrease cell surface
and total levels of CD28 in CD4+ T cells. Specifically, we have shown cell surface CD28
downregulation with VSV-G pseudotyped NL4.3 in Sup-T1 cells and primary cells, as well
as downregulation in primary CD4+ T cells infected with replication competent NL4.3
(Figure 2.1; Figure 2.21). These results are consistent with observations made in cell lines
transiently transfected with Nef and/or Vpu expression vectors. Downregulation of
endogenous CD28 was previously illustrated in Jurkat cells transiently expressing Nef [39].
Moreover, Haller et al. completed a large screen for receptors downregulated by Nef and
Vpu through transient expression of the viral proteins in A3.01 T lymphocytes and
observed downregulation of CD28 by both HIV-1 Nef and Vpu [19]. Interestingly, the
latter study demonstrated varying degrees of downregulation by HIV-1 Nef and Vpu, as
well as SIVmac239 Nef, with respect to the multiple tested receptors [19]. Accordingly, we
observed that the effects of Nef and Vpu on CD28 are subtler than the effects of Nef and
Vpu on other receptors, namely MHC-I and CD4 (Figure 2.13; Figure 2.16), but
nevertheless in the presence of Nef and Vpu this receptor traffics to a degradative
lysosomal compartment and decreased total CD28 protein levels are observed (Figure 2.8;
Figure 2.9; Figure 2.10). Moreover, it has been shown that there are varying degrees of
CD28 downregulation by various Nef proteins, with HIV-2 and certain SIV derived Nef
proteins exhibiting more efficient downregulation than HIV-1 Nef proteins [19, 63].
However, this does not limit the potential physiological relevance of CD28 downregulation
by HIV-1 and our current findings demonstrate mechanistic details of Nef and Vpu-
mediated CD28 downregulation.
Our results demonstrate that slight increases in cell surface CD28 levels can be observed
in cells infected with viruses lacking Nef and Vpu (dVpu dNef) in comparison to
uninfected cells (Figure 2.1B, E). This suggests that HIV-1 proteins other than Nef and
Vpu may also be contributing to changes in cell surface CD28 levels upon infection. The
latter effects may be due to events independent of membrane trafficking and could be the
consequence of changes at the level of CD28 transcription. Notably, CD28 expression
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levels may be affected at the transcriptional level via the HIV-1 tat protein, as it interacts
with the Sp1 transcription factor which positively affects CD28 transcription [64, 65].
Given that Nef and Vpu collaborate to mediate the downregulation and degradation of
cellular receptors, specifically CD4 [27, 66-68], we hypothesized that the observed
decreases in total CD28 levels in the presence of Nef and Vpu were due to CD28
degradation. This is supported by our observation that CD28 co-localizes with the
lysosome marker LAMP-1 in the presence of the viral proteins (Figure 2.9). Moreover, we
found that we could limit Nef- and Vpu-mediated decreases in total CD28 and CD4 protein
levels upon treatment with ammonium chloride (Figure 2.10; Figure 2.11). The effects of
ammonium chloride on CD4 downregulation are of interest given the previously reported
Vpu-dependent proteasomal degradation of CD4 [69]. In our experiments, ammonium
chloride may be playing a non-specific role by impacting cellular physiology by affecting
general cell acidification. Importantly, we have used the specific endosomal acidification
inhibitors Bafilomycin A1 and Concanamycin A to provide a more unambiguous
mechanistic explanation of Nef- and Vpu-dependent effects on CD28 (Figure 2.10),
thereby bypassing the more general effects of using ammonium chloride. The specific
inhibition of the vacuolar ATPase with these molecules limits the co-localization of CD28
with LAMP-1 in Nef and Vpu-expressing cells (Figure 2.10).
We also report how CD28 depletion involves the distinct ability of Nef and Vpu to interact
with host cellular proteins. Indeed, cell surface CD28 downregulation was inhibited by
mutation of Nef LL165 and DD175, while maintaining the ability to downregulate MHC-I
(Figure 2.13; Figure 2.14). The findings that Nef LL165 and DD175 are critical for cell
surface CD28 downregulation is in accordance with previously published reports [39, 70],
and suggests that the Nef LL165 and DD175 motif binding proteins AP-2 [49] and the
vacuolar ATPase [50] may be critical for cell surface downregulation by Nef. Interestingly,
mutation of the G2, LL165 and DD175 motifs abolished Nef-dependent decreases in total
levels of cellular CD28 in the absence of Vpu (Figure 2.14), while mutation of the LL165
motif did not significantly alter total CD28 levels in the presence of Vpu (Figure 2.13).
This may be due to Vpu’s ability to compensate for specific Nef mutations. Our observed
increases in total cellular CD28 when the ability of Nef to interact with the vacuolar
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ATPase is inhibited further supports the idea that Nef mediates degradation of CD28
(DD175GA; Fig. 5C). The Nef DD175 motif is also critical for Nef-mediated lysosomal
degradation of the co-inhibitory receptor CTLA-4 [71], which interestingly, competes with
CD28 for binding the same receptors, B7.1/B7.2 [72]. Overall, this suggests that as with
cell surface downregulation, the downregulation of total CD28 levels is dependent on
specific Nef: host cell protein interaction motifs.
We have also provided mechanistic details on how Vpu modulates CD28 expression and
localization within HIV-1 infected cells. Intriguingly, the mutations exhibited different
effects on CD28 and CD4 downregulation (Figure 2.15; Figure 2.16), indicating that CD28
downregulation by Vpu may have evolved independently. Specifically, mutation of the
Vpu W22, S52/56 and LV64 motifs inhibited the ability of Vpu to downregulate cell surface
CD4 (Figure 2.16), in agreement with previous reports [52-54]. In contrast, mutation of the
highly conserved W22 did not inhibit cell surface downregulation of CD28 (Figure 2.15A,
B), suggesting that this residue is dispensable for CD28 downregulation despite being
implicated in the downregulation of CD4 and BST-2 [53, 56, 73, 74].
Similarly to CD4 downregulation, mutation of Vpu S52/S56 and LV64 inhibited cell surface
downregulation of CD28 (Figure 2.15A, B). Interestingly, these Vpu motifs play distinct
roles with AP-1 and AP-2, in addition to contributing to mechanisms that promote the
degradation of cellular proteins. Indeed, the non-phosphorylatable mutants of Vpu (Vpu
S52/56N) do not recruit AP-1 and AP-2 and subsequently fail to downregulate BST-2 [36],
whereas Vpu LV64 promotes the degradation of BST-2 via phagocytosis [75]. Thus, the
ability of Vpu to downregulate CD28 from the cell surface may require AP-1 or AP-2
binding to the LV64 motif in a Vpu S52/S56 dependent manner to exclude CD28 from the
cell surface post-endocytosis or prior to anterograde transport.
Interestingly, only the S52/56N mutations in Vpu inhibited Vpu-mediated decreases in total
CD28, whereas the W22, S52/56 and LV64 motifs were all necessary to decreasing total CD4
(Figure 2.15C; Figure 2.16). The adaptor protein-binding motif in Vpu (LV64) was critical
for cell surface downregulation, but dispensable for decreasing total CD28 (Figure 2.15B,
C). This motif may be unnecessary for reducing total CD28 levels as additional host cell
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binding factors, either known or currently unidentified, may compensate for mutation of
the LV64 motif. Overall, the mechanism of Vpu-mediated CD28 cell surface
downregulation is discrete from that which mediates total decreases in cellular CD28, and
this mechanism is distinct from Vpu-mediated downregulation of total CD4.
In addition to utilizing mutations in host cell binding motifs to shed light on the
mechanisms of Nef- and Vpu-mediated CD28 downregulation, we employed shRNA
knock-downs to potentially implicate specific host cellular proteins in CD28
downregulation. Interestingly, despite the fact that mutation of the Nef and Vpu AP-2
binding sites abolished the ability of these proteins to downregulate cell surface CD28
(Figure 2.13; Figure 2.14; Figure 2.15), shRNA depletion of the AP-2 α and μ subunits in
CD4+ Sup-T1 cells did not alter the ability of Nef or Vpu to downregulate surface CD28
relative to cells expressing a non-specific control shRNA (Figure 2.17). The incomplete
depletion of these AP-2 subunits may be insufficient to completely abolish the ability of
AP-2 to mediate trafficking. Alternatively, the dileucine binding motifs in Nef and Vpu
may be facilitating hijacking of AP-1, rather than AP-2, to downregulate CD28. The
dispensability of AP-2 for cell surface CD28 downregulation is supported by our finding
that a dominant negative SNX9 protein, which inhibits AP-2 dependent trafficking events
[58], did not inhibit Nef-mediated cell surface CD28 downregulation (Figure 2.18).
However, despite our findings that suggest AP-2 and SNX9 may be dispensable for cell
surface CD28 downregulation, it is possible that upon AP-2 depletion or inhibition of
SNX9 trafficking pathways additional membrane trafficking proteins compensate for these
losses to facilitate CD28 downregulation. This is supported by the redundant functions
observed for SNX9 and the related SNX18 protein [76, 77]. Moreover, shRNA-mediated
knock-down of SNX9 inhibited Nef-mediated decreases in total protein levels of
overexpressed epitope tagged CD28 in HeLa and Sup-T1 cells (Figure 2.19; Figure 2.20).
This discrepancy between the effects of SNX9 on Nef-mediated cell surface and total
protein downregulation may be due to differences that occur as a result of overexpressing
CD28 versus examining endogenous CD28. Indeed, our observations made using over-
expressed CD28 were often much less reproducible than our findings with endogenous
CD28. Alternatively, SNX9, and perhaps AP-2, may be dispensable for mediating
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decreases in cell surface CD28, but are required for degradation of CD28, as CD28
degradation may occur independently of cell surface downregulation.
We demonstrated that the ability of Vpu to downregulate cell surface and total CD28 is not
specific to the laboratory adapted NL4.3 strain Vpu protein. Specifically, a subtype C
consensus Vpu protein and a subtype B reference strain (B.US.86.JRFL) protein
downregulated cell surface and total CD28 in infected Sup-T1 cells (Figure 2.21).
Contrastingly, the Vpu protein derived from a subtype C reference strain
(C.BR.92.92BR025) did not downregulate CD28 (Figure 2.21), which may be attributable
in part to decreased expression (Figure 2.21D). Interestingly, in a separate study we found
that this subtype C reference strain encodes a non-functional Nef protein and has previously
been shown to have lower fitness when compared to other well described subtype reference
strains [78, 79]. Moreover, we found that in infected CD4+ PBMCs both the NL4.3 and
B.US.86.JRFL derived Vpu proteins were capable of downregulating cell surface CD28
(Figure 2.21E, F, G). However, unlike in Sup-T1 cells, PBMCs infected with virus
encoding the consensus C Vpu protein did not exhibit downregulation (Figure 2.21G). The
observed difference between Sup-T1 cells and CD4+ PBMCs may be due to intrinsic
differences between Sup-T1 and primary cells, as well as potential differences in the
expression pattern of the various Vpu proteins in different cell types. Interestingly, upon
examination of the amino acid sequences of these various Vpu proteins, we observed that
they exhibit conservation at the S52/56 motif we demonstrated to be critical for NL4.3 Vpu-
mediated CD28 downregulation (Figure 2.15; Figure 2.24). However, the B.JFRL, CC and
C.BR92025 Vpu proteins all lack the LV64 motif found in the NL4.3 Vpu protein, which
was critical for NL4.3 Vpu cell surface CD28 downregulation (Figure 2.15B; Figure 2.24).
Therefore, perhaps non-NL4.3 Vpu proteins contain unique residues which influence
CD28 downregulation ability. Nonetheless, the ability of distinct Vpu proteins to
downregulate cell surface and total CD28 suggests that this function is conserved to some
extent, despite high HIV-1 diversity [80].
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Figure 2.24 Protein sequence alignment of the Vpu proteins which were tested for
their ability to downregulate CD28
The protein sequences of the NL4.3, B.JFRL, CC and C.BR92025 Vpu proteins were
aligned using Clustal Omega [81]. The residues in NL4.3 Vpu that were mutated and
tested for their effects on CD28 downregulation are indicated.
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HIV-1 has evolved multiple means of downregulating CD28, which are in part genetically
separable from CD4 and MHC-I downregulation, implying that CD28 downregulation is
critical during infection. Moreover, we hypothesize that the function of CD28
downregulation is relevant during infection and that CD28 downregulation by Nef and Vpu
may alter cell activation through CD28 receptor stimulation. Indeed, we observed that in
primary CD4+ T cells infected with pseudotyped virus encoding Vpu alone (dNef), a
significantly smaller proportion of infected cells secreted IL-2 upon CD3/CD28
stimulation, relative to cells infected with virus encoding Nef and Vpu (NL4.3; Figure
2.23B). In contrast, cells infected with virus lacking Vpu (dVpu) displayed an increase in
cell activation (Figure 2.23), indicating that Nef alone increases CD28 stimulation
responsiveness.
The observed differences in cell activation in the presence or absence of Nef and Vpu may
be partially attributable to other reported functions of these viral proteins. Namely, Nef
alters the subcellular localization of the T cell receptor and immunological synapse
associated kinase Lck, leading to a physical separation of the T cell receptor from the
immunological synapse [6, 82]. Nef also binds and activates the serine/threonine kinase
PAK2, which induces T cell activation [62]. Nef thus alters the activation status of cells,
perhaps inducing the correct balance between activating and inhibitory signaling, thereby
enabling optimal viral replication without inducing anergy or activation induced cell death
[83]. However, when compared to Nef, little is known regarding how Vpu alters T cell
activation. Nonetheless, Vpu may interfere with T cell activation and IL-2 production
through its ability to inhibit NF-κB [84]. Ultimately, the evolution of CD28 downregulation
may be one component of the multitude of alterations within infected cells that act to attain
optimal levels of cell activation.
The role or function of CD28 downregulation in vivo remains elusive, however we
speculate it may play a role in cell activation. CD28 is critical for T cell activation,
providing co-stimulatory signaling necessary for activation upon binding to CD80/CD86
[4, 5] and in the absence of CD28, T cell receptor ligation can lead to an unresponsive,
anergic state [85]. The importance of CD28 in vivo has been demonstrated through the use
of anti-CD28 therapies to induce immunosuppression following transplantation, in patients
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with autoimmune diseases (reviewed in [86]) and during cancer immunotherapies via
CD28 activation [87]. In addition, reductions in IL-2 levels, as observed in the presence of
Vpu (Figure 2.23), may lead to impairments in T cell survival, proliferation and function
(reviewed in [88]). Interestingly, decreases in IL-2 levels are observed in HIV infected
individuals [89, 90], and clinical trials have examined the benefit of administering
recombinant IL-2 in combination with anti-retroviral therapy [91]. IL-2 has also been
associated with reactivation of latently infected cells [92, 93]. Therefore, it is conceivable
that CD28 downregulation alters responsiveness and activation of infected T cells.
Alterations in the activation status of infected cells, which may in part be achieved through
CD28 downregulation, could have effects in vivo during HIV-1 infection. Upon HIV-1
infection, a transcriptionally silent latent reservoir of cells is formed, which currently
present the largest obstacle for achieving a HIV-1 cure [94]. A decrease in cell activation
as a result of alterations in cell surface CD28 levels may allow an infected cell to enter a
transcriptionally silent, latent state. Indeed, CD28 activation is capable of inducing HIV-1
transcription and replication, even in the absence of TCR activation [95]. Moreover, viral
microRNA-mediated silencing of genes that contribute to cellular activation, which may
include CD28 [96], have been suggested to be important for the development of latency
[97]. Furthermore, CD28 activation is utilized to reactivate latent cells, as mTOR, a kinase
activated downstream of CD28 signaling [98], was identified as a critical controller of
HIV-1 latency [99].
Overall, we have demonstrated that the HIV-1 Nef and Vpu accessory proteins
downregulate CD28 from the cell surface and at the cellular level. This effect is observed
with more than just the laboratory adapted NL4.3 strain, indicating this function is
conserved. We propose that the decreases in total cellular CD28 may be potentiated by
lysosomal degradation of CD28. Moreover, our analysis of Nef and Vpu mutants suggest
that the Nef: vacuolar ATPase interaction and phosphorylation of Vpu S52/56 are both
critical for downregulation of cell surface and total protein levels of this key co-stimulatory
molecule. Finally, the presence or absence of Nef and Vpu modulates the ability of cells to
respond to CD28-mediated stimulation.
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2.4 Materials and Methods
2.4.1 DNA constructs
2.4.1.1 Expression vectors
To facilitate efficient overexpression of epitope tagged CD28, the human CD28 gene was
amplified out of the MegaMan Human Transcriptome Library (Agilent Technologies,
Santa Clara, CA) and subsequently amplified with a forward primer containing the CD8α
signal peptide and a reverse primer encoding the FLAG epitope tag. The CD8α signal
peptide-CD28-FLAG encoding DNA was subsequently sub-cloned into the peGFP-N1
expression vector (Clontech, Mountain View, CA) in which eGFP was removed by
restriction digestion. For expression of Nef-eGFP and eGFP, the NL4.3 nef gene was
subcloned into the peGFP-N1 expression vector or this vector was left unmodified. To test
the expression of the non-NL4.3 Vpu proteins the Vpu encoding fragments were PCR
amplified and sub-cloned into the peGFP-N1 expression vector (Clontech).
2.4.1.2 Lentiviral vectors
Viral infection plasmids used for VSV-G pseudotyped lentivirus production were
engineered from the previously described pNL4.3 dG/P eGFP or pNL4.3 dG/P eGFP dNef
backbones [100, 101]. To make viral constructs lacking Vpu, HIV-1NL4-3 vpu/nef/UD
Deletion Mutant (p230-11; NIH-AIDS reagents catalog number 2535) was digested with
EcoRI and BamHI and the fragment encoding the vpu gene was sub-cloned into pNL4.3
dG/P eGFP or pNL4.3 dG/P eGFP dNef. NL4.3 based lentiviral vectors encoding
mStrawberry, CD28-FLAG and SNX9 were produced through insertion into a previously
described vector encoding truncated gag and pol genes and the foot and mouth disease 2A
(F2A) site [43]. Specifically, to make a viral vector for overexpression of CD28-FLAG,
CD8α-CD28-FLAG from the aforementioned expression vector was PCR amplified and
sub-cloned into the F2A-containing lentiviral vectors of interest. For the expression of
wild-type or dominant negative SNX9, the full SNX9 gene or only residues 2 through 182,
respectively, were PCR amplified from a SNX9/pGEX-KG vector obtained from R.
Prekeris (University of Colorado, Aurora, CO) with a forward primer encoding the FLAG
epitope.
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Mutations in the NL4.3 nef gene were produced by site directed mutagenesis within a pN1
expression vector encoding NL4.3 Nef. Mutagenesis primers were created with the Agilent
Technologies QuickChange Primer Design program (Agilent Technologies). The nef genes
containing mutations were subsequently PCR amplified with primers encoding XmaI and
NotI cut sites, and were inserted into a previously described vector encoding XmaI and
NotI cut sites flanking the nef gene [43]. Mutations in the NL4.3 vpu gene were produced
by site directed mutagenesis in a pN1 Vpu-FLAG expression vector. Vpu genes encoding
mutations were subsequently PCR amplified and inserted into pRECnfl HIV-1
dVpu/URA3 (obtained from Eric Arts, University of Western Ontario), using a previously
described yeast recombination system [102]. The vpu-encoding fragment was then sub-
cloned into a NL4.3 backbone lacking a functional nef gene (pNL4.3 dG/P eGFP dNef)
using the EcoRI and BamHI restriction sites. Vpu genes derived from a subtype C reference
strain (C.BR.92.92BR025), subtype B reference strain (B.US.86.JRFL) or a consensus C
protein synthesized using Invitrogen GeneArt Synthesis (Thermo Fisher Scientific,
Mississauga, ON) were inserted into the pRECnfl HIV-1 dVpu/URA3 vector followed by
sub-cloning into pNL4.3 dG/P eGFP dNef, as described above.
Viral infection plasmids utilized to make replication competent NL4.3 were engineered
from the previously described pNL4.3 vector [101]. A Nef deficient plasmid (pNL4.3
dNef) was obtained from Gary Thomas (University of Pittsburgh) and Vpu (dVpu) and Nef
and Vpu (dVpu dNef) deficient plasmids were produced by subcloning the vpu encoding
portion of HIV-1NL4-3 vpu/nef/UD Deletion Mutant into pNL4.3 or pNL4.3 dNef, as
described above.
2.4.1.3 shRNA encoding vectors
The shRNA encoding lentiviral vectors encoding a non-specific (catalog number
TR30021) or SNX9 (catalog number TL307006D) targeting shRNA were obtained from
OriGene (OriGene Technologies, Inc., Rockville, MD). The lentiviral vectors encoding
AP-2 targeting shRNAs were produced as previously described [83].
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2.4.2 Cell culture
HEK293T (American Type Culture Collection (ATCC)), HeLa (ATCC) and U87 CD4+
CXCR4+ (National Institutes of Health-AIDS Research and Reference Reagent program;
catalog number 4036) cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 4 mM L-glutamine, 4500 mg/L glucose and sodium pyruvate
(HyClone, Logan, UT) and supplemented with 1% Penicillin-Streptomycin (Hyclone) and
10% fetal bovine serum (FBS; Wisent, St-Bruno, QC). Sup-T1 cells were maintained in
Roswell Park Memorial Institute media (RPMI) 1640 media with L-glutamine
supplemented with 100 g/ml penicillin-streptomycin, 1% sodium pyruvate, 1% non-
essential amino acids, 2 mM L-glutamine (Hyclone) and 10% FBS. All cell lines were
grown at 37°C in the presence of 5% CO2 and sub-cultured in accordance with supplier’s
recommendations.
Primary peripheral blood mononuclear cells were isolated from four healthy donors by
density centrifugation using Histopaque (Sigma-Aldrich, Oakville, ON) and
cryopreserved. Upon revival, cells were maintained in RPMI with L-glutamine
supplemented with 100 μg/ml penicillin-streptomycin, 1% sodium pyruvate, 1% non-
essential amino acids, 2 mM L-glutamine (Hyclone) and 10% FBS at 37°C and 5% CO2 in
the presence or absence of IL-2 and phytohemagglutinin (PHA) stimulation, as indicated.
CD4+ T cells were purified from PBMCs using the MojoSortTM human CD4 T cell isolation
kit (BioLegend, San Diego, CA) and Magnetic-activated cell sorting cell separation
columns (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s protocol.
For ammonium chloride treatment, cells were pelleted and culture medium was replaced
with complete RPMI containing or lacking 40 mM ammonium chloride (Sigma-Aldrich)
in phosphate buffered saline (PBS) twenty-four hours prior to analysis. For vacuolar
ATPase inhibitor treatment, cells were pelleted and culture medium was replaced with
complete RPMI containing vehicle, 100 nM Concanamycin A (Santa Cruz Biotechnology,
Dallas, TX) or 100 nM Bafilomycin A1 (Sigma-Aldrich) in DMSO 5 hours prior to
analysis.
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HeLa cells stably expressing shRNA with a non-specific target or targeting SNX9 were
produced by transduction with shRNA-encoding lentiviral vectors. Briefly, HeLa cells
were transduced with VSV-G pseudotyped lentivirus produced as described below with
pGFP-C-shLenti plasmid encoding a scrambled shRNA (TR30021, Origene) or a SNX9
targeting shRNA (TL307006D, Origene). Transduced cells were selected for using 0.8
ug/mL puromycin and clones were selected. Cells were subsequently maintained in
DMEM containing 0.4 ug/mL puromycin.
2.4.3 Transfections and Infections
2.4.3.1 Transfections
To examining CD28 expression levels in HeLa cells in the presence or absence of Nef,
HeLa cells were transfected with the appropriate plasmids using PolyJet (FroggaBio, North
York, ON), as per the manufacturer’s protocol.
2.4.3.2 Lentivirus production
For VSV-G pseudotyped lentivirus production, HEK293T cells were triply transfected
with the lentiviral vector of interest, the VSV-G envelope-encoding pMD2.G plasmid
(Addgene; catalog number 12259) and pCMV-DR8.2 (Addgene; catalog number 12263)
using PolyJet (FroggaBio) as per the manufacturer’s protocol. Forty-eight hours post
transfection, lentivirus was harvested via cell culture supernatant clarification by spinning
at 1500 x g, followed by 20 m filtration. For production of NL4.3 provirus, viral vectors
were transfected into HEK293T cells using PolyJet. Forty-eight hours post-transfection,
cell supernatant was applied to U87 CD4+ CXCR4+ cells to propagate the virus. Cell
supernatant was harvested four to six days post infection as described above. Viruses were
stored in 20% FBS at -80C.
2.4.3.3 Lentivirus transduction
For infection of Sup-T1 cells with VSV-G pseudotyped NL4.3 viruses to examine effects
on endogenous CD28, 8 x 105 Sup-T1 cells were pelleted and re-suspended in the
appropriate volume of pseudovirus in 20% FBS, 8 g/mL polybrene and brought to one
mL with 10% complete RPMI. Forty-eight hours post infection, cells were analyzed via
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flow cytometry, microscopy or Western blotting. For infection of peripheral blood
mononuclear cells with VSV-G pseudotyped viruses, cells were cultured for three days in
10 ng/mL IL-2 (PeproTech, Rocky Hill, NJ) and 5 μg/mL PHA (Sigma-Aldrich). Cells
were then pelleted and re-suspended in the appropriate volume of pseudovirus containing
8 g/mL polybrene. Cells were subsequently spinoculated for four hours at 2880 x g at
room temperature, re-suspended in fresh RPMI and incubated for two days prior to
analysis. For infection of primary CD4+ T cells with replication competent NL4.3 viruses,
cells were cultured for three days in 10 ng/mL IL-2 and 5 g/mL PHA-L. CD4+ T cells
were then purified as described above and re-suspended in the appropriate volume of virus
in 20% FBS and 8 g/mL polybrene. Subsequently, cells were spinoculated for four hours
at 2880 x g at room temperature, re-suspended in fresh complete RPMI containing 5 ng/mL
IL-2 and 5 g/mL PHA-L. Two days post-infection, cells were pelleted and re-suspended
in fresh media containing PHA and IL-2 and four days post-infection cells were fixed and
stained. For infection of purified CD4+ T cells with VSV-G pseudotyped virus, cells were
cultured for three days in 10 ng/mL IL-2 and 5 g/mL PHA-L. CD4+ T cells were then
purified as described above and re-suspended in the appropriate volume of virus in 20%
FBS and 8 g/mL polybrene. Subsequently, cells were spinoculated for four hours at 2880
x g at room temperature, re-suspended in fresh complete 10% RPMI and analyzed forty-
eight hours post-infection.
For examination of the effects of AP-2 μ or α knock-down on endogenous CD28
downregulation, 1 x 106 Sup-T1 cells were suspended in 8 g/mL polybrene and virus
preparations in 20% FBS produced as described above from pGFP-C-shLenti vectors
encoding different shRNAs. Cells were then spinoculated for four hours at 2880 x g at
room temperature and re-suspended in fresh RPMI. The next day, cells were pelleted and
re-suspended in 10% RPMI (mock infection) or pNL4.3 dG/P F2A-mStrawberry Nef/dNef
pseudovirions in 20% FBS and 8 g/mL polybrene. Forty-eight hours later, cells were
analyzed via flow cytometry or Western blotting. For examination of the effects of SNX9
knock-down on CD28-FLAG, cells were infected as above with pGFP-C-shLenti vectors
encoding NS or SNX9 targeting shRNAs, infected the next day with viruses encoding
CD28-FLAG and Nef or no Nef, and forty-eight hours later analyzed by Western blot.
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2.4.4 Flow cytometry analysis of cell surface receptors
Cells were analyzed using a BD Biosciences FACSCanto SORP (BD Biosciences, San
Jose, CA). Data analysis was performed using FlowJo software (version 9.6.4, FlowJo
LLC, Ashland, OR).
2.4.4.1 Sup-T1 cells
For cell surface staining of Sup-T1 cells, forty-eight hours post infection, cells were washed
twice with PBS, followed by staining for 20 minutes at room temperature with 1:6000
Zombie RedTM (BioLegend) or 1:6000 Zombie NIRTM (BioLegend) in PBS, where
appropriate. Cells were then washed in FACS buffer (1% FBS, 50 mM
ethylenediaminetetraacetic acid (EDTA) in PBS) and fixed in 1% paraformaldehyde (PFA)
for 20 minutes at room temperature. Cells were subsequently washed twice with FACS
buffer and stained with the appropriate antibodies by rocking for 40 minutes at room
temperature. Cells were then washed twice with FACS buffer and re-suspended in PBS.
For staining of CD28 and CD4, 1:25 APC-conjugated mouse-anti-CD28 (clone CD28.2,
BioLegend) and 1:25 APC-conjugated mouse-anti-CD4 (clone OKT4, BioLegend) were
utilized, respectively. For cell surface MHC-I staining, cells were stained with W6/32 (anti-
MHC-I, pan-selective for HLA-A, B and C, provided by D. Johnson, Oregon Health and
Science University), washed twice with FACS buffer, incubated with an APC-conjugated
species specific secondary antibody, followed by washing with FACS buffer and re-
suspending in PBS. For analysis of total CD28 or CD4 levels, cells were prepared as above,
but were permeabilized and blocked prior to staining. Specifically, after fixation, cells were
permeabilized by incubation with 0.5% saponin for 15 minutes at room temperature. Cells
were then washed with 1% FBS, 0.1% saponin, 5 mM EDTA and blocked for thirty
minutes with blocking buffer (10% FBS, 0.1% saponin, 5 mM EDTA in PBS). Cells were
incubated with primary antibody diluted in blocking buffer followed by washing with 1%
FBS, 0.1% saponin, 5 mM EDTA and re-suspending in PBS.
2.4.4.2 Primary cells
For cell surface staining of peripheral blood mononuclear cells infected with VSV-G
pseudotyped NL4.3 encoding eGFP, cells were fixed in 2% PFA for 20 minutes at room
136
temperature, followed by washing twice with cell staining buffer (BioLegend). Cells were
then incubated for 40 minutes at room temperature with the appropriate fluorescently
labeled primary antibodies (1:50 APC-Cy7 conjugated anti-CD4 (clone OKT4,
BioLegend), 1:25 APC conjugated anti-CD28 (clone CD28.2, BioLegend)). Cells were
then washed twice with cell staining buffer and re-suspended in PBS prior to analysis.
For analysis of total CD28 in primary CD4+ T cells infected with VSV-G pseudotyped
NL4.3 provirus, cells were washed twice with cell staining buffer (BioLegend) and fixed
and permeabilized in BD Cytofix/Cytoperm solution (BD Biosciences). Cells were
subsequently washed twice with Perm/Wash buffer (BD Biosciences) and stained for 30
minutes at 4°C for CD28 level analysis (1:25 APC conjugated anti-CD28 (clone CD28.2,
BioLegend)). Cells were then washed, re-suspended in PBS and analyzed.
For analysis of cell surface CD28 on primary CD4+ T cells infected with replication
competent NL4.3 provirus, cells were washed twice with cell staining buffer (BioLegend)
and incubated for 30 minutes at 4°C with fluorophore conjugated primary antibodies
against the appropriate cell surface antigen (1:25 APC conjugated anti-CD28 (clone
CD28.2, BioLegend)). Cells were then washed twice with cell staining buffer and then
fixed and permeabilized in BD Cytofix/Cytoperm solution (BD Biosciences, San Jose,
CA). Cells were subsequently washed twice with Perm/Wash buffer (BD Biosciences) and
stained for 30 minutes at 4°C for p24 level analysis (1:50 anti-p24; clone KC57, Beckman
Coulter). Cells were then washed, re-suspended in PBS and analyzed. The following
isotype control antibodies were used in lieu of primary antibody as required: APC-Cy7
conjugated mouse IgG2b, κ isotype (clone MOPC-21, BioLegend), APC mouse IgG1 κ
isotype control (clone MOPC-21, BioLegend), PE mouse IgG1κ (clone: MOPC-21,
BioLegend).
2.4.5 Protein analysis
Infected Sup-T1 cells and HeLa or HEK293T cells transfected as described above were
lysed forty-eight hours post transduction or transfection for protein expression analysis.
Briefly, cells were pelleted and lysed by rocking in lysis buffer (0.5 M HEPES, 1.25 M
NaCl, 1 M MgCl2, 0.25M EDTA, 0.1% Triton X-100 and 1X complete Protease Inhibitor
137
Tablets (Roche, Indianapolis, IN)) for one hour at 4C. The supernatant was then clarified
by spinning at 16100 x g for 30 minutes at 4C, mixed with SDS-PAGE sample buffer
(0.312 M Tris pH 6.8, 2.6 M 2-Mercaptoethanol, 50% glycerol, 10% SDS) and boiled at
95C for 10 minutes. Samples were run on 12 or 14% SDS-PAGE gels followed by
transferring to nitrocellulose. Membranes were blocked with 5% milk in TBST (50 mM
Tris, 150 mM NaCl, 0.1% Tween 20) for one hour at room temperature, followed by
incubation overnight at 4C with the appropriate primary antibody in 5% milk in TBST.
The following primary antibodies were used: 1:1000 rabbit anti-Nef polyclonal antibody
(NIH-AIDS Research and Reference Reagent program, catalog number 2949), 1:2000
rabbit anti-GFP polyclonal antibody (Clontech), 1:5000 rabbit anti-Vpu polyclonal
antibody (NIH-AIDS Research and Reference Reagent program, catalog number 969),
1:2000 mouse anti-Actin monoclonal antibody (Thermo Fisher Scientific, 1:1000 rabbit
anti-GAPDH polyclonal antibody (Thermo Fisher Scientific), 1:500 rabbit anti-AP2 μ (BD
Biosciences), 1:1000 rabbit anti-AP-2 α (Sigma-Aldrich), 1:800 rabbit anti-SNX9 (Sigma-
Aldrich), 1:1000 rat anti-DYKDDDK (BioLegend). The next day, membranes were
washed three times with TBST and incubated for two hours at room temperature with the
appropriate species specific HRP-conjugated secondary antibodies (1:2000, Thermo Fisher
Scientific) in 5% milk in TBST. Blots were subsequently washed and developed using ECL
substrates (Millipore, Etobicoke, ON) and a C-DiGit chemiluminescence Western blot
scanner (LI-COR Biosciences, Lincoln, NE). Relative intensities of protein bands were
determined using LI-COR Image Studio software.
2.4.6 Microscopy
For microscopy experiments, cells were infected and treated with inhibitors as described
above. Cells were then adhered to poly-L-lysine (Sigma-Aldrich) coated coverslips and
fixed in 2% PFA for 10 minutes at room temperature. Cells were subsequently washed
twice with PBS and permeabilized with methanol for 20 minutes. Cells were then washed
twice with PBS and blocked in 2% bovine serum albumin (BSA) in PBS for 1 hour at room
temperature. Cells were subsequently stained for two hours with mouse anti-CD28
(Thermo Fisher Scientific) and rabbit anti-LAMP-1 (Developmental Studies Hybridoma
Bank, University of Iowa) diluted at 1:200 in 0.2% BSA in PBS, washed twice with 0.2%
138
BSA in PBS and incubated for one and a half hours with the appropriate fluorophore
conjugated secondary antibodies (Alexa-Fluor-647 conjugated anti-mouse and Cy3-
conjugated anti-rabbit; Jackson ImmunoResearch, West Grove, PA) at 1:400 in 0.2% BSA
in PBS. Finally, cells were washed twice with PBS and mounted on coverslips with DAPI
Fluoromount-G (SouthernBiotech, Birmingham, AL). Cells were imaged on a Leica
DMI6000 B at 63X or 100X magnification using the FITC, Cy3, Cy5 and DAPI filter
settings and imaged with a Hamamatsu Photometrics Delta Evolve camera. Images were
subsequently deconvolved using the Advanced Fluorescence Deconvolution application
(Lecia, Wetzlar, Germany) on the Leica Application Suite software. Co-localization
analysis was conducted using Mander’s Coefficient from the ImageJ plugin JACoP, as
described previously [103].
2.4.7 Cell activation analysis
To examine activation in infected cells, cryopreserved PBMCs were revived and rested for
6 hours in complete RPMI media (without PHA/IL-2) at 37º C and 5% CO2. CD4+ T cells
were then purified as described above and spinoculated with the appropriate VSV-G
pseudotyped NL4.3 virus encoding both Nef and Vpu (NL4.3 dG/P eGFP), encoding Vpu
alone (NL4.3 dG/P eGFP dNef), encoding Nef alone (NL4.3 dG/P eGFP dNef), or lacking
both Nef and Vpu (NL4.3 dG/P eGFP dVpu dNef). After spinoculation, cells were
incubated in complete 10% RPMI for 24 hours prior to incubating with anti-CD3 (10
μg/ml, clone OKT3, BioLegend) immobilized on a plate and soluble anti-CD28 (5μg/ml,
clone CD28.2, BioLegend). After 24 hours of activation, cells were treated with Brefeldin
A (1:1000 dilution, BD Biosciences) for 12 hours before proceeding for intracellular IL-2
staining.
For intracellular staining of IL-2, infected CD4+ T cells were fixed using Cytofix/Cytoperm
solution (BD Biosciences) for 20 minutes at 4ºC, washed twice with Perm/Wash buffer
(BD Biosciences) and stained for intracellular IL-2 with an APC-conjugated anti-IL-2
antibody (clone MQ1-17H12, BD Biosciences), or the appropriate isotype control (APC
Rat IgG2a, κ isotype control, clone R35-95, BD Biosciences), for 1 hour at 4ºC (1:20
dilution in Perm/Wash buffer). Cells were then washed twice using Perm/Wash buffer prior
to analysis by flow cytometry. In order to determine the percentage of the infected cells
139
that were IL-2 positive, the percentage of GFP+ IL-2+ cells (Q2, Figure 2.23) was divided
by the total percentage of infected cells (sum of GFP+ IL-2+ (Q2, Fig. 8) and GFP+ IL-2-
(Q3, Figure 2.23) and multiplied by 100.
2.4.8 Data and Statistical analysis
2.4.8.1 Flow cytometry data analysis
For analyses of flow cytometry data obtained for Sup-T1 cells infected with VSV-G
pseudotyped NL4.3 encoding Vpu and various Nef mutations, relative levels of receptors
were determined by normalizing geometric mean fluorescence intensity after gating on
infected (GFP+) cells. For all other analysis of cell surface receptors on Sup-T1 cells
infected with a single VSV-G pseudotyped virus, relative levels of receptors were
determined by normalizing geometric mean fluorescence intensity after gating on single,
live (Zombie RedTM-), infected (GFP+) cells. Relative levels of CD28 on primary CD4+ T
cells infected with replication competent NL4.3 were determined by normalizing
geometric mean fluorescence intensity after gating on single, p24 high and CD28+ cells.
Relative levels of CD28 on VSV-G pseudotyped NL4.3 infected PBMCs, were determined
by normalizing geometric mean fluorescence intensity after gating on CD4+ and infected
(GFP+) lymphocytes. For all cell surface or total receptor analysis by flow cytometry, the
geometric mean fluorescence intensity of the control in each experiment (left-most sample
on each graph) was set to 1 and the other sample MFIs were calculated relative to the
control having an MFI of 1.
To calculate the relative increase in intracellular staining upon ammonium chloride
treatment, the geometric mean fluorescence intensity of the live and infected cells treated
with ammonium chloride was divided by the geometric mean fluorescence intensity of cells
that were untreated. This ratio was then normalized such that the fold increase in MFI for
cells infected with NL4.3 was equal to one.
For analysis of cell surface levels of CD28 on Sup-T1 cells infected with VSV-G
pseudotyped viruses encoding shRNAs and eGFP and mStrawberry and Nef and/or Vpu,
geometric mean fluorescence intensity of CD28 was determined after gating on single, live
(Zombie NIRTM-), doubly infected (mStrawberry+ GFP+) cells. The relative Nef or Vpu-
140
mediated downregulation of cell surface CD28 in the presence of the shRNA vectors was
then determined based on the MFI of cells infected with virus encoding versus lacking the
accessory protein of interest. The downregulation of CD28 was then normalized to the
downregulation mediated by the same viral protein in the presence of the non-specific
shRNA.
To analyze the effects of overexpression of wild-type SNX9 or dominant negative SNX9,
levels of CD28 downregulation were calculated by determining geometric mean
fluorescence intensity after gating on single, live (Zombie RedTM-), infected (GFP+) cells
and normalizing the levels on cells expressing Nef-eGFP to those expressing Nef G2A-
eGFP. Downregulation levels were then compared between cells transduced with virus
encoding wild-type SNX9 or dominant negative SNX9.
2.4.8.2 Statistical analyses
All statistics for analysis of receptor levels were conducted using a one-way analysis of
variance with Bonferroni’s multiple comparison test. For analysis of CD28: LAMP-1 co-
localization, the mean Manders’ overlap coefficients were compared using a one-way
analysis of variance with Bonferroni’s multiple comparison test to compare wild-type
infected cells to cells infected with viruses encoding or lacking Nef and/or Vpu (Fig. 3) or
wild-type infected cells treated with vehicle to cells treated with various inhibitors (Fig. 4).
Alternatively, for analysis of the percentage of IL-2 positive infected cells, a paired two-
tailed t-test was used. All statistical tests were completed using Graph Pad Prism (Graph
Pad Software Inc., La Jolla, CA).
2.5 References
1. Warrington R, Watson W, Kim HL, Antonetti FR. An introduction to immunology
and immunopathology. Allergy Asthma Clin Immunol. 2011;7 Suppl 1:S1.
2. Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-
inhibition. Nat Rev Immunol. 2013;13(4):227-42.
3. Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol.
2009;27:591-619.
4. Weiss A, Manger B, Imboden J. Synergy between the T3/antigen receptor complex
and Tp44 in the activation of human T cells. J Immunol. 1986;137(3):819-25.
141
5. Jenkins MK, Taylor PS, Norton SD, Urdahl KB. CD28 delivers a costimulatory
signal involved in antigen-specific IL-2 production by human T cells. J Immunol.
1991;147(8):2461-6.
6. Thoulouze MI, Sol-Foulon N, Blanchet F, Dautry-Varsat A, Schwartz O, Alcover
A. Human immunodeficiency virus type-1 infection impairs the formation of the
immunological synapse. Immunity. 2006;24(5):547-61.
7. Simmons A, Gangadharan B, Hodges A, Sharrocks K, Prabhakar S, Garcia A, et
al. Nef-mediated lipid raft exclusion of UbcH7 inhibits Cbl activity in T cells to
positively regulate signaling. Immunity. 2005;23(6):621-34.
8. Haller C, Rauch S, Michel N, Hannemann S, Lehmann MJ, Keppler OT, et al. The
HIV-1 pathogenicity factor Nef interferes with maturation of stimulatory T-
lymphocyte contacts by modulation of N-Wasp activity. J Biol Chem.
2006;281(28):19618-30.
9. de Waal Malefyt R, Yssel H, Spits H, de Vries JE, Sancho J, Terhorst C, et al.
Human T cell leukemia virus type I prevents cell surface expression of the T cell
receptor through down-regulation of the CD3-gamma, -delta, -epsilon, and -zeta
genes. J Immunol. 1990;145(7):2297-303.
10. Koga Y, Oh-Hori N, Sato H, Yamamoto N, Kimura G, Nomoto K. Absence of
transcription of lck (lymphocyte specific protein tyrosine kinase) message in IL-2-
independent, HTLV-I-transformed T cell lines. J Immunol. 1989;142(12):4493-9.
11. Ingham RJ, Raaijmakers J, Lim CS, Mbamalu G, Gish G, Chen F, et al. The
Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases
used by the T cell receptor. J Biol Chem. 2005;280(40):34133-42.
12. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, et al. Restoring
function in exhausted CD8 T cells during chronic viral infection. Nature.
2006;439(7077):682-7.
13. Muthumani K, Choo AY, Shedlock DJ, Laddy DJ, Sundaram SG, Hirao L, et al.
Human immunodeficiency virus type 1 Nef induces programmed death 1
expression through a p38 mitogen-activated protein kinase-dependent mechanism.
J Virol. 2008;82(23):11536-44.
14. El-Far M, Ancuta P, Routy JP, Zhang Y, Bakeman W, Bordi R, et al. Nef promotes
evasion of human immunodeficiency virus type 1-infected cells from the CTLA-4-
mediated inhibition of T-cell activation. J Gen Virol. 2015;96(Pt 6):1463-77.
15. Chaudhry A, Das SR, Jameel S, George A, Bal V, Mayor S, et al. A two-pronged
mechanism for HIV-1 Nef-mediated endocytosis of immune costimulatory
molecules CD80 and CD86. Cell Host Microbe. 2007;1(1):37-49.
16. Sugden SM, Bego MG, Pham TN, Cohen EA. Remodeling of the host cell plasma
membrane by HIV-1 Nef and Vpu: a strategy to ensure viral fitness and persistence.
Viruses. 2016;8(3):67.
142
17. Pawlak EN, Dikeakos JD. HIV-1 Nef: a master manipulator of the membrane
trafficking machinery mediating immune evasion. Biochim Biophys Acta.
2015;1850(4):733-41.
18. Guenzel CA, Herate C, Benichou S. HIV-1 Vpr-a still "enigmatic multitasker".
Front Microbiol. 2014;5:127.
19. Haller C, Muller B, Fritz JV, Lamas-Murua M, Stolp B, Pujol FM, et al. HIV-1 Nef
and Vpu are functionally redundant broad-spectrum modulators of cell surface
receptors, including tetraspanins. J Virol. 2014;88(24):14241-57.
20. Roeth JF, Williams M, Kasper MR, Filzen TM, Collins KL. HIV-1 Nef disrupts
MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J Cell Biol.
2004;167(5):903-13.
21. Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y. Structural basis of evasion
of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol. 2012;19(7):701-
6.
22. Dirk BS, Pawlak EN, Johnson AL, Van Nynatten LR, Jacob RA, Heit B, et al. HIV-
1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and
recycling. Sci Rep. 2016;6:37021.
23. Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D. HIV-1 Nef protein
protects infected primary cells against killing by cytotoxic T lymphocytes. Nature.
1998;391(6665):397-401.
24. Ren X, Park SY, Bonifacino JS, Hurley JH. How HIV-1 Nef hijacks the AP-2
clathrin adaptor to downregulate CD4. Elife. 2014;3:e01754.
25. Chaudhuri R, Lindwasser OW, Smith WJ, Hurley JH, Bonifacino JS.
Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent
on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. J
Virol. 2007;81(8):3877-90.
26. Ross TM, Oran AE, Cullen BR. Inhibition of HIV-1 progeny virion release by cell-
surface CD4 is relieved by expression of the viral Nef protein. Curr Biol.
1999;9(12):613-21.
27. Wildum S, Schindler M, Munch J, Kirchhoff F. Contribution of Vpu, Env, and Nef
to CD4 down-modulation and resistance of human immunodeficiency virus type 1-
infected T cells to superinfection. J Virol. 2006;80(16):8047-59.
28. Michel N, Allespach I, Venzke S, Fackler OT, Keppler OT. The Nef protein of
human immunodeficiency virus establishes superinfection immunity by a dual
strategy to downregulate cell-surface CCR5 and CD4. Curr Biol. 2005;15(8):714-
23.
29. Pham TN, Lukhele S, Hajjar F, Routy JP, Cohen EA. HIV Nef and Vpu protect
HIV-infected CD4+ T cells from antibody-mediated cell lysis through down-
modulation of CD4 and BST2. Retrovirology. 2014;11:15.
143
30. Veillette M, Desormeaux A, Medjahed H, Gharsallah NE, Coutu M, Baalwa J, et
al. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by
antibody-dependent cell-mediated cytotoxicity. J Virol. 2014;88(5):2633-44.
31. Piguet V, Gu F, Foti M, Demaurex N, Gruenberg J, Carpentier JL, et al. Nef-
induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal
targeting signal through the binding of beta-COP in endosomes. Cell.
1999;97(1):63-73.
32. Jia X, Weber E, Tokarev A, Lewinski M, Rizk M, Suarez M, et al. Structural basis
of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor
protein complex 1. Elife. 2014;3:e02362.
33. Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, et al.
The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated
from the cell surface by the viral Vpu protein. Cell Host Microbe. 2008;3(4):245-
52.
34. Mitchell RS, Katsura C, Skasko MA, Fitzpatrick K, Lau D, Ruiz A, et al. Vpu
antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-
lysosomal trafficking. PLoS Pathog. 2009;5(5):e1000450.
35. Mangeat B, Gers-Huber G, Lehmann M, Zufferey M, Luban J, Piguet V. HIV-1
Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its
beta-TrCP2-dependent degradation. PLoS Pathog. 2009;5(9):e1000574.
36. Kueck T, Foster TL, Weinelt J, Sumner JC, Pickering S, Neil SJ. Serine
Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction
with Clathrin Adaptors. PLoS Pathog. 2015;11(8):e1005141.
37. Margottin F, Bour SP, Durand H, Selig L, Benichou S, Richard V, et al. A novel
human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to
the ER degradation pathway through an F-box motif. Mol Cell. 1998;1(4):565-74.
38. Leonard JA, Filzen T, Carter CC, Schaefer M, Collins KL. HIV-1 Nef disrupts
intracellular trafficking of major histocompatibility complex class I, CD4, CD8,
and CD28 by distinct pathways that share common elements. J Virol.
2011;85(14):6867-81.
39. Swigut T, Shohdy N, Skowronski J. Mechanism for down-regulation of CD28 by
Nef. EMBO J. 2001;20(7):1593-604.
40. Norment AM, Lonberg N, Lacy E, Littman DR. Alternatively spliced mRNA
encodes a secreted form of human CD8 alpha. Characterization of the human CD8
alpha gene. J Immunol. 1989;142(9):3312-9.
41. de Souza AJ, Oriss TB, O'Malley K J, Ray A, Kane LP. T cell Ig and mucin 1 (TIM-
1) is expressed on in vivo-activated T cells and provides a costimulatory signal for
T cell activation. Proc Natl Acad Sci U S A. 2005;102(47):17113-8.
42. Wu J, Song Y, Bakker AB, Bauer S, Spies T, Lanier LL, et al. An activating
immunoreceptor complex formed by NKG2D and DAP10. Science.
1999;285(5428):730-2.
144
43. Dirk BS, Jacob RA, Johnson AL, Pawlak EN, Cavanagh PC, Van Nynatten L, et
al. Viral bimolecular fluorescence complementation: a novel tool to study
intracellular vesicular trafficking pathways. PLoS One. 2015;10(4):e0125619.
44. Mindell JA. Lysosomal acidification mechanisms. Annu Rev Physiol. 2012;74:69-
86.
45. Cardelli JA, Richardson J, Miears D. Role of acidic intracellular compartments in
the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium
chloride and chloroquine differentially affect proteolytic processing and sorting. J
Biol Chem. 1989;264(6):3454-63.
46. Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y. Bafilomycin A1, a
specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein
degradation in lysosomes of cultured cells. J Biol Chem. 1991;266(26):17707-12.
47. Drose S, Bindseil KU, Bowman EJ, Siebers A, Zeeck A, Altendorf K. Inhibitory
effect of modified bafilomycins and concanamycins on P- and V-type
adenosinetriphosphatases. Biochemistry. 1993;32(15):3902-6.
48. Kaminchik J, Bashan N, Pinchasi D, Amit B, Sarver N, Johnston MI, et al.
Expression and biochemical characterization of human immunodeficiency virus
type 1 nef gene product. J Virol. 1990;64(7):3447-54.
49. Bresnahan PA, Yonemoto W, Ferrell S, Williams-Herman D, Geleziunas R, Greene
WC. A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4
downregulation and binds the AP-1 clathrin adaptor. Curr Biol. 1998;8(22):1235-
8.
50. Geyer M, Yu H, Mandic R, Linnemann T, Zheng YH, Fackler OT, et al. Subunit H
of the V-ATPase binds to the medium chain of adaptor protein complex 2 and
connects Nef to the endocytic machinery. J Biol Chem. 2002;277(32):28521-9.
51. Noviello CM, Benichou S, Guatelli JC. Cooperative binding of the class I major
histocompatibility complex cytoplasmic domain and human immunodeficiency
virus type 1 Nef to the endosomal AP-1 complex via its mu subunit. J Virol.
2008;82(3):1249-58.
52. Hill MS, Ruiz A, Schmitt K, Stephens EB. Identification of amino acids within the
second alpha helical domain of the human immunodeficiency virus type 1 Vpu that
are critical for preventing CD4 cell surface expression. Virology. 2010;397(1):104-
12.
53. Tiganos E, Friborg J, Allain B, Daniel NG, Yao XJ, Cohen EA. Structural and
functional analysis of the membrane-spanning domain of the human
immunodeficiency virus type 1 Vpu protein. Virology. 1998;251(1):96-107.
54. Margottin F, Benichou S, Durand H, Richard V, Liu LX, Gomas E, et al. Interaction
between the cytoplasmic domains of HIV-1 Vpu and CD4: role of Vpu residues
involved in CD4 interaction and in vitro CD4 degradation. Virology.
1996;223(2):381-6.
145
55. Tervo HM, Homann S, Ambiel I, Fritz JV, Fackler OT, Keppler OT. beta-TrCP is
dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction
to HIV-1 release. Retrovirology. 2011;8:9.
56. Pang X, Hu S, Li J, Xu F, Mei S, Zhou J, et al. Identification of novel key amino
acids at the interface of the transmembrane domains of human BST-2 and HIV-1
Vpu. Retrovirology. 2013;10:84.
57. Kueck T, Neil SJ. A cytoplasmic tail determinant in HIV-1 Vpu mediates targeting
of tetherin for endosomal degradation and counteracts interferon-induced
restriction. PLoS Pathog. 2012;8(3):e1002609.
58. Lundmark R, Carlsson SR. Sorting nexin 9 participates in clathrin-mediated
endocytosis through interactions with the core components. J Biol Chem.
2003;278(47):46772-81.
59. Badour K, McGavin MK, Zhang J, Freeman S, Vieira C, Filipp D, et al. Interaction
of the Wiskott-Aldrich syndrome protein with sorting nexin 9 is required for CD28
endocytosis and cosignaling in T cells. Proc Natl Acad Sci U S A.
2007;104(5):1593-8.
60. HIV type 1 variation in World Health Organization-sponsored vaccine evaluation
sites: genetic screening, sequence analysis, and preliminary biological
characterization of selected viral strains. WHO Network for HIV Isolation and
Characterization. AIDS Res Hum Retroviruses. 1994;10(11):1327-43.
61. June CH, Ledbetter JA, Gillespie MM, Lindsten T, Thompson CB. T-cell
proliferation involving the CD28 pathway is associated with cyclosporine-resistant
interleukin 2 gene expression. Mol Cell Biol. 1987;7(12):4472-81.
62. Olivieri KC, Mukerji J, Gabuzda D. Nef-mediated enhancement of cellular
activation and human immunodeficiency virus type 1 replication in primary T cells
is dependent on association with p21-activated kinase 2. Retrovirology. 2011;8:64.
63. Yu H, Khalid M, Heigele A, Schmokel J, Usmani SM, van der Merwe J, et al.
Lentiviral Nef proteins manipulate T cells in a subset-specific manner. J Virol.
2015;89(4):1986-2001.
64. Lin CJ, Tam RC. Transcriptional regulation of CD28 expression by CD28GR, a
novel promoter element located in exon 1 of the CD28 gene. J Immunol.
2001;166(10):6134-43.
65. Seve M, Favier A, Osman M, Hernandez D, Vaitaitis G, Flores NC, et al. The
human immunodeficiency virus-1 Tat protein increases cell proliferation, alters
sensitivity to zinc chelator-induced apoptosis, and changes Sp1 DNA binding in
HeLa cells. Arch Biochem Biophys. 1999;361(2):165-72.
66. Geleziunas R, Bour S, Wainberg MA. Cell surface down-modulation of CD4 after
infection by HIV-1. FASEB J. 1994;8(9):593-600.
67. Rhee SS, Marsh JW. Human immunodeficiency virus type 1 Nef-induced down-
modulation of CD4 is due to rapid internalization and degradation of surface CD4.
J Virol. 1994;68(8):5156-63.
146
68. Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeficiency virus
type 1 Vpu protein induces rapid degradation of CD4. J Virol. 1992;66(12):7193-
200.
69. Magadan JG, Perez-Victoria FJ, Sougrat R, Ye Y, Strebel K, Bonifacino JS.
Multilayered mechanism of CD4 downregulation by HIV-1 Vpu involving distinct
ER retention and ERAD targeting steps. PLoS Pathog. 2010;6(4):e1000869.
70. Heigele A, Schindler M, Gnanadurai CW, Leonard JA, Collins KL, Kirchhoff F.
Down-modulation of CD8alphabeta is a fundamental activity of primate lentiviral
Nef proteins. J Virol. 2012;86(1):36-48.
71. El-Far M, Isabelle C, Chomont N, Bourbonniere M, Fonseca S, Ancuta P, et al.
Down-regulation of CTLA-4 by HIV-1 Nef protein. PLoS One. 2013;8(1):e54295.
72. Alegre ML, Frauwirth KA, Thompson CB. T-cell regulation by CD28 and CTLA-
4. Nat Rev Immunol. 2001;1(3):220-8.
73. Vigan R, Neil SJ. Determinants of tetherin antagonism in the transmembrane
domain of the human immunodeficiency virus type 1 Vpu protein. J Virol.
2010;84(24):12958-70.
74. Magadan JG, Bonifacino JS. Transmembrane domain determinants of CD4
Downregulation by HIV-1 Vpu. J Virol. 2012;86(2):757-72.
75. Madjo U, Leymarie O, Fremont S, Kuster A, Nehlich M, Gallois-Montbrun S, et
al. LC3C Contributes to Vpu-Mediated Antagonism of BST2/Tetherin Restriction
on HIV-1 Release through a Non-canonical Autophagy Pathway. Cell Rep.
2016;17(9):2221-33.
76. Park J, Kim Y, Lee S, Park JJ, Park ZY, Sun W, et al. SNX18 shares a redundant
role with SNX9 and modulates endocytic trafficking at the plasma membrane. J of
Cell Sci. 2010;123(10):1742-50.
77. Mygind KJ, Storiko T, Freiberg ML, Samsoe-Petersen J, Schwarz J, Andersen OM,
et al. Sorting nexin 9 (SNX9) regulates levels of the transmembrane disintegrin and
metalloproteinase (ADAM)-9 at the cell surface. J Biol Chem. 2018.
78. Johnson AL, Dirk BS, Coutu M, Haeryfar SM, Arts EJ, Finzi A, et al. A Highly
Conserved Residue in HIV-1 Nef Alpha Helix 2 Modulates Protein Expression.
mSphere. 2016;1(6).
79. Quinones-Mateu ME, Ball SC, Marozsan AJ, Torre VS, Albright JL, Vanham G,
et al. A dual infection/competition assay shows a correlation between ex vivo
human immunodeficiency virus type 1 fitness and disease progression. J Virol.
2000;74(19):9222-33.
80. Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, et al. Nef-
mediated suppression of T cell activation was lost in a lentiviral lineage that gave
rise to HIV-1. Cell. 2006;125(6):1055-67.
81. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable
generation of high-quality protein multiple sequence alignments using Clustal
Omega. Mol Syst Biol. 2011;7:539.
147
82. Pan X, Rudolph JM, Abraham L, Habermann A, Haller C, Krijnse-Locker J, et al.
HIV-1 Nef compensates for disorganization of the immunological synapse by
inducing trans-Golgi network-associated Lck signaling. Blood. 2012;119(3):786-
97.
83. Jacob RA, Johnson AL, Pawlak EN, Dirk BS, Van Nynatten LR, Haeryfar SMM,
et al. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-
mediated CD4+ T cell apoptosis. Virology. 2017;509:1-10.
84. Bour S, Perrin C, Akari H, Strebel K. The human immunodeficiency virus type 1
Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated
degradation of Ikappa B. J Biol Chem. 2001;276(19):15920-8.
85. Harding FA, McArthur JG, Gross JA, Raulet DH, Allison JP. CD28-mediated
signalling co-stimulates murine T cells and prevents induction of anergy in T-cell
clones. Nature. 1992;356(6370):607-9.
86. Esensten JH, Helou YA, Chopra G, Weiss A, Bluestone JA. CD28 Costimulation:
From Mechanism to Therapy. Immunity. 2016;44(5):973-88.
87. Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, et al. Rescue
of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science.
2017.
88. Gaffen SL, Liu KD. Overview of interleukin-2 function, production and clinical
applications. Cytokine. 2004;28(3):109-23.
89. Clerici M, Hakim FT, Venzon DJ, Blatt S, Hendrix CW, Wynn TA, et al. Changes
in interleukin-2 and interleukin-4 production in asymptomatic, human
immunodeficiency virus-seropositive individuals. J Clin Invest. 1993;91(3):759-
65.
90. Orsilles MA, Pieri E, Cooke P, Caula C. IL-2 and IL-10 serum levels in HIV-1-
infected patients with or without active antiretroviral therapy. APMIS.
2006;114(1):55-60.
91. Group I-ES, Committee SS, Abrams D, Levy Y, Losso MH, Babiker A, et al.
Interleukin-2 therapy in patients with HIV infection. N Engl J Med.
2009;361(16):1548-59.
92. Chun TW, Engel D, Mizell SB, Ehler LA, Fauci AS. Induction of HIV-1 replication
in latently infected CD4+ T cells using a combination of cytokines. J Exp Med.
1998;188(1):83-91.
93. Wang FX, Xu Y, Sullivan J, Souder E, Argyris EG, Acheampong EA, et al. IL-7 is
a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of
infected individuals on virally suppressive HAART. J Clin Invest.
2005;115(1):128-37.
94. Margolis DM, Garcia JV, Hazuda DJ, Haynes BF. Latency reversal and viral
clearance to cure HIV-1. Science. 2016;353(6297):aaf6517.
148
95. Asjo B, Cefai D, Debre P, Dudoit Y, Autran B. A novel mode of human
immunodeficiency virus type 1 (HIV-1) activation: ligation of CD28 alone induces
HIV-1 replication in naturally infected lymphocytes. J Virol. 1993;67(7):4395-8.
96. Couturier JP, Root-Bernstein RS. HIV may produce inhibitory microRNAs
(miRNAs) that block production of CD28, CD4 and some interleukins. J Theor
Biol. 2005;235(2):169-84.
97. Weinberg MS, Morris KV. Are viral-encoded microRNAs mediating latent HIV-1
infection? DNA Cell Biol. 2006;25(4):223-31.
98. Mondino A, Mueller DL. mTOR at the crossroads of T cell proliferation and
tolerance. Semin Immunol. 2007;19(3):162-72.
99. Besnard E, Hakre S, Kampmann M, Lim HW, Hosmane NN, Martin A, et al. The
mTOR Complex Controls HIV Latency. Cell Host Microbe. 2016;20(6):785-97.
100. Husain M, Gusella GL, Klotman ME, Gelman IH, Ross MD, Schwartz EJ, et al.
HIV-1 Nef induces proliferation and anchorage-independent growth in podocytes.
J Am Soc Nephrol. 2002;13(7):1806-15.
101. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, et al.
Production of acquired immunodeficiency syndrome-associated retrovirus in
human and nonhuman cells transfected with an infectious molecular clone. J Virol.
1986;59(2):284-91.
102. Dudley DM, Gao Y, Nelson KN, Henry KR, Nankya I, Gibson RM, et al. A novel
yeast-based recombination method to clone and propagate diverse HIV-1 isolates.
Biotechniques. 2009;46(6):458-67.
103. Bolte S, Cordelieres FP. A guided tour into subcellular colocalization analysis in
light microscopy. J Microsc. 2006;224(Pt 3):213-32.
149
Chapter 3
3 Defining the role of the novel interaction between HIV-1
Nef and the membrane trafficking protein SNX18
3.1 Introduction
In order for viruses to persist within their hosts, they must evade host anti-viral immune
responses. This is particularly critical for viruses that cause chronic infections, such as
HIV-1, which fails to be eliminated by the immune response upon establishment of
infection. Key to the ability of HIV-1 to evade the immune response are the actions of the
accessory protein Nef. Nef mediates multiple functions to facilitate the evasion of both
innate and adaptive immune responses (reviewed in [1]). A central mechanism by which
Nef enables evasion of immune responses is via the reduction of cell surface levels of
MHC-I molecules [2]. Briefly, cell surface MHC-I molecules present antigens to the
adaptive immune cells CD8+ cytotoxic T cells, which secrete cytotoxic enzymes to induce
the death of cells presenting viral antigens on MHC-I molecules [3]. Therefore, as a result
of the Nef-dependent reduction of cell surface MHC-I molecules, cells infected with HIV-
1 encoding a functional nef gene are less effectively killed by CD8+ cytotoxic T cells [4].
Moreover, increased downregulation of MHC-I by Nef correlates with faster disease
progression and higher viral loads in rhesus macaques infected with the related simian
immunodeficiency virus [5-8].
Nef mediates its functions, including decreasing surface MHC-I molecules, by interacting
with host cell proteins and subverting their functions. Specifically, many Nef-dependent
functions require Nef to alter the subcellular localization of various host cell proteins by
hijacking host proteins implicated in cellular membrane trafficking. A well characterized
example of this is the hijacking of the membrane trafficking adaptor proteins AP-1 and
AP-2. Indeed, Nef binds to both AP-1 and AP-2 to connect host cell cargo to the trafficking
machinery, whereby these Nef: adaptor protein: cargo complexes mediate the transport of
cellular cargo to specific subcellular locations [9-12]. However, in order for cargo to be
trafficked to different locations within the cell a multitude of host cellular machinery are
necessary, as vesicular trafficking is complex and generally requires many factors [13],
150
leading to the possibility that Nef recruits one or more additional factors to facilitate cargo
trafficking.
An additional protein that may facilitate Nef-dependent cargo trafficking is SNX18, which
was shown to form a complex with Nef in infected HeLa cells [14]. SNX18 is a member
of a subgroup of sorting nexin proteins that contain an SH3 domain, which also includes
the proteins SNX9 and SNX33 (SNX30) [15]. Together, these sorting nexin proteins
facilitate membrane trafficking by mediating the formation of tubulated membranes [15,
16], which may ultimately form vesicles. In addition, SNX9, SNX18 and SNX33 have been
reported to interact with and recruit additional membrane trafficking proteins, thereby
making these key regulators of membrane trafficking events. Notably, the SH3 domains of
SNX18, SNX9 and SNX33 interact with the GTPase dynamin-II [15, 17, 18], which
mediates the scission of membranes to form vesicles during membrane trafficking [19].
Interestingly, SNX9 and SNX18 also interact with the Nef-binding vesicular adaptor
proteins. Specifically, SNX9 immunoprecipitated with AP-2 and SNX18 with AP-1 from
K562 and HeLa cells, respectively [15, 20]. Moreover, endogenous SNX18 co-localizes
with the Nef and AP-1 binding protein PACS-1 in HeLa cells [15]. Given that the classical
trafficking organelles, clathrin coated vesicles, contain upwards of 200 discrete proteins
which can contribute to their trafficking within cells [21], the relatively small number of
host cell proteins currently characterized as essential for Nef-mediated trafficking of host
cell cargo are undoubtedly not sufficient to facilitate membrane trafficking. Therefore, we
hypothesize that additional host cell factors are required during Nef-mediated trafficking
events and have focused on SNX18 and SNX9 as potential candidates.
Herein, we investigate the SNX18: Nef interaction further by mapping the protein domains
implicated in this complex using in vitro protein binding assays. We demonstrate that Nef
interacts directly with SNX18, but not the related protein SNX9, and map this interaction
to the SH3 domain of SNX18 and the PxxP75 motif of Nef. Moreover, we investigate the
role of the Nef: SNX18 interaction in cells, specifically by examining the requirement for
this interaction for Nef-mediated MHC-I downregulation. Our findings suggest that
SNX18 may be important for Nef to mediate decreases in cell surface MHC-I in T cells.
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3.2 Results
3.2.1 SNX18 and Nef interact in vitro through a SNX18 SH3 domain: Nef PxxP motif
interaction
We first sought to map the interaction between Nef and members of the sorting nexin
family of proteins, in addition to determining if this interaction is direct. Interestingly, the
HIV-1 Nef protein contains a motif (PxxP75) implicated in direct interactions with SH3-
domain containing proteins [22-25], and these same SH3 domains are present in the sorting
nexin proteins SNX18 and SNX9. We first tested if Nef and SNX18 or SNX9 interact
directly. To monitor this, in vitro capture assays were performed using bacterially produced
and subsequently purified proteins. Specifically, purified His6-Nef (SF2) was incubated
with glutathione conjugated beads and increasing concentrations of GST-tagged SNX18
and SNX9 or GST alone. In the presence of GST-SNX18, His6-Nef was detected via
Western blot in the pulled-down proteins, unlike in the presence of SNX9 (Figure 3.1A).
Relative to our control, GST, significantly greater SNX18, but not SNX9, pulled down
Nef, and there was greater His6-Nef detected when an increased amount of SNX18 bait
protein was utilized (Figure 3.1B). Overall, this suggests that SNX18 interacts with Nef in
vitro, thus demonstrating a direct interaction, while the related protein SNX9 does not
interact with Nef.
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Figure 3.1 GST-SNX18, but not GST-SNX9, pulls down His6-Nef
Various GST-tagged proteins and His6-Nef protein were bacterially produced and
subsequently purified. His6-Nef was then pulled down by sepharose beads bound with
the indicated amounts of GST, GST-SNX18 or GST-SNX9 and detected by Western
blot. (A) Anti-His6 Western blot of 5% of input (top); Anti-His6 Western blot of protein
remaining associated with glutathione sepharose after washing with 500 mM sodium
chloride (middle); Ponceau S stain of membrane from pulled down proteins (bottom).
(B) Mean (+/-SD) band intensity of relative His6-Nef pulled down by GST, GST-SNX18
or GST-SNX9. Mean His6-Nef binding was compared using a one-way Anova and the
Bonferroni’s multiple comparisons test was utilized to compare binding to GST. Assays
were performed in quadruplicate. (SD: standard deviation; * p≤ 0.05; **** p≤ 0.0001;
WB: Western blot)
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Next, we mapped the SNX18: Nef interaction interface. To determine if the SH3 domain
of SNX18 mediates the Nef: SNX18 interaction, as it does with the SFK interaction with
Nef [26], a GST-tagged variant of the SH3 domain of SNX18 was bacterially produced
and subsequently purified, along with a GST-tagged version of SNX18 that is devoid of its
SH3 domain (SH3; Figure 3.2A). Upon performing in vitro pull-down assays, we
observed that His6-Nef was indeed pulled down by the SH3 domain of SNX18, but not by
the SH3 protein (Figure 3.2B, C). This result suggests that the SH3 domain is necessary
and sufficient for interacting with Nef in vitro. Moreover, to further test the specificity of
the SNX18 SH3 domain in mediating a direct interaction with Nef, we designed a GST-
tagged chimeric protein in which the SH3 domain of the SNX18 protein was replaced with
the SH3 domain of SNX9 (Figure 3.2A), which fails to bind Nef in vitro (Figure 3.1), and
tested for its ability to pull down His6-Nef. Interestingly, the SNX9 SH3/SNX18 chimera
failed to pull down His6-Nef in an in vitro binding assay, in a similar manner to the GST
protein negative control (Figure 3.2D, E), confirming the specificity and importance of the
SNX18 SH3 domain for binding to Nef in vitro.
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Figure 3.2 The direct Nef: SNX18 interaction is dependent on the SH3 domain
GST-tagged bait proteins and His6-Nef were bacterially produced and subsequently
purified. His6-Nef was then pulled-down by glutathione-sepharose beads coated with the
indicated GST-tagged proteins and detected by Western blot. (A) Schematics indicating
the GST-tagged constructs utilized as bait proteins. (B) His6-Nef was captured by
glutathione sepharose bound GST, GST-tagged SH3 domain of SNX18 or GST-tagged
ΔSH3 (SNX18 lacking the SH3 domain) and detected by Western blotting. Anti-His6
Western blot of 5% of input (top); Anti-His6 Western blot of protein remaining
associated with glutathione sepharose after washing with 500 mM sodium chloride
(middle); Ponceau S stain of membrane from pulled down proteins (bottom). (C) Mean
band intensity of relative His6-Nef pulled down by GST, GST-SH3 or GST-ΔSH3.
Assays were performed multiple times (n=7) and the results are presented as the mean
(+/-SD). Mean His6-Nef binding was compared using a one-way Anova and the
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Bonferroni’s multiple comparisons test was utilized to compare binding in all conditions
versus GST. (E) Anti-His6 Western blot of 5% of input (top); Anti-His6 Western blot of
protein remaining associated with glutathione sepharose after washing with 500 mM
sodium chloride (bottom). (F) Mean band intensity of relative His6-Nef pulled down by
GST, GST-SNX18, GST-SNX9 or a GST-SH3 domain chimera comprised of the SH3
domain of SNX9 fused to the entire SNX18 protein except for the SH3 domain. Assays
were performed in triplicate and results are presented as the mean (+/-SD). Mean His6-
Nef binding was compared using a one-way Anova and the Bonferroni’s multiple
comparisons test was utilized to compare binding in all conditions versus GST. (SD:
standard deviation; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001; **** p≤ 0.0001; WB: Western
blot)
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It has been previously demonstrated that Nef interactions that are dependent on SH3
domains are mediated by the Nef PxxP75 motif [23, 26], which has defined roles in AIDS
pathogenesis and immune evasion [26-30]. To test for the requirement of the Nef PxxP75
motif for interacting with the SNX18 SH3 domain and thereby establishing a Nef motif
implicated in the Nef: SNX18 interaction, we repeated our in vitro protein pull-down assay
using purified His6-Nef or His6-Nef AxxA75, a Nef mutant that fails to bind SFKs ([26];
Figure 3.3). We observed that significantly less His6-Nef AxxA75 was pulled down by the
GST-tagged SNX18 SH3 domain when compared to wild-type His6-Nef (Figure 3.3A, B).
Overall, this data demonstrates that SNX18 and Nef interact directly in vitro, unlike the
related SNX9 protein which fails to bind Nef. Moreover, mapping of this interaction
revealed its dependence on the SH3 domain of SNX18 and the Nef PxxP75 motif.
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Figure 3.3 In vitro protein capture of His6-Nef and a Nef mutant, His6-Nef AxxA75,
by the SH3 domain of SNX18
Bacterially produced and subsequently purified GST-tagged SH3 domain of SNX18 was
bound to sepharose beads and tested for its ability to pull down wild-type or AxxA75
mutant His6-tagged Nef. (A) His6-Nef or His6-Nef AxxA75 was captured by glutathione-
sepharose bound GST or GST tagged SH3 domain of SNX18 and detected by Western
blotting. Top: Anti-His6 Western blot of 5% of input; bottom: Anti-His6 Western blot of
protein remaining associated with glutathione sepharose after washing with 500 mM
sodium chloride. (B) Mean (+/-SD) band intensity of hexa-histidine tagged protein
pulled down by GST-tagged SNX18 SH3 domain, relative to protein pulled down by
GST. Assays were performed in triplicate and the relative mean band intensities were
compared using a Student’s t-test. (SD: standard deviation; **= p≤ 0.01; WB: Western
blot)
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3.2.2 SNX18 and Nef interact in cells
We next sought to define the molecular determinants implicated in the Nef: SNX18
interaction in cells. Our group previously described the detection of a close association
between SNX18 and Nef within cells using the technique of Bimolecular Fluorescence
Complementation (BiFC) [14], which reconstitutes a split fluorophore to demonstrate a
protein-protein interaction. Therefore, with the molecular details of the Nef: SNX18
interaction interface acquired in vitro (Figure 3.3), we wished to validate the previously
observed Nef: SNX18 BiFC signal. To test this, we expressed vectors encoding wild-type
or the PxxP75 mutant Nef (NL4.3) protein fused to a C-terminal fragment of the Venus
fluorophore (Nef-VC or Nef AxxA75-VC) and SNX18 fused to a N-terminal fragment of
Venus (SNX18-VN) in HeLa cells and examined the reconstitution of the fluorophore using
wide field microscopy (Figure 3.4). We observed that the mean fluorescence intensity of
the Nef: SNX18 BiFC signal was significantly reduced with the mutant Nef (Nef AxxA75-
VC), compared to wild type Nef (Figure 3.4B). Moreover, expression of the Nef-VC and
Nef AxxA75-VC proteins was confirmed by Western blotting (Figure 3.4C). These findings
suggest that the Nef: SNX18 interaction occurs within cells and is mediated at least in part
by the Nef PxxP75 motif.
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Figure 3.4 Mutation of the Nef PxxP75 motif reduces SNX18: Nef BiFC intensity
HeLa cells were co-transfected with vectors encoding SNX18-VN-FLAG and either Nef-
VC or Nef AxxA75-VC. Twenty-four hours post transfection, cells were incubated in the
dark for 1 hour to mature the fluorophore, followed by fixation in 4% PFA and
subsequent visualization by wide field microscopy. (A) Representative images of
160
transfected cells illustrating Nef: SNX18 BiFC signal (left), anti-FLAG staining (middle)
and merge (right). (B) Mean (+/-SD) fluorescence intensity of non-transfected (NT),
SNX18-VN-FLAG and Nef-VC transfected, or SNX18-VN-FLAG and Nef AxxA75-VC
transfected cells (n=30). The mean fluorescence intensities were compared using a one-
way ANOVA followed by Tukey’s multiple comparisons test. (C) Western blots
demonstrating expression of Nef-VC and Nef AxxA75-VC. (*** p≤ 0.001; NT: non-
transfected; Mr: molecular weight)
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3.2.3 SNX18 is required for Nef-dependent reductions in cell surface MHC-I
We next sought to identify if the Nef: SNX18 interaction is required for Nef-mediated
decreases in cell surface MHC-I, a key mechanism utilized by HIV-1 to evade immune
surveillance. Indeed, it has been previously demonstrated that the Nef PxxP75 motif which
mediates SNX18 binding also binds SFKs to trigger an intracellular signaling cascade
which downregulates cell surface MHC-I [30, 31]. To test this, CD4+ Sup-T1 T cells were
transduced with lentiviral vectors encoding both GFP and shRNA sequences targeting
SNX18 or an irrelevant non-specific shRNA. In addition, these same Sup-T1 cells were
transduced with a modified HIV-1 NL4.3 vector encoding the fluorescent protein
mStrawberry in addition to encoding (Nef) or lacking Nef (dNef). Cell surface MHC-I
levels on dual infected (GFP+ mStrawberry+) cells were quantified by flow cytometry
(Figure 3.5A). We observed a small, but significant, increase in the amount of cell surface
MHC-I in the presence of Nef in cells infected with virus decreasing SNX18 expression,
compared to cells expressing the non-specific shRNA (Figure 3.5B, C), suggesting that
Nef-mediated decreases in cell surface MHC-I are dependent on SNX18 expression.
Moreover, efficient knock-down of SNX18 in Sup-T1 cells was confirmed using Western
blot analyses of infected cells (Figure 3.5D). Indeed, we observed a significant decrease in
cellular SNX18 levels in cells transduced with SNX18 targeting shRNAs, compared to
non-specific shRNA (Figure 3.5E). Overall, these findings suggest that the presence of
SNX18 is important for the ability of Nef to decrease cell surface MHC-I levels.
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Figure 3.5 Knock-down of SNX18 reduces Nef-mediated MHC-I downregulation
CD4+ Sup-T1 T cells were infected with viruses encoding both GFP and non-specific or
SNX18 targeting shRNAs in addition to a virus encoding mStrawberry and encoding or
lacking Nef. Infected cells were stained for cell surface MHC-I (HLA-A, B, C) and
analyzed by flow cytometry. (A) Illustration of gating strategy. Dual infected (GFP+
mStrawberry+) cells were gated on and cell surface MHC-I was quantified on dual
infected cells. (B) Representative histograms illustrating cell surface MHC-I levels on
dual infected cells infected with non-specific (NS) shRNA or SNX18 targeting (anti-
SNX18) shRNA and with virus encoding (Nef; blue) or lacking (dNef; orange) Nef. (red:
isotype stained cells) (C) Relative mean (+/-SD) cell surface MHC-I in cells infected
163
with virus encoding Nef and a non-specific (NS) or SNX18 (anti-SNX18) targeting
shRNA. (n=16) Relative means were compared using a paired Student’s t-test. (D)
Western blot illustrating endogenous levels of SNX18 in uninfected Sup-T1 cells (UI)
or cells infected with viruses encoding a non-specific (NS) or SNX18 (anti-SNX18)
targeting shRNA. (E) Quantification of relative mean (+/-SD) SNX18 levels in
uninfected Sup-T1 cells (UI) or cells infected with viruses encoding a non-specific (NS)
or SNX18 (anti-SNX18) targeting shRNA detected by Western blots (n=8). Mean
SNX18 levels were compared using a one-way Anova and Dunnett’s multiple
comparisons test. (GFP: green fluorescent protein; MHC-I: major histocompatibility
complex type I; Mr: molecular weight; * p≤ 0.05; ** p≤ 0.01; SD: standard deviation;
UI: uninfected; SNX18: sorting nexin 18)
164
3.2.4 SNX18 is unlikely to be required for Nef-mediated secretion in extracellular
vesicles
In order to decrease cell surface levels of MHC-I, Nef facilitates the endocytosis and
intracellular sequestration of cell surface MHC-I [32]. However, it has also been reported
that Nef can facilitate the secretion of extracellular vesicles containing MHC-I via
exocytosis [33, 34]. To perform this function, Nef activates host cell signaling proteins to
form the Nef-associated kinase complex [35], which facilitates the complex’s interaction
with the scaffold protein Paxillin [34]. Interestingly, Paxillin has been previously shown to
interact with the SNX18-binding metalloproteinases ADAM-10 and ADAM-17 to promote
the inclusion of Nef, ADAM-17 and ADAM-10 into extracellular vesicles [34, 36].
Therefore, we hypothesized that SNX18 may contribute to Nef-mediated decreases in cell
surface MHC-I by facilitating the secretion of extracellular vesicles. Thus, we examined if
the SNX18: Nef BiFC complex localizes to the late endosome or mutivesicular body
(MVB) compartment, which contains vesicles that can serve as precursors for extracellular
vesicles [37]. Specifically, to label the MVB compartment we utilized an antibody that
recognizes the tetraspanin protein CD63. We observed that the Nef: SNX18 BiFC signal
co-localized with CD63 in HeLa cells (Figure 3.6). Interestingly, the SNX18: Nef BiFC
signal co-localized with CD63 to a greater extent than GPF-tagged SNX18 or the related
protein SNX9, which does not bind Nef (Figure 3.1), in the absence of Nef (Figure 3.6).
Therefore, given that neither SNX9 or SNX18 localize to CD63 positive compartments in
the absence of Nef, this suggests that at least in the case of SNX18, this membrane
trafficking protein is being diverted to MVBs in the presence of Nef.
165
Figure 3.6 The SNX18: Nef BiFC complex co-localizes with CD63
HeLa cells were transfected with vectors encoding eGFP, SNX9-eGFP, SNX18-eGFP or
SNX18-VN and Nef-VC. Twenty-four hours post transfection, cells were incubated in the
dark to mature the fluorophore, stained for endogenous CD63 and visualized using wide
field microscopy. (A) Representative images of transfected cells. Puncta where SNX18:
Nef BiFC and CD63 overlap are denoted by arrows. Scale bar = 10 μm. (B) Mean (+/-SD)
Pearson correlation coefficient between signal in the FITC (eGFP/BiFC) and Cy3 (CD63)
channels (n≥7). Means were compared using one way ANOVA followed by Tukey’s
eGFP
SNX18
-eG
FP
SNX9-
eGFP
Nef
:SNX18
BiF
C
0.0
0.2
0.4
0.6
0.8
1.0
Pe
ars
on
co
rrela
tio
n
Co
eff
icie
nt
n.s
***
A.
B.
166
multiple comparisons test. (*** p≤ 0.001; eGFP: enhanced green fluorescent protein; SD:
standard deviation)
167
Next, we wanted to determine if SNX18 is required for Nef-mediated secretion of
extracellular vesicles. To investigate this, stable HEK293T cell lines expressing shRNA
targeting SNX18 (293TαSNX18) or a non-specific target control (293TNS) were produced.
These cells were then transfected with the pNL4.3 dG/P eGFP lentiviral vector. The
extracellular vesicles from this transfected cell culture supernatant were purified and
probed for their content (Figure 3.7). Specifically, the cell lysate and purified extracellular
vesicles were probed for the viral proteins Nef and Vpu, which were previously shown to
be secreted into extracellular vesicles, and specific markers for intracellular proteins
(Calnexin, Actin) and extracellular vesicles (TSG-101). In addition, we probed for the
metalloprotease ADAM-17, which has been previous implicated in the sorting of Nef into
extracellular vesicles [34]. Through five independent experiments, a significant difference
in the amount of Nef secreted from 293TαSNX18 or 293TNS cells was not observed (Figure
3.7B). However, upon Western blotting for Nef in the extracellular vesicle preparations,
an additional protein band was consistently observed. Moreover, Nef exhibited an altered
migration pattern, as was seen in the extracellular fraction for the HIV-1 protein Vpu and
for ADAM-17.
168
Figure 3.7 Extracellular vesicles purified from Nef-expressing SNX18 knock-down
cells
HEK293T cells stably expressing non-targeting (NS) or SNX18 targeting (anti-SNX18)
shRNAs were transfected with a vector encoding all HIV-1 viral accessory proteins
(NL4.3 dG/P eGFP). Forty-eight hours post transfection, extracellular vesicles in cell
169
culture supernatants were purified using a commercial purification reagent (exoquick)
and the associated proteins analyzed by Western blotting. (A) Representative Western
blots illustrating cellular and extracellular vesicle proteins. (B) Mean relative
extracellular Nef (+/-SD) from five independent experiments. Mean relative extracellular
Nef was not significantly different, as determined using a Student’s t-test. (Mr: molecular
weight, SD: standard deviation)
170
3.3 Discussion
In the present work, we identify and investigate the underlying mechanism of a novel Nef:
host protein interaction, the interaction between Nef and SNX18. Using in vitro protein
capture assays, we have shown that Nef binds directly to SNX18 (Figure 3.1). This
interaction was dependent on the SH3 domain of SNX18 and was specific to SNX18, as
the SH3 domain from a related family member, SNX9, could not mediate binding to Nef
(Figure 3.2). Moreover, the Nef: SNX18 interaction was reduced upon mutation of the Nef
PxxP75 motif both in vitro and in mammalian cells (Figure 3.3; Figure 3.4). Notably, while
mutation of the Nef PxxP75 motif inhibited complex formation with full length SNX18
within cells (Figure 3.4), the examination of the effects of this Nef mutation on binding in
vitro was limited to the interaction with the SH3 domain of SNX18 (Figure 3.3). Therefore,
future experiments to examine Nef AxxA75 binding to full length SNX18 protein in vitro
may be of value. The finding that this interaction required the SH3 domain of SNX18 and
the PxxP75 motif of Nef is consistent with the previously defined role of SH3 domains in
binding to the Nef polyproline motif [26], an interaction critical for the immunoevasive
effects of Nef [30]. Indeed, the Nef PxxP75 motif is critical for interacting with the SH3
domains of multiple SFKs to mediate the downregulation of cell surface MHC-I [30, 31].
Thus, the use of the Nef PxxP75 motif to hijack two different host proteins, SFKs and
SNX18, by recognizing a common SH3 domain, exemplifies the amazing ability of viral
proteins to efficiently mediate their functions by expanding their repertoire of binding
partners. Moreover, given that proteins containing SH3 domains classically interact with
other proteins via polyproline rich interaction interfaces [38], this is a prime example of
viruses evolving the ability to mimic host cellular interactions while maintaining a degree
of binding specificity, as SH3 domain binding was specific to SNX18 (Figure 3.2). Given
that the affinity of previously identified Nef polyproline helix: SH3 domain interactions
have been shown to be goverened by a specific region of the SH3 domain, known as the
RT-loop [24, 39], future studies to investigate the residues within the SNX18 RT-loop that
faciliate Nef binding may be of interest. The specificity of Nef: host protein interactions
that required the Nef polyproline motif may also be mediated by differences in subcellular
localization. Specifically, upon the localization of Nef to the TGN, wherein SFKs are
enriched, Nef can interact with and activate a signaling cascade that initiates MHC-I
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downregulation [30]. Concomitantly, Nef may interact with SNX18 at peripheral endocytic
compartments. This multi-functionality of a viral protein interaction motif is not unlike that
seen with host cellular proteins, including SNX18, whereby, in uninfected cells the SH3
domain of SNX18 is implicated in binding to the polyproline rich domains of the proteins
N-WASP and dynamin-II [15, 40].
In order to investigate the functional consequence of Nef: SNX18 interactions in cells, we
utilized shRNA to silence SNX18 expression in T cells. We observed a reduction in Nef’s
ability to downregulate cell surface MHC-I in the absence of SNX18 (Figure 3.5). Notably,
the effect of our SNX18 knock-down on MHC-I downregulation was small. These studies
may be limited by the functional redundancy between SNX18 and the related protein SNX9
[40, 41], whereby SNX9 may compensate for the knock-down of SNX18 despite the lack
of a direct Nef: SNX9 interaction. Specifically, a recent study illustrated that knock-down
of both SNX18 and SNX9 was required to inhibit the proper trafficking of the
metalloproteinase ADAM9, an effect not observed upon knock-down of the proteins
independently [41]. Thus, future studies examining MHC-I downregulation upon reduction
of both SNX18 and SNX9 may be of value. Additionally, the knock-down of SNX18
observed via Western blot was variable and limited (Figure 3.5E), with some experiments
exhibiting reductions in uninfected (UI) cell SNX18 protein levels relative to cells
transduced with lentiviral vectors encoding non-specific (NS) shRNA. These observed
decreases in SNX18 protein levels within uninfected cells may be a result of global changes
in cells resulting from lentiviral vector infection. These experiments could be improved
through creation of SNX18 knock-out cell lines utilizing CRISPR/Cas9 technology [42].
The observed inhibition of Nef-mediated MHC-I downregulation upon knock-down of
SNX18 is not likely due to SNX18 facilitating Nef-mediated extracellular vesicle release,
as SNX18 was dispensable for Nef-induced extracellular vesicle release in HEK293T cells
(Figure 3.7B). However, we did note molecular weight shifts in multiple proteins when
comparing migration patterns in the cellular fraction vis-a-vis the extracellular fraction
(Figure 3.7A), as has been seen previously observed with Nef in extracellular vesicle
preparations [43, 44]. These differences in Nef, Vpu and ADAM-17 migration patterns
may reflect post-translation modifications which act as signals to mediate the incorporation
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of these proteins into intraluminal vesicles destined to be secreted from the cell [45].
However, we have not examined the physical properties or biogenesis pathway of our
extracellular vesicle preparations, thus we cannot conclude that we are examining
extracellular vesicles derived from the secretion of intraluminal vesicles.
As an alternative to mediating decreases in cell surface MHC-I through secretion of
extracellular vesicles, the Nef: SNX18 interaction may be facilitating the endocytosis of
MHC-I which is transiting from the cell surface to the TGN for intracellular sequestration,
a virus induced activity which limits antigen presentation [32, 46, 47]. It has been
previously established that to downregulate MHC-I, Nef and AP-1 form a ternary complex
with the cytoplasmic tail of MHC-I [11, 12]. Additionally, it is proposed that PACS-1,
which can bind both Nef and AP-1 and co-localizes with SNX18 [15, 32, 48, 49], facilitates
this interaction and mediates proper trafficking of the Nef: AP-1: MHC-I ternary complex
[32, 47]. Since SNX18 binds AP-1 and Nef, via the SH3 domain and low complexity
domain, respectively ([15]; Figure 3.2), it is plausible that SNX18 may be recruited to and
interact with the Nef: AP-1: MHC-I ternary complex. Notably, due to the ability of SNX18
to dimerize [15, 40], it is conceivable that despite the potential for competition between
Nef and other polyproline rich proteins for SH3 domain binding, a single SNX18 dimer
could bind to both Nef and an additional interaction partner simultaneously (Figure 3.8).
There are numerous mechanisms by which Nef-bound SNX18 could facilitate the
endocytic trafficking of MHC-I. Firstly, SNX18 contains a BAR domain, which can
dimerize with other BAR domains into crescent shaped structures which forms a concave
“bent” surface capable of interacting with membranes, thereby contributing to overall
membrane bending [50]. Membrane bending is a hallmark state of lipid bilayers that
facilitates the scission of membranes, thereby ensuring the formation of small vesicles that
bud from one membrane enclosed structure to transport cargo from one location to another
[50]. Moreover, SNX18 can promote the scission of membranes by interacting with
dynamin-II [15], which uses the energy from GTP hydrolysis to enable membrane scission
[19]. Furthermore, SNX18 interacts with N-WASP [40], the recruitment of which may
facilitate changes in actin dynamics to promote vesicular trafficking [51]. Ultimately, we
propose that the interaction between Nef and SNX18 may be facilitating the vesicular
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Figure 3.8 Proposed model for the role of SNX18 in Nef-mediated MHC-I
trafficking
SNX18 can dimerize via its BAR domain and interact with curved membranes. The
SNX18 low-complexity domain interacts with AP-1, which also forms a ternary complex
with Nef and the cytoplasmic tail of MHC-I. Moreover, the Nef PxxP75 motif interacts
with the SH3 domain of SNX18. Nef and AP-1 also interact with PACS-1, which can
co-localize with SNX18, to facilitate MHC-I trafficking. While one SNX18 monomer in
a single heterodimer interacts with Nef, the other can interact with additional host
cellular trafficking proteins through its SH3 domain, including dynamin-II or N-WASP,
to further promote membrane trafficking.
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In conclusion, our results identify a novel Nef: host protein interaction, specifically
between Nef and SNX18, that may contribute to the ability of Nef to evade the immune
system via MHC-I downregulation. This interaction is mediated by a highly conserved
polyproline motif in Nef and therefore may be conserved across the diverse HIV-1 clades.
The finding that additional Nef: host protein interactions may mediate immune evasion,
highlights the complexity of the mechanism evolved by the HIV-1 virus to persist within
the host and contribute to pathogenesis.
3.4 Material and Methods
3.4.1 DNA vectors
Plasmids encoding GST-tagged SNX18 and SNX9 in pGEX-6P1 and pGEX-KG
backbones, respectively, were kindly provided by R. Prekeris (University of Colorado,
Aurora, CO). GST-SNX18 SH3 domain and GST-SNX18 ΔSH3 expression vectors were
produced by PCR amplification of the SNX18 SH3 domain (residues: 1-61) or low
complexity through BAR domains (residues: 62-624) with primers encoding EcoRI and
NotI restriction sites, followed by insertion into pGEX-4T1. A vector encoding the GST-
tagged SNX9 SH3 domain- SNX18 chimeric protein was produced by insertion of a
HindIII restriction site immediately downstream of the SH3 domain (residue 63) of SNX18
using site directed mutagenesis with mutagenesis primers created with the Agilent
Technologies QuikChange Primer Design program (Agilent Technologies, Santa Clara,
CA). The SNX18 SH3 domain was subsequently replaced with PCR amplified SNX9 SH3
domain (residues: 1-61) using the inserted HindIII restriction site. The bacterial expression
vector for production of His6-Nef encoded hexa-histidine tagged Nef from the HIV-1 SF2
strain and was provide by T. Smithgall (University of Pittsburgh, Pittsburgh, PA). A His6-
Nef AxxA75 mutant was produced by site directed mutagenesis, as above.
The SNX18 gene was sub-cloned into a peGFP-N1 backbone, in which the eGFP
fluorophore was replaced with an N-terminal portion of Venus (VN 1-173) FLAG tagged
on the N-terminus. The Nef-VC expression vector was previously described [46] and a Nef
AxxA75-VC expression vector was produced using site directed mutagenesis, as described
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above. The SNX18 and SNX9 genes were sub-cloned into peGFP-N1 to produce SNX18-
eGFP and SNX9-eGFP expression vectors.
Viral infection plasmids were engineered via insertion of the mStrawberry encoding region
of the previously described pNL4.3 F2A-mStrawberry vector into pNL4.3 dG/P eGFP or
pNL4.3 dG/P eGFP dNef backbones [14, 52, 53]. Lentiviral vectors encoding shRNAs
were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD) and are
described below.
3.4.2 Bacterial protein expression and purification
E. coli BL21(DE3) were transformed with expression vectors encoding GST or hexa-
histidine tagged proteins of interest and cultured in LB media containing the appropriate
antibiotic to an optical density at 600 nanometers (OD600) between 0.5-0.7. GST tagged
sorting nexin proteins or domains of interest were subsequently induced with 0.1 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) (Thermo Fisher Scientific, Mississauga,
ON) overnight at 30° C, while GST and His6-Nef were induced for four hours with 1.0 mM
IPTG at 37° C and 1.5 mM IPTG at 30° C, respectively. Induced E. coli BL21 (DE3)
transformed with GST-tagged protein expression vectors were pelleted at 3,840 x g, re-
suspended in lysis buffer [0.1 M sodium phosphate (pH 7.2), 1 M sodium chloride, 10 mM
benzamidine, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol
(DTT)], and lysed using a French press. The lysate was ultra-centrifuged at 256,630 x g to
separate the soluble and insoluble lysate components. The supernatant was incubated with
glutathione sepharose 4B (GE Healthcare, Baie-D’Urfé, QC), with rocking for one hour at
4° C. The resin was then pelleted, the supernatant removed, and the resin washed to remove
unbound or non-specifically bound proteins. Briefly, the resin was washed sequentially as
follows: two washes with 12 resin volumes of wash buffer 1 [0.1 M sodium phosphate
buffer (pH 7.2), 1 M sodium chloride, 0.5 mM EDTA, 1 mM DTT], three washes with 12
resin volumes of wash buffer 2 [0.1 M sodium phosphate buffer (pH 7.2), 150 mM sodium
chloride, 1 mM DTT], and two washes with one resin volume of elution buffer [50 mM
Tris buffer (pH 8.0), 15 mM reduced glutathione]. The elution buffer washes were pooled
and proteins were further purified using a size exclusion column and fast protein liquid
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chromatography such that eluted proteins were in 20 mM Tris buffer (pH 8.0), 150 mM
sodium chloride, 1 mM DTT, and 0.5 mM EDTA.
The induced E. coli BL21 (DE3) transformed with His6-Nef expression vectors were
pelleted at 3,840 x g, re-suspended in lysis buffer [50 mM sodium phosphate (pH 8.0), 200
mM sodium chloride, 10 mM imidazole], lysed using a French press, and the lysate ultra-
centrifuged at 256,630 x g. The supernatant was then incubated with nickel-nitriloacetic
acid (Ni2+-NTA) agarose (5 PRIME, Mississauga, ON) for one hour at 4°C with rocking.
The elution of proteins bound to the Ni2+-NTA agarose resin was performed using buffer
containing imidazole. Specifically, the resin was pelleted and washed sequentially as
follows: two washes with 12 resin volumes of wash buffer 1 [50 mM sodium phosphate
buffer (pH 8.0), 200 mM sodium chloride, 20 mM imidazole], three washes with 12 resin
volumes of wash buffer 2 [50 mM sodium phosphate buffer (pH 8.0), 200 mM sodium
chloride, 100 mM imidazole], and one wash with 6 resin volumes of wash buffer 3 [50 mM
sodium phosphate buffer (pH 8.0), 200 mM sodium chloride, 250 mM imidazole]. The
protein eluted in the 100 and 250 mM imidazole washes was concentrated using 10,000 Da
cut-off centricon centrifugal filters (EMD Millipore, Billerica, MA) and stored at -80ºC.
3.4.3 Protein capture assay
In vitro captures of His6-Nef were performed using the various GST-tagged bait proteins.
Variable amounts of bait protein (GST-tagged proteins), as quantified by Lowry assay,
were incubated with rocking for 45 minutes at room temperature with glutathione
sepharose in binding buffer [20 mM Tris (pH 8.0), 150 mM sodium chloride, 0.5 mM
EDTA, 0.1% NP-40 alternative]. Subsequently, 8 µg His6-Nef was then combined with
each bait protein, at which time a loading control sample was obtained. After incubating
for one hour at room temperature with rocking the resin was pelleted at 1,200 x g and
washed three times with 25 resin volumes of wash buffer [20 mM Tris (pH 8.0), 500 mM
sodium chloride, 0.1 mM EDTA, 0.1% NP-40 alternative], and re-suspended in SDS-
PAGE sample buffer for Western blot analysis.
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3.4.4 Cell culture
HEK293T (ATCC) cells and HeLa cells (ATCC) were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) containing 4 mM L-glutamine, 4500 mg/L glucose and sodium
pyruvate (HyClone, Logan, UT) and supplemented with 1% Penicillin-Streptomycin
(Hyclone) and 10% fetal bovine serum (FBS; Wisent, St-Bruno, QC). Sup-T1 cells were
maintained in Roswell Park Memorial Institute media (RPMI) 1640 media with L-
glutamine supplemented with 100 g/ml penicillin-streptomycin, 1% sodium pyruvate, 1%
non-essential amino acids, 2 mM L-glutamine (Hyclone) and 10% FBS. All cell lines were
grown at 37°C in the presence of 5% CO2 and sub-cultured in accordance with supplier’s
recommendations.
HEK293T cells stably expressing shRNAs against a non-specific target or SNX18 were
produced by transduction with shRNA-encoding lentiviral vectors. Briefly, HEK293T cells
were transduced with VSV-G pseudotyped lentivirus produced as described below with
pGFP-C-shLenti plasmid encoding a scrambled shRNA (TR30021, Origene) or a SNX18
targeting shRNA (TL307432D, Origene). Transduced cells were selected for using 0.8
ug/mL puromycin and clones were selected. Cells were subsequently maintained in
DMEM containing 0.4 ug/mL puromycin.
3.4.5 Transfections and virus production
Transfection of HeLa cells for microscopy and Western blotting was performed using
PolyJet (FroggaBio, North York, ON) as per the manufacturer’s protocol. For VSV-G
pseudotyped lentivirus production, HEK293T cells were triply transfected with the NL4.3
or shRNA encoding vector of interest, the VSV-G envelope-encoding pMD2.G plasmid
(Addgene; catalog number 12259) and pCMV-DR8.2 (Addgene; catalog number 12263)
using PolyJet (FroggaBio) as per the manufacturer’s protocol. Seventy-two hours post
transfection, lentivirus was harvested via cell culture supernatant clarification by spinning
at 1500 x g, followed by 20 m filtration. Viruses were stored in 20% FBS at -80C.
3.4.6 Western blot analysis
For Western blotting of whole cell lysates, cells were pelleted and lysed by rocking in lysis
buffer [0.5 M HEPES, 1.25 M NaCl, 1 M MgCl2, 0.25M EDTA, 0.1% Triton X-100 and
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1X complete Protease Inhibitor Tablets (Roche, Indianapolis, IN)] for one hour at 4C. The
supernatant was then clarified by spinning at 16,100 x g for 30 minutes at 4C, combined
with SDS-PAGE sample buffer (0.312 M Tris pH 6.8, 3.6 M 2-Mercaptoethanol, 50%
glycerol, 10% SDS) and boiled at 95C for 10 minutes.
All Western blots were completed by running samples on 10 or 12% SDS-PAGE gels,
followed by transferring to a nitrocellulose membrane. Membranes were blocked with 5%
milk in TBST (50 mM Tris, 150 mM NaCl, 0.1% Triton-X) for one hour at room
temperature, followed by incubation overnight at 4C with the appropriate primary
antibody in 5% milk in TBST. The next day membranes were washed three times with
TBST and incubated for two hours at room temperature with the appropriate species
specific HRP-conjugated secondary antibodies (1:2000, Thermo Fisher Scientific) in 5%
milk in TBST. Blots were subsequently washed and developed using ECL substrates
(Millipore, Etobicoke, ON) and a C-DiGit chemiluminescence Western blot scanner (LI-
COR Biosciences, Lincoln, NE). The following primary antibodies were utilized: 1:100
THETM His antibody (GenScript, Piscataway, NJ), 1:1000 rabbit anti-Nef polyclonal
antibody (NIH-AIDS Research and Reference Reagent program; catalog number 2949),
1:5000 rabbit anti-Vpu polyclonal antibody (NIH-AIDS Research and Reference Reagent
program; catalog number 969), 1:2000 mouse anti-Actin monoclonal antibody (Thermo
Fisher Scientific), 1:2000 rabbit anti-Calnexin (abcam, Cambridge, MA; catalog number
ab22595), 1:1000 rabbit anti-ADAM17 (abcam; catalog number ab2051), 1:1000 mouse
anti-TSG-101 (abcam; catalog number ab83), 1:1000 rabbit anti-SNX18 (Sigma-Aldrich,
Oakville Ontario; catalog number HPA037800).
3.4.7 Microscopy
For microscopy experiments, cells were plated on cover slips and transfected with the
expression vectors of interest using PolyJet, as described above. Twenty-four hours post
transfection cells were incubated at room temperature for 2 hours to mature the fluorophore
and fixed with 4% paraformaldehyde. Cells were subsequently washed twice with PBS and
permeabilized and blocked in 2% bovine serum albumin (BSA) and 0.1% Triton-X in PBS
for 2 hour at room temperature. Cells were subsequently stained for two hours with anti-
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DYKDDDK (BioLegend, San Diego, CA) or anti-CD63 (Developmental Studies
Hybridoma Bank, The University of Iowa) diluted down to in 2% BSA in PBS, washed
twice with 0.2% BSA in PBS and incubated for one and a half hours with the appropriate
fluorophore conjugated secondary antibodies (Alexa-Fluor-647 conjugated anti-mouse or
anti-Rat; Jackson ImmunoResearch, West Grove, PA) in 0.2% BSA in PBS. Finally, cells
were washed twice with PBS and mounted on coverslips with DAPI Fluoromount-G
(SouthernBiotech, Birmingham, AL). Cells were imaged on a Leica DMI6000 B at 63X
or 100X magnification using the FITC, Cy5 and DAPI filter settings and imaged with a
Hamamatsu Photometrics Delta Evolve camera. Images were subsequently de-
convolved using the Advanced Fluorescence Deconvolution application on the Leica
Application Suite software (Lecia, Wetzlar, Germany). The mean fluorescence intensity
of the fluorescence signal was determined using image J [54] and co-localization
analysis was conducted using Pearson correlation coefficient between signal in the FITC
(eGFP/BiFC) and Cy3 (CD63) channel using the ImageJ plugin JACoP, as described
previously [55].
3.4.8 Extracellular vesicle purification
HEK293T cells stably expressing a non-specific or SNX18 targeting shRNA were
transfected with the pNL4.3 dG/P eGFP vector using PolyJet, as described above. Forty-
eight hours post transfection the cell culture medium was removed and processed to purify
extracellular vesicles, while the cells were washed with 1 x PBS followed by lysis and
preparation for Western blotting, as described above. To purify extracellular vesicles,
culture supernatant was pelleted at 3000 x g for 15 minutes and the supernatant was run
through a 2 m filter. Culture supernatant was then concentrated using a 10,000 Da cut-off
PeirceTM protein concentrator (Thermo Fisher Scientific). The extracellular vesicles were
then precipitated from concentrated supernatants using ExoQuick exosome precipitation
solution (FroggaBio), as per the manufactures protocol. Briefly, the exosome precipitation
solution was mixed with the culture supernatant and incubate at 4ºC overnight prior to
pelleting extracellular vesicles and removing supernatant via subsequent spins at 1500 x g
for 30 mins and 5 mins. Pelleted vesicles were then re-suspended in RIPA-Doc buffer
(140 mM NaCl, 10 mM NaH2PO4, 1% NP-40, 0.05% SDS, 0.5% sodium deoxycholate)
181
and mixed with SDS-PAGE sample buffer (0.312 M Tris pH 6.8, 3.6 M 2-
Mercaptoethanol, 50% glycerol, 10% SDS) and boiled at 95C for 10 mins.
3.4.9 Transient shRNA knock-down experiments
For the dual infection of Sup-T1 cells with VSV-G pseudotyped viruses, 1 x 106 Sup-T1
cells were suspended in 8 g/mL polybrene and virus preparations in 20% FBS produced
as described above from pGFP-C-shLenti vectors encoding different shRNAs. Cells were
then spinoculated for four hours at 2880 x g at room temperature and re-suspended in fresh
RPMI. The next day, cells were pelleted and re-suspended in 10% RPMI (mock infection)
or pNL4.3 dG/P F2A-mStrawberry Nef/dNef pseudovirions in 20% FBS and 8 g/mL
polybrene. Forty-eight hours later, cells were analyzed via flow cytometry or Western
blotting.
3.4.10 Flow cytometry
For cell surface staining of MHC-I on transduced Sup-T1 cells, cells were washed twice
with PBS, followed by fixing in 1% paraformaldehyde for 20 minutes at room temperature.
Cells were subsequently washed twice with FACS buffer (1% FBS, 50 mM
ethylenediaminetetraacetic acid (EDTA) in PBS) and stained with the appropriate antibody
by rocking for 40 minutes at room temperature. Cells were then washed twice with FACS
buffer and re-suspended in PBS. For staining of MHC-I 1:25 APC-Cy7-conjugated mouse-
anti-HLA-A, B, C (clone W6/32, BioLegend) or an isotype control antibody (APC-Cy7
conjugated mouse IgG2b, κ isotype, clone MOPC-21, BioLegend) was utilized. Cells were
analyzed using a BD Biosciences FACSCanto SORP (BD Biosciences). Data analysis was
performed using FlowJo software (version 9.6.4, FlowJo LLC, Ashland, OR).
3.4.11 Data analysis
3.4.11.1 In vitro protein capture assays
For in vitro pull-down assays, Western blots were acquired using a C-DiGit
chemiluminescent blot scanner (LI-COR) and band intensities were quantified using Image
Studio (LI-COR). The band intensities were normalized to the highest intensity in each
assay and the mean normalized intensities were analyzed by one-way analysis of variance
182
followed by Bonferroni’s multiple comparison test (Figures 3.1B, 3.2C, E) or a Student’s
t-test (Figure 3.3B).
3.4.11.2 Microscopy
For analysis of Nef: SNX18 BiFC and CD63 overlap, the mean (+/-SD) Pearson correlation
coefficient between signals in the FITC (eGFP/BiFC) and Cy3 (CD63) were determined
using the ImageJ plugin JACoP, as described previously [55]. Means were compared
using an Ordinary one-way analysis of variance followed by Tukey’s multiple
comparison test. The mean fluorescence intensity of Nef-VC: SNX18-VN and Nef
AxxA75-VC: SNX18-VN BiFC signal was compared using an unpaired Student’s t-test.
3.4.11.3 Transient shRNA knock-down experiments
For analyses of flow cytometry data obtained for dual infected Sup-T1 cells, double
positive (GFP+ mStrawberry+) cells we gated on based on fluorescence minus one controls.
The relative levels of MHC-I on double positive cells were determined by normalizing MFI
levels on cells infected with virus encoding Nef to those infected with virus lacking Nef
(dNef), where cell surface levels of MHC-I in cells infected with dNef virus was set to 100.
The relative levels of MHC-I on cells infected with virus encoding Nef was then compared
between cells infected with virus encoding a non-specific and SNX18-targeting shRNA
using a paired t-test.
To quantify the amount of SNX18 in Sup-T1 cells transduced with virus encoding a non-
specific and SNX18-targeting shRNA, SNX18 and actin band intensities obtained by
Western blotting, as described above, were quantified using Image Studio (LI-COR). The
intensity of the SNX18 protein bands were normalized to actin levels from the same sample
and normalized to the SNX18 intensity in cells transduced with virus encoding a non-
specific shRNA. The mean relative SNX18 levels were then analyzed by one-way analysis
of variance followed by Dunnett’s multiple comparison test.
3.4.11.4 Extracellular vesicle purifications
To quantify the amount of Nef in the extracellular vesicle purifications from transfected
cells, the Western blot band intensities were quantified using Image Studio (LI-COR). The
183
intensity of Nef from the extracellular fraction was normalized to the intracellular levels of
Nef. The relative levels of extracellular Nef in 293TαSNX18 knock-down cells was than
normalized to those in 293TNS cells from the same experiment. Mean levels of extracellular
Nef purified from 293TαSNX18 and 293TNS cell supernatants was then compared using a
Student’s t-test.
3.4.11.5 Statistical analysis
All statistical tests were completed using Graph Pad Prism (Graph Pad Software Inc., La
Jolla, CA).
3.5 References
1. Sugden SM, Bego MG, Pham TN, Cohen EA. Remodeling of the host cell plasma
membrane by HIV-1 Nef and Vpu: a strategy to ensure viral fitness and persistence.
Viruses. 2016;8(3):67.
2. Pawlak EN, Dikeakos JD. HIV-1 Nef: a master manipulator of the membrane
trafficking machinery mediating immune evasion. Biochim Biophys Acta.
2015;1850(4):733-41.
3. Wong P, Pamer EG. CD8 T cell responses to infectious pathogens. Annu Rev
Immunol. 2003;21:29-70.
4. Ali A, Ng HL, Dagarag MD, Yang OO. Evasion of cytotoxic T lymphocytes is a
functional constraint maintaining HIV-1 Nef expression. Eur J Immunol.
2005;35(11):3221-8.
5. Munch J, Stolte N, Fuchs D, Stahl-Hennig C, Kirchhoff F. Efficient class I major
histocompatibility complex down-regulation by simian immunodeficiency virus
Nef is associated with a strong selective advantage in infected rhesus macaques. J
Virol. 2001;75(21):10532-6.
6. Friedrich TC, Piaskowski SM, Leon EJ, Furlott JR, Maness NJ, Weisgrau KL, et
al. High Viremia Is Associated with High Levels of In Vivo Major
Histocompatibility Complex Class I Downregulation in Rhesus Macaques Infected
with Simian Immunodeficiency Virus SIVmac239. J Virol. 2010;84(10):5443-7.
7. Schindler M, Schmokel J, Specht A, Li H, Munch J, Khalid M, et al. Inefficient
Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4(+)
T cells in natural SIV infection. PLoS Pathog. 2008;4(7).
8. Swigut T, Alexander L, Morgan J, Lifson J, Mansfield KG, Lang S, et al. Impact
of Nef-mediated downregulation of major histocompatibility complex class I on
immune response to simian immunodeficiency virus. J Virol. 2004;78(23):13335-
44.
184
9. Ren X, Park SY, Bonifacino JS, Hurley JH. How HIV-1 Nef hijacks the AP-2
clathrin adaptor to downregulate CD4. Elife. 2014;3:e01754.
10. Chaudhuri R, Mattera R, Lindwasser OW, Robinson MS, Bonifacino JS. A Basic
Patch on alpha-Adaptin Is Required for Binding of Human Immunodeficiency
Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex. J Virol.
2009;83(6):2518-30.
11. Noviello CM, Benichou S, Guatelli JC. Cooperative binding of the class I major
histocompatibility complex cytoplasmic domain and human immunodeficiency
virus type 1 Nef to the endosomal AP-1 complex via its mu subunit. J Virol.
2008;82(3):1249-58.
12. Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y. Structural basis of evasion
of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol. 2012;19(7):701-
6.
13. Bonifacino JS, Glick BS. The mechanisms of vesicle budding and fusion. Cell.
2004;116(2):153-66.
14. Dirk BS, Jacob RA, Johnson AL, Pawlak EN, Cavanagh PC, Van Nynatten L, et
al. Viral bimolecular fluorescence complementation: a novel tool to study
intracellular vesicular trafficking pathways. PLoS One. 2015;10(4):e0125619.
15. Haberg K, Lundmark R, Carlsson SR. SNX18 is an SNX9 paralog that acts as a
membrane tubulator in AP-1-positive endosomal trafficking. J Cell Sci.
2008;121(Pt 9):1495-505.
16. Pylypenko O, Lundmark R, Rasmuson E, Carlsson SR, Rak A. The PX-BAR
membrane-remodeling unit of sorting nexin 9. EMBO J. 2007;26(22):4788-800.
17. Schobel S, Neumann S, Hertweck M, Dislich B, Kuhn PH, Kremmer E, et al. A
novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage
of the amyloid precursor protein. J Biol Chem. 2008;283(21):14257-68.
18. Lundmark R, Carlsson SR. Sorting nexin 9 participates in clathrin-mediated
endocytosis through interactions with the core components. J Biol Chem.
2003;278(47):46772-81.
19. Hinshaw JE. Dynamin and its role in membrane fission. Annu Rev Cell Dev Biol.
2000;16:483-+.
20. Lundmark R, Carlsson SR. The beta-appendages of the four adaptor-protein (AP)
complexes: structure and binding properties, and identification of sorting nexin 9
as an accessory protein to AP-2. Biochem J. 2002;362:597-607.
21. Takamori S, Holt M, Stenius K, Lemke EA, Gronborg M, Riedel D, et al. Molecular
anatomy of a trafficking organelle. Cell. 2006;127(4):831-46.
22. Trible RP, Emert-Sedlak L, Smithgall TE. HIV-1 Nef selectively activates Src
family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction. J Biol
Chem. 2006;281(37):27029-38.
185
23. Lee CH, Saksela K, Mirza UA, Chait BT, Kuriyan J. Crystal structure of the
conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell.
1996;85(6):931-42.
24. Lee CH, Leung B, Lemmon MA, Zheng J, Cowburn D, Kuriyan J, et al. A single
amino acid in the SH3 domain of Hck determines its high affinity and specificity
in binding to HIV-1 Nef protein. EMBO J. 1995;14(20):5006-15.
25. Arold S, Franken P, Strub MP, Hoh F, Benichou S, Benarous R, et al. The crystal
structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role
for this complex in altered T cell receptor signaling. Structure. 1997;5(10):1361-
72.
26. Saksela K, Cheng GH, Baltimore D. Proline-Rich (Pxxp) Motifs in Hiv-1 Nef Bind
to Sh3 Domains of a Subset of Src Kinases and Are Required for the Enhanced
Growth of Nef(+) Viruses but Not for down-Regulation of Cd4. EMBO J.
1995;14(3):484-91.
27. Hanna Z, Weng XD, Kay DG, Poudrier J, Lowell C, Jolicoeur P. The pathogenicity
of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice
is abolished by mutation of its SH3-binding domain, and disease development is
delayed in the absence of Hck. J Virol. 2001;75(19):9378-92.
28. Briggs SD, Sharkey M, Stevenson M, Smithgall TE. SH3-mediated Hck tyrosine
kinase activation and fibroblast transformation by the Nef protein of HIV-1. J Biol
Chem. 1997;272(29):17899-902.
29. Wiskerchen M, ChengMayer C. HIV-1 Nef association with cellular serine kinase
correlates with enhanced virion infectivity and efficient proviral DNA synthesis.
Virology. 1996;224(1):292-301.
30. Hung CH, Thomas L, Ruby CE, Atkins KM, MorriS NP, Knight ZA, et al. HIV-1
nef assembles a src family Kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-
surface MHC-I. Cell Host Microbe. 2007;1(2):121-33.
31. Atkins KM, Thomas L, Youker RT, Harriff MJ, Pissani F, You HH, et al. HIV-1
Nef binds PACS-2 to assemble a multikinase cascade that triggers major
histocompatibility complex class I (MHC-I) down-regulation - Analysis using short
interfering RNA and knock-out mice. J Biol Chem. 2008;283(17):11772-84.
32. Dikeakos JD, Thomas L, Kwon G, Elferich J, Shinde U, Thomas G. An interdomain
binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to
down-regulate MHC-I. Mol Biol Cell. 2012;23(11):2184-97.
33. Lee JH, Schierer S, Blume K, Dindorf J, Wittki S, Xiang W, et al. HIV-Nef and
ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with
Immune Pathogenesis in Chronic HIV Infection. EBioMedicine. 2016;6:103-13.
34. Lee JH, Wittki S, Brau T, Dreyer FS, Kratzel K, Dindorf J, et al. HIV Nef, paxillin,
and Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases. Mol
Cell. 2013;49(4):668-79.
186
35. Witte V, Laffert B, Rosorius O, Lischka P, Blume K, Galler G, et al. HIV-1 Nef
mimics an integrin receptor signal that recruits the polycomb group protein Eed to
the plasma membrane. Mol Cell. 2004;13(2):179-90.
36. Ebsen H, Lettau M, Kabelitz D, Janssen O. Identification of SH3 domain proteins
interacting with the cytoplasmic tail of the a disintegrin and metalloprotease 10
(ADAM10). PLoS One. 2014;9(7):e102899.
37. Simons M, Raposo G. Exosomes - vesicular carriers for intercellular
communication. Curr Opin Cell Biol. 2009;21(4):575-81.
38. Saksela K, Permi P. SH3 domain ligand binding: What's the consensus and where's
the specificity? FEBS Lett. 2012;586(17):2609-14.
39. Jung J, Byeon IJ, Ahn J, Gronenborn AM. Structure, dynamics, and Hck interaction
of full-length HIV-1 Nef. Proteins. 2011;79(5):1609-22.
40. Park J, Kim Y, Lee S, Park JJ, Park ZY, Sun W, et al. SNX18 shares a redundant
role with SNX9 and modulates endocytic trafficking at the plasma membrane. J
Cell Sci. 2010;123(10):1742-50.
41. Mygind KJ, Storiko T, Freiberg ML, Samsoe-Petersen J, Schwarz J, Andersen OM,
et al. Sorting nexin 9 (SNX9) regulates levels of the transmembrane disintegrin and
metalloproteinase (ADAM)-9 at the cell surface. J Biol Chem. 2018.
42. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-
scale CRISPR-Cas9 knockout screening in human cells. Science.
2014;343(6166):84-7.
43. Sami Saribas A, Cicalese S, Ahooyi TM, Khalili K, Amini S, Sariyer IK. HIV-1
Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-
mediated neurotoxicity. Cell Death Dis. 2017;8(1):e2542.
44. Lee JH, Ostalecki C, Zhao Z, Kesti T, Bruns H, Simon B, et al. HIV Activates the
Tyrosine Kinase Hck to Secrete ADAM Protease-Containing Extracellular
Vesicles. EBioMedicine. 2018;28:151-61.
45. Moreno-Gonzalo O, Villarroya-Beltri C, Sanchez-Madrid F. Post-translational
modifications of exosomal proteins. Front Immunol. 2014;5.
46. Dirk BS, Pawlak EN, Johnson AL, Van Nynatten LR, Jacob RA, Heit B, et al. HIV-
1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and
recycling. Sci Rep. 2016;6:37021.
47. Dikeakos JD, Atkins KM, Thomas L, Emert-Sedlak L, Byeon IJL, Jung JW, et al.
Small Molecule Inhibition of HIV-1-Induced MHC-I Down-Regulation Identifies
a Temporally Regulated Switch in Nef Action. Mol Bio Cell. 2010;21(19):3279-
92.
48. Piguet V, Wan L, Borel C, Mangasarian A, Demaurex N, Thomas G, et al. HIV-1
Nef protein binds to the cellular protein PACS-1 to downregulate class I major
histocompatibility complexes. Nat Cell Biol. 2000;2(3):163-7.
187
49. Crump CM, Xiang Y, Thomas L, Gu F, Austin C, Tooze SA, et al. PACS-1 binding
to adaptors is required for acidic cluster motif-mediated protein traffic. EMBO J.
2001;20(9):2191-201.
50. Frost A, Unger VM, De Camilli P. The BAR Domain Superfamily: Membrane-
Molding Macromolecules. Cell. 2009;137(2):191-6.
51. Takenawa T, Suetsugu S. The WASP-WAVE protein network: connecting the
membrane to the cytoskeleton. Nat Rev Mol Cell Bio. 2007;8(1):37-48.
52. Husain M, Gusella GL, Klotman ME, Gelman IH, Ross MD, Schwartz EJ, et al.
HIV-1 Nef induces proliferation and anchorage-independent growth in podocytes.
J Am Soc Nephrol. 2002;13(7):1806-15.
53. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, et al.
Production of acquired immunodeficiency syndrome-associated retrovirus in
human and nonhuman cells transfected with an infectious molecular clone. J Virol.
1986;59(2):284-91.
54. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image
analysis. Nat Methods. 2012;9(7):671-5.
55. Cordelieres FP, Bolte S. Experimenters' guide to colocalization studies: finding a
way through indicators and quantifiers, in practice. Method Cell Biol.
2014;123:395-408.
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Chapter 4
4 General Discussion
4.1 Thesis overview
The work presented in this thesis aimed to provide novel insights into the mechanisms by
which the HIV-1 accessory proteins Nef and Vpu alter the host cellular environment. We
posited that the functions of Nef and Vpu are mediated through the ability of these viral
proteins to hijack host cellular proteins, specifically those involved in the trafficking of
cargo to different cellular locales. In Chapter 2, we observed Nef- and Vpu-mediated
downregulation of the immune cell co-stimulatory molecule CD28 and also implicated host
cell protein binding motifs located within the viral proteins in this process. In Chapter 3,
we identified a novel host cell binding partner of Nef, the membrane trafficking regulator
SNX18, which we propose is hijacked by Nef via a host cell protein binding motif to
mediate dysregulation of the immune cell receptor MHC-I.
4.1.1 Chapter 2: Findings, limitations and future work
In Chapter 2, we set out to examine how the HIV-1 accessory proteins alter the co-
stimulatory molecule CD28. Prior studies suggested that Nef downregulates cell surface
levels of CD28 [1, 2], however it was unknown how Nef was mediating this process.
Moreover, the cell surface downregulation of CD28 by the accessory protein Vpu had only
been previously documented in a large-scale screen of receptors that are modulated by Nef
and Vpu [3]. Given that Nef and Vpu alter the subcellular localization of other cell surface
receptors by hijacking cellular trafficking machinery, we hypothesized that this is how Nef
and Vpu mediate their effects on CD28. Overall, the work in this chapter has given us an
unprecedented view of the mechanism of Nef- and Vpu-mediated downregulation of
endogenous CD28 in CD4+ T cells. Moreover, this is the first examination of Vpu-mediated
CD28 downregulation.
4.1.1.1 The subcellular fate of CD28 upon HIV-1 infection
We observed that in infected cells HIV-1 Nef and Vpu mediate decreases in cell surface
levels of CD28, as well as decreases in total CD28 protein levels (Figure 2.1; Figure 2.8).
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These decreases in CD28 were accompanied by increased CD28 localization to lysosomal
compartments (Figure 2.9). Moreover, our results support a mechanism whereby total
CD28 protein levels are decreased by the viral proteins in an endosomal acidification-
dependent manner (Figure 2.10). Thus, we concluded that the mechanism by which Nef
and Vpu decrease cell surface CD28 is mediated, at least in part, by decreasing total protein
levels via mis-localizing CD28 to acidified degradative compartments. Similar
mechanisms are utilized by Nef and Vpu to downregulate cell surface CD4 and BST-2,
respectively [4-9].
4.1.1.2 Nef and Vpu motifs in CD28 downregulation
We hypothesized that Nef and Vpu mediate their effects within the cell by hijacking
cellular trafficking proteins. Therefore, we examined the consequences of introducing
mutations in motifs that were previously implicated in mediating interactions between the
viral proteins and host cellular proteins. We observed that mutations in specific virus: host
protein interaction motifs inhibited downregulation of CD28 at both the cell surface and
total protein levels, namely mutation of the Nef LL165 and DD175 motifs (Figure 2.13;
Figure 2.14) and the Vpu S52/56 motif (Figure 2.15). Given that these motifs mediate
interactions with host cell proteins to downregulate other cellular receptors [10-14], these
findings support our hypothesis that CD28 downregulation is facilitated by Nef and Vpu
hijacking host cellular trafficking proteins. To more directly test this hypothesis, we sought
to knock-down or inhibit specific cellular trafficking proteins and examined the effects on
CD28 downregulation. We did not observe any impairment in Nef-mediated cell surface
CD28 downregulation upon knock-down of the membrane trafficking adaptor AP-2 α and
μ subunits or overexpression of a dominant negative form of the membrane trafficking
regulator SNX9 (Figure 2.17; Figure 2.18). As described in section 2.3, these experiments
may be limited by the fact that host cellular trafficking proteins and pathways can be
redundant or capable of compensating for the loss of specific trafficking proteins. Indeed,
previous work suggests that upon knock-out of adaptor protein subunits, namely AP-1µ,
cells may compensate for this loss by upregulating other membrane trafficking proteins
[15]. Further studies would benefit from simultaneous knock-down or inhibition of
multiple membrane trafficking proteins, though this may be technically difficult.
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Moreover, the effects of knock-downs may be increasingly apparent if more efficient
decreases in protein levels are obtained, which could be achieved through the use of gene
editing technologies to knock-out genes of interest [16, 17]. Nonetheless, it would be useful
to validate our AP-2 knock-downs or SNX9 dominant negative protein by determining if
they impede the Nef-mediated downregulation of receptors believed to be downregulated
in an AP-2-dependent manner. In addition to the trafficking mediators explored herein, the
importance of the Nef LL165 and DD175 motifs and the Vpu S52/56 motif also suggest
potential involvement of AP-1, LC3C and β-TrCP in Vpu-mediated downregulation [13,
18, 19], and the v-ATPase in Nef-mediated CD28 downregulation [20, 21]. Thus, these
host proteins represent strong candidates for further exploration.
4.1.1.3 Conservation of Vpu-mediated CD28 downregulation
In addition to exploring the mechanism of Nef- and Vpu-mediated CD28 downregulation,
we sought to determine if the ability of Vpu to downregulate CD28 was a feature unique
to the HIV-1 lab adapted strain NL4.3, or if Vpu proteins from other HIV-1 viruses can
downregulate CD28. This is important as the HIV-1 epidemic is comprised of a large array
of diverse viruses [22-24]. Thus, if Vpu-mediated CD28 downregulation is observed for
multiple distinct viruses, our findings may be more applicable to the epidemic as a whole.
We observed that the downregulation of CD28 in CD4+ Sup-T1 cells can be mediated by
Vpu proteins derived from the HIV-1 strain B.JFRL and a consensus C Vpu protein (Figure
2.21). However, our analysis was limited to a small number of Vpu proteins. Furthermore,
we examined the ability of these Vpu proteins to downregulate CD28 in the context of a
viral backbone derived from NL4.3. Therefore, it would be worthwhile for future studies
to examine Vpu-mediated downregulation of CD28 by a variety of viruses using full length
molecular clones. Additionally, the dileucine-like motif of NL4.3 Vpu was required for
cell surface CD28 downregulation (Figure 2.15). However, this motif is absent in the
B.JRFL Vpu protein which also mediated efficient CD28 downregulation (Figure 2.21;
Figure 2.24). Thus, it will be important to examine if the host cell proteins required to
mediate CD28 downregulation by these different Vpu proteins are equivalent. Nonetheless,
the finding that non-NL4.3 Vpu proteins can downregulate CD28 validates that Vpu-
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mediated downregulation of CD28 could be a universal feature across the epidemic,
supporting the importance of future research.
4.1.1.4 The function of CD28 downregulation
Given the central role of CD28 in signaling to mediating T cell activation, we proposed
that CD28 downregulation by the viral proteins may impact the activation of T cells. We
observed that in stimulated cells Nef promoted increased activation, while Vpu inhibited
activation (Figure 2.23). These effects could be due to changes in the cell surface levels of
CD28, but may also be attributable to the numerous other effects of the viral proteins on
immune cell activation, as described in section 1.5.5. Future experiments are necessary to
more conclusively link downregulation of CD28 to cell activation; however, this is
challenging given the fact that there are no known mutations in the viral proteins that can
be made to specifically negate CD28 downregulation without impeding other functions.
Thus, it is difficult to inhibit CD28 downregulation without hindering other viral protein
functions which could impact cellular activation. Nevertheless, it would be interesting for
future experiments to determine if the amount of phosphorylated, and thus signaling
competent, CD28 differs in stimulated CD4+ T cells infected with virus encoding or lacking
Nef and Vpu. Overall, we postulate that changes in cell surface CD28 mediated by the viral
proteins contribute to the overall dysregulation of cellular activation by limiting detection
of extrinsic stimuli.
4.1.1.5 Redundancy in CD28 downregulation ability
The ability of two HIV-1 proteins to counteract the same cell surface molecules
underscores the importance of this function. In this manner, HIV-1 has a back-up plan to
ensure that CD28 is downregulated to some extent. Future experiments may be useful to
determine if there are differences in CD28 downregulation at different times during
infection. In our experiments in Sup-T1 cells, we examined downregulation at forty-eight
hours post infection. However, Nef is expressed early during infection, even from
unintegrated HIV-1 DNA [25], while Vpu is a “late gene”, as its translation is dependent
on prior expression of the viral Rev protein [26]. Therefore, it would be interesting to
examine whether the effects of Vpu are more pronounced if the infection proceeds for an
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increased amount of time, or if downregulation by Nef and Vpu differ over the course of a
number of days. Moreover, given that vpu and nef genes can compensate for each other,
we could examine whether there are viruses encoding Nef proteins that poorly
downregulate CD28, but encode Vpu proteins that compensate for this defect by
downregulating CD28 more efficiently, or vice-versa. Overall, the ability of multiple viral
proteins to alter cell surface expression of CD28 suggests that this function is significant.
4.1.1.6 Summary and future directions
In taking together the data presented in Chapter 2 and previous findings, we propose a
model of how Nef and Vpu modulate CD28 within infected CD4+ T cells (Figure 4.1). Nef
decreases both cell surface and total cellular protein CD28 levels (Figure 2.1; Figure 2.8).
It remains unknown if Nef targets CD28 at the cell surface, though it has been proposed
that Nef accelerates the internalization of cell surface resident CD28 [1]. Moreover, it
remains unclear if Nef mediated downregulation of CD28 occurs through binding to the
cytoplasmic tail of CD28, similar to how Nef downregulates MHC-I and CD4 [27-29]. We
observed that mutation of the Nef LL165 or DD175 motifs inhibits the ability of Nef to
downregulate CD28 (Figure 2.14), suggesting that downregulation of CD28 by Nef may
require host cellular proteins known to bind to Nef via these motifs, such as AP-2 or v-
ATPase [20, 30]. However, future studies are required to confirm if these proteins are
necessary for downregulation of CD28. Finally, we observed that in the absence of Nef
there is less CD28 trafficked to LAMP-1 positive compartments (Figure 2.9), suggesting
that Nef mediates trafficking to, and potentially degradation of CD28 within, lysosomes.
Future studies to better map the trafficking pathway and proteins required for CD28
downregulation by Nef are required. In the case of Vpu, our findings indicate that the S52/56
motif, which mediates binding to β-TrCP [13], is critical for decreasing cell surface and
total cellular CD28 protein levels (Figure 2.15). Thus, the mechanism of Vpu-mediated
CD28 downregulation may require β-TrCP recruitment. However, it remains unknown if
Vpu is hijacking CD28 at the endoplasmic reticulum, as it does CD4 [13, 31], or at the cell
surface or intracellular compartments, as it does BST-2 [4, 32]. Moreover, future work is
needed to determine if Vpu interacts with CD28, to identify additional host cellular proteins
necessary for Vpu-mediated CD28 downregulation, and to further understand how Vpu
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contributes to increased lysosomal localization of CD28 (Figure 2.9). Given the importance
of CD28 in cell activation [33, 34], Nef- and Vpu-mediated decreases in cellular CD28
levels may play a significant role in altering the activation status of the cell. We observed
opposite effects of Nef and Vpu on cellular activation (Figure 2.23), which may be a result
of changes in cellular CD28 levels in addition to the other effects on cellular activation that
are mediated by the accessory proteins (Section 1.5.5). Overall, changes in cellular
activation are important, as they can influence antiviral responses and HIV-1 viral gene
transcription [35-37].
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Proposed models of CD28 downregulation by (a) Nef and (b) Vpu. (A) Newly translated
CD28 is trafficked to the cell surface via the TGN. In the presence of Nef, CD28 may be
targeted at the cell surface to increase endocytosis [1], contributing to decreases in cell
surface levels of CD28. The LL165 and DD175 motifs, which facilitate binding of Nef to
AP-2 and v-ATPase [20, 30], are critical for CD28 downregulation. In the presence of
Nef, CD28 localizes to lysosomes. (B) Vpu mediates decreases in cell surface and total
CD28, but it remains unknown if CD28 is targeted by Vpu shortly after translation in the
ER, at the cell surface, or at intracellular compartments. The β-TrCP-binding motif of
Vpu, S52/56, is critical for Vpu-mediated downregulation of CD28. In the presence of
Vpu, CD28 localizes to lysosomes.
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4.1.2 Chapter 3: Findings, limitations and future work
We propose that HIV-1 Nef and Vpu mediate their functions by interacting with host
cellular membrane trafficking proteins and in Chapter 3 we characterize a novel Nef: host
protein interaction and examine its importance in Nef-mediated MHC-I downregulation.
Given the sheer complexity of membrane trafficking, we hypothesized that numerous host
cell proteins are critical for Nef to downregulate MHC-I, including proteins not yet
implicated in this process. We were specifically interested in the sorting nexin proteins,
which stems from our previously published work that illustrates a SNX18: Nef complex is
formed that localizes to compartments containing AP-1 [38], a trafficking adaptor that Nef
hijacks to downregulate MHC-I [27].
4.1.2.1 The Nef: SNX18 interaction interface
In this chapter, we illustrated for the first time that SNX18 is a direct Nef binding partner
(Figure 3.1). We have mapped the interaction between SNX18 and Nef to the SH3 domain
of SNX18 (Figure 3.2). Moreover, the in vitro direct binding, as well as the interaction
between SNX18 and Nef in cells, was dependent on the Nef PxxP75 motif (Figure 3.3;
Figure 3.4), which is unsurprising given its documented role in interacting with other SH3
domain containing proteins [39, 40]. Notably, the interaction between SNX18 and Nef in
cells, as quantified by BiFC fluorescence intensity, was not completely abolished by
mutation of the Nef PxxP75 motif (Figure 3.4). This indicates that there may be residues
within Nef outside of this motif that contribute to this interaction, or that additional host
cell proteins may facilitate this interaction in cells. A candidate host cell protein that could
facilitate SNX18 and Nef binding is AP-1, which binds to both SNX18 and Nef [27, 41,
42], and was previously found to co-localize with the Nef: SNX18 complex in cells [38].
Therefore, it would be of interest for future studies to determine if mutation of the Nef
EEEE65 motif, which mediates AP-1 interactions [27, 43], alters the intensity of the Nef:
SNX18 BiFC signal. Moreover, it would be interesting to determine if Nef inhibitors such
as 2c, which targets the Nef PxxP75: SFK SH3 domain interaction interface [44], influence
the Nef: SNX18 interaction, which could contribute to the ability of these inhibitors to
block Nef-mediated MHC-I downregulation [44]. In summary, we have identified a novel
Nef-binding host protein which we posit may facilitate Nef-mediated functions.
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4.1.2.2 The Nef: SNX18 interaction in MHC-I downregulation
Overall, we propose that the Nef: SNX18 interaction functions to facilitate Nef-mediated
MHC-I downregulation. This is supported by our findings that the shRNA knock-down of
SNX18 reduced Nef-mediated MHC-I downregulation (Figure 3.5). While the inhibition
of downregulation was consistent, the change was small. However, as discussed in section
3.3, given that SNX18 and SNX9 exhibit some redundant functions we cannot exclude the
possibility that despite the lack of a direct SNX9: Nef interaction SNX9 may compensate
for the loss of SNX18, thereby reducing the effect of SNX18 knock-down on Nef-mediated
MHC-I downregulation. Future experiments could examine if knock-down of both SNX18
and SNX9 leads to a greater deficit in Nef-mediated MHC-I downregulation. Moreover,
while we would like to illustrate that a physical interaction between SNX18 and Nef is
explicitly required for Nef-mediated MHC-I downregulation it is technically challenging
given the fact that mutations in the interaction interface also inhibit other protein
interactions. Indeed, mutation of the Nef PxxP75 motif has already been implicated in
inhibiting MHC-I downregulation through its abolition of the Nef: SFK interaction [45,
46]. Nonetheless, our data suggest SNX18 plays a role in decreasing cell surface levels of
MHC-I in the presence of Nef.
Given our interest in the role of host cell proteins in the mechanisms of Nef function, we
sought to define SNX18’s specific role during MHC-I downregulation. Our findings
indicated that Nef-containing extracellular vesicles are secreted despite reductions in
SNX18 protein levels (Figure 3.7). Thus, we do not believe that SNX18 is promoting
decreased cell surface levels of MHC-I by facilitating Nef-mediated secretion of vesicles
containing MHC-I. However, as described in section 3.3, future experiments to define the
components within our extracellular vesicle purifications would be beneficial to determine
the purity of our preparations, as we have not fully defined the characteristics of our
purified content, nor have we defined the biogenesis pathway from which these vesicles
originated. This could be completed by electron microscopy or nanoscale flow cytometry
to determine the size and physical properties of the purified content. In addition, we
observed that the SNX18: Nef BiFC complex was present at CD63 positive compartments
(Figure 3.6). Co-localization of the SNX18: Nef BiFC signal with the CD63 marker could
198
suggest that this interaction is occurring at locations where vesicles destined for secretion
accumulate, however late endosomes marked by CD63 can also contain cargo destined for
other intracellular compartments. Therefore, the SNX18: Nef interaction may simply be
occurring at late endosomes, a compartment through which Nef and MHC-I traffic during
MHC-I downregulation [47]. In this way, SNX18 may function to mediate trafficking of
MHC-I from the cell surface to the TGN, where MHC-I is sequestered in the presence of
Nef [44, 47, 48]. Future experiments are necessary to further map the cellular location of
the SNX18: Nef interaction and examine if Nef-mediated sequestration of MHC-I in the
TGN is altered in the absence of SNX18. Ultimately, we propose that the binding of Nef
to SNX18 facilitates Nef-mediated mis-localization of MHC-I within infected cells.
4.1.2.3 Summary and future directions
The data presented in Chapter 3 sheds light on the mechanism of how Nef contributes to
decreases in cell surface levels of MHC-I, which prevents CD8+ cytotoxic T cell detection
of virally infected CD4+ T cells (Figure 4.2; [49, 50]). Nef promotes endocytosis of cell
surface MHC-I through an Arf1- or Arf6-dependent mechanism [51, 52]. Trafficking of
MHC-I away from the cell surface is dependent on Nef’s ability to bind AP-1 and PACS-
1 [53, 54]. This process may be further facilitated by the membrane trafficking mediator
SNX18, which we have identified as a novel Nef-binding partner (Figure 3.1). This
interaction is mediated by the PxxP75 motif of Nef binding to the SNX18 SH3 domain
(Figure 3.2; Figure 3.3). SNX18, like Nef, binds to AP-2 [30]. Upon reductions in SNX18
protein levels, Nef-mediated MHC-I downregulation is reduced (Figure 3.5). However,
future studies are needed to confirm the importance of SNX18 in MHC-I downregulation.
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Figure 4.2 Model of Nef-mediated evasion of cytotoxic T cell responses
Nef mediates decreases in CD4+ T cell surface levels of MHC-I, preventing detection by
CD8+ cytotoxic T cells (left; [49, 50]). MHC-I downregulation is facilitated by Arf1- or
Arf6-mediated endocytosis of cell surface MHC-I (right; [51, 52]). MHC-I is then
trafficked to the trans-Golgi network in a manner dependent on Nef, PACS-1 and AP-1
[53, 54]. This trafficking step may be facilitated by SNX18 binding to Nef via a
PxxP75:SH3 domain interaction.
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4.2 Significance
Overall, this thesis provides novel insight into how host cellular receptors are mis-localized
by the HIV-1 accessory proteins Nef and Vpu. These alterations in the location of host cell
proteins undoubtedly alter how cells function during HIV-1 infection. In the case of the
receptors of interest to this work, MHC-I and CD28, the decreases in cell surface levels of
these immune cell receptors may alter immune responses, despite the effects on these
receptors being modest. Indeed, it is well established that HIV-1-mediated decreases in cell
surface levels of MHC-I inhibit detection and killing of virally infected cells [50].
Specifically, cells infected with HIV-1 encoding a functional nef gene are more effeciently
killed by CTLs than by cells infected with HIV-1 enconding Nef unable to downregulate
MHC-I [50, 55]. Moreover, clinical evidence indicates that MHC-I downregulation ability
may correlate with HIV-1 disease progression [56, 57]. In contrast, decreases in cell surface
CD28 likely inhibit CD28 receptor activation. This may contribute to inhibiting the
responsiveness of cells to external stimuli, in conjunction with the countless other Nef- and
Vpu-mediated modifications described in section 1.5.5 that influence immune cell
activation. Changes in cellular activation are important during HIV-1 infection, given that
HIV-1 transcription is driven by transcription factors that are activated during cell
activation, thus influencing viral replication (reviewed in [58]). The importance of altering
the subcellular localization of these receptors during a HIV-1 infection is supported by the
functional redundancy in Nef and Vpu, whereby both viral proteins contribute to decreases
in cell surface MHC-I and CD28. In this way, the virus has a contingency plan to ensure
these functions are fulfilled.
4.2.1 Implications for HIV-1 treatment
The functional redundancy of Nef and Vpu emphasizes their significance during infection
and implies they could be effective therapeutic targets. However, the development of HIV-
1 therapeutics against the accessory proteins Nef and Vpu has been limited. This is likely
owing to the fact that therapeutics inhibiting Nef and Vpu would need to be utilized in
combination with other anti-retroviral therapies, as HIV-1 can still replicate in the absence
of these proteins. Moreover, given that Nef and Vpu are not enzymes, they cannot be
counteracted by targeting catalytic sites or inhibiting enzymatic activity, as has been done
201
for most of the previously developed anti-retrovirals [59]. However, Nef and Vpu
functions, including MHC-I and CD28 downregulation, can be targeted based on our
knowledge of their interactions with host cell proteins. Specifically, as druggable targets
we can utilize the interaction interfaces between the virus and host proteins that are crucial
for accessory protein functions. By preventing these binding events through the use of
small molecule inhibitors, we can prevent Nef- and Vpu-mediated functions. Importantly,
the Nef and Vpu host cell protein binding motifs are good targets as they are generally
made up of conserved residues [60-64]. This conservation would allow for targeting of
these accessory proteins in a manner applicable across the HIV-1 epidemic.
In addition to being potential targets for new HIV-1 therapeutics, the presence of these
proteins needs to be accounted for when developing HIV-1 cure strategies, as the functions
of Nef and Vpu alter the ability of the immune system to detect and eliminate HIV-1
infection. Obtaining a cure for HIV-1 for those that are currently infected is of importance
due to the large cost of remaining on life-long anti-retroviral treatment and the co-
morbidities associated with HIV-1 infection [65, 66]. Currently, the greatest challenge to
curing HIV-1 infections is the presence of latently infected cells which remain “hidden”
from both the immune system and anti-retroviral therapy by remaining in a resting state
where HIV-1 gene expression does not occur [67]. Nevertheless, these latently infected
cells can be reactivated to begin producing virus. Thus, these cells must be eradicated in
order to eliminate HIV-1 infection. A major approach being used to eliminate latently
infected cells is reactivating or “shocking” latently infected cells, such that they begin
expressing viral proteins. This process is followed by elimination or “killing” of these
reactivated cells via the immune system or viral cytopathic effects [68]. HIV-1 cure
approaches such as this shock-and-kill method that rely on the immune system to eliminate
the virus will need to address the fact that if the viral accessory proteins are present, they
will continue to inhibit the ability of the immune system to eliminate the virus. In this way,
the use of inhibitors to block the immune evasion capabilities of the accessory proteins has
been suggested to be critical for successful elimination of latently infected cells [69].
It has been proposed that molecules such as the Nef: SFK interaction inhibitor 2c, which
inhibits Nef-mediated MHC-I downregulation [44], are good candidates to consider for
202
preventing immune evasion during attempts to eliminate latently infected cells [69].
However, we must also consider the fact that the virus has a back-up plan, with Vpu also
contributing to MHC-I downregulation [70, 71]. In addition to inhibiting Nef-mediated
MHC-I downregulation, the targeting of Nef and Vpu’s ability to hide from the immune
system has also been facilitated through the use of small molecules known as CD4
mimetics. Specifically, Nef- and Vpu-mediated CD4 downregulation prevents the
exposure of CD4-induced epitopes on cell surface envelope to ADCC-mediating antibodies
which serves as a signal for NK cell-mediated lysis of infected cells [72, 73]. CD4 mimetics
can overcome this process by mimicking the presence of cell surface CD4. Specifically,
they bind to cell surface envelope and stimulate exposure of CD4-induced epitopes, thereby
increases NK cell-mediated killing of infected cells [74].
Ultimately, if we are to successfully cure HIV-1 by eliminating the latent reservoir, we
need to consider how we can overcome the incredibly effective and redundant abilities of
the HIV-1 accessory proteins Nef and Vpu to evade the immune system.
4.2.2 Concluding remarks
The ability of HIV-1 Nef and Vpu to exhibit functional redundancy is through their shared
ability to usurp broad cellular mechanisms that determine protein localization within the
cell. Both Nef and Vpu bind to and hijack host cellular proteins to link their target proteins,
often cell surface molecules, with the machinery that alter their primary location in the cell.
This frequently results in the sequestration of target proteins intracellularly or their
trafficking to sites of protein degradation. The viral proteins facilitate this by mimicking
canonical host cell protein signals that determine protein localization, such as those that
mediate binding of membrane trafficking adaptors or the ubiquitin ligase machinery.
Ultimately, functions similar to those exhibited by HIV-1 Nef and Vpu allow for viruses
to commandeer host cell machinery to make cells optimally efficient viral production
factories.
The study of viruses has broad implications. Notably, researching the fundamental
mechanisms of clinically relevant viruses is critical to improving prevention, detection and
treatment of pathogenic viruses. Additionally, since viruses require host cells to replicate,
203
these basic studies also enhance our understanding of fundamental host cell functions.
Therefore, virology research inevitably leads to an increased understanding of cellular
biology and biochemistry. In this way, viruses and viral proteins can also serve as research
tools, effectively expanding our understanding of how cellular processes are mediated in
the absence of viral infection.
4.3 References
1. Swigut T, Shohdy N, Skowronski J. Mechanism for down-regulation of CD28 by
Nef. EMBO J. 2001;20(7):1593-604.
2. Leonard JA, Filzen T, Carter CC, Schaefer M, Collins KL. HIV-1 Nef disrupts
intracellular trafficking of major histocompatibility complex class I, CD4, CD8,
and CD28 by distinct pathways that share common elements. J Virol.
2011;85(14):6867-81.
3. Haller C, Muller B, Fritz JV, Lamas-Murua M, Stolp B, Pujol FM, et al. HIV-1 Nef
and Vpu are functionally redundant broad-spectrum modulators of cell surface
receptors, including tetraspanins. J Virol. 2014;88(24):14241-57.
4. Douglas JL, Viswanathan K, McCarroll MN, Gustin JK, Fruh K, Moses AV. Vpu
directs the degradation of the human immunodeficiency virus restriction factor
BST-2/Tetherin via a {beta}TrCP-dependent mechanism. J Virol.
2009;83(16):7931-47.
5. Iwabu Y, Fujita H, Kinomoto M, Kaneko K, Ishizaka Y, Tanaka Y, et al. HIV-1
accessory protein Vpu internalizes cell-surface BST-2/tetherin through
transmembrane interactions leading to lysosomes. J Biol Chem.
2009;284(50):35060-72.
6. Rollason R, Dunstan K, Billcliff PG, Bishop P, Gleeson P, Wise H, et al. Expression
of HIV-1 Vpu leads to loss of the viral restriction factor CD317/Tetherin from lipid
rafts and its enhanced lysosomal degradation. PLoS One. 2013;8(9):e75680.
7. Schubert U, Anton LC, Bacik I, Cox JH, Bour S, Bennink JR, et al. CD4
glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu
protein requires the function of proteasomes and the ubiquitin-conjugating
pathway. J Virol. 1998;72(3):2280-8.
8. Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeficiency virus
type 1 Vpu protein induces rapid degradation of CD4. J Virol. 1992;66(12):7193-
200.
9. Rhee SS, Marsh JW. Human immunodeficiency virus type 1 Nef-induced down-
modulation of CD4 is due to rapid internalization and degradation of surface CD4.
J Virol. 1994;68(8):5156-63.
10. Mangasarian A, Piguet V, Wang JK, Chen YL, Trono D. Nef-induced CD4 and
major histocompatibility complex class I (MHC-I) down-regulation are governed
204
by distinct determinants: N-terminal alpha helix and proline repeat of Nef
selectively regulate MHC-I trafficking. J Virol. 1999;73(3):1964-73.
11. Jia X, Weber E, Tokarev A, Lewinski M, Rizk M, Suarez M, et al. Structural basis
of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor
protein complex 1. Elife. 2014;3:e02362.
12. Grzesiek S, Stahl SJ, Wingfield PT, Bax A. The CD4 determinant for
downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding
surface by NMR. Biochemistry. 1996;35(32):10256-61.
13. Margottin F, Bour SP, Durand H, Selig L, Benichou S, Richard V, et al. A novel
human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to
the ER degradation pathway through an F-box motif. Mol Cell. 1998;1(4):565-74.
14. Mangeat B, Gers-Huber G, Lehmann M, Zufferey M, Luban J, Piguet V. HIV-1
Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its
beta-TrCP2-dependent degradation. PLoS Pathog. 2009;5(9):e1000574.
15. Navarro Negredo P, Edgar JR, Wrobel AG, Zaccai NR, Antrobus R, Owen DJ, et
al. Contribution of the clathrin adaptor AP-1 subunit mu1 to acidic cluster protein
sorting. J Cell Biol. 2017;216(9):2927-43.
16. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-
scale CRISPR-Cas9 knockout screening in human cells. Science.
2014;343(6166):84-7.
17. Wang H, Hu YC, Markoulaki S, Welstead GG, Cheng AW, Shivalila CS, et al.
TALEN-mediated editing of the mouse Y chromosome. Nat Biotechnol.
2013;31(6):530-2.
18. Kueck T, Foster TL, Weinelt J, Sumner JC, Pickering S, Neil SJ. Serine
Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction
with Clathrin Adaptors. PLoS Pathog. 2015;11(8):e1005141.
19. Madjo U, Leymarie O, Fremont S, Kuster A, Nehlich M, Gallois-Montbrun S, et
al. LC3C Contributes to Vpu-Mediated Antagonism of BST2/Tetherin Restriction
on HIV-1 Release through a Non-canonical Autophagy Pathway. Cell Rep.
2016;17(9):2221-33.
20. Geyer M, Yu H, Mandic R, Linnemann T, Zheng YH, Fackler OT, et al. Subunit H
of the V-ATPase binds to the medium chain of adaptor protein complex 2 and
connects Nef to the endocytic machinery. J Biol Chem. 2002;277(32):28521-9.
21. Lu X, Yu H, Liu SH, Brodsky FM, Peterlin BM. Interactions between HIV1 Nef
and vacuolar ATPase facilitate the internalization of CD4. Immunity.
1998;8(5):647-56.
22. Hemelaar J. Implications of HIV diversity for the HIV-1 pandemic. J Infect.
2013;66(5):391-400.
23. Hemelaar J. The origin and diversity of the HIV-1 pandemic. Trends Mol Med.
2012;18(3):182-92.
205
24. Louwagie J, McCutchan FE, Peeters M, Brennan TP, Sanders-Buell E, Eddy GA,
et al. Phylogenetic analysis of gag genes from 70 international HIV-1 isolates
provides evidence for multiple genotypes. AIDS. 1993;7(6):769-80.
25. Wu Y, Marsh JW. Selective transcription and modulation of resting T cell activity
by preintegrated HIV DNA. Science. 2001;293(5534):1503-6.
26. Arrigo SJ, Chen IS. Rev is necessary for translation but not cytoplasmic
accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 1991;5(5):808-
19.
27. Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y. Structural basis of evasion
of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol. 2012;19(7):701-
6.
28. Preusser A, Briese L, Baur AS, Willbold D. Direct in vitro binding of full-length
human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain. J
Virol. 2001;75(8):3960-4.
29. Rossi F, Gallina A, Milanesi G. Nef-CD4 physical interaction sensed with the yeast
two-hybrid system. Virology. 1996;217(1):397-403.
30. Ren X, Park SY, Bonifacino JS, Hurley JH. How HIV-1 Nef hijacks the AP-2
clathrin adaptor to downregulate CD4. Elife. 2014;3:e01754.
31. Binette J, Dube M, Mercier J, Halawani D, Latterich M, Cohen EA. Requirements
for the selective degradation of CD4 receptor molecules by the human
immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.
Retrovirology. 2007;4:75.
32. Tokarev AA, Munguia J, Guatelli JC. Serine-threonine ubiquitination mediates
downregulation of BST-2/tetherin and relief of restricted virion release by HIV-1
Vpu. J Virol. 2011;85(1):51-63.
33. Jenkins MK, Taylor PS, Norton SD, Urdahl KB. CD28 delivers a costimulatory
signal involved in antigen-specific IL-2 production by human T cells. J Immunol.
1991;147(8):2461-6.
34. Weiss A, Manger B, Imboden J. Synergy between the T3/antigen receptor complex
and Tp44 in the activation of human T cells. J Immunol. 1986;137(3):819-25.
35. Asjo B, Cefai D, Debre P, Dudoit Y, Autran B. A novel mode of human
immunodeficiency virus type 1 (HIV-1) activation: ligation of CD28 alone induces
HIV-1 replication in naturally infected lymphocytes. J Virol. 1993;67(7):4395-8.
36. Nabel G, Baltimore D. An inducible transcription factor activates expression of
human immunodeficiency virus in T cells. Nature. 1987;326(6114):711-3.
37. Kinoshita S, Su L, Amano M, Timmerman LA, Kaneshima H, Nolan GP. The T
cell activation factor NF-ATc positively regulates HIV-1 replication and gene
expression in T cells. Immunity. 1997;6(3):235-44.
206
38. Dirk BS, Jacob RA, Johnson AL, Pawlak EN, Cavanagh PC, Van Nynatten L, et
al. Viral bimolecular fluorescence complementation: a novel tool to study
intracellular vesicular trafficking pathways. PLoS One. 2015;10(4):e0125619.
39. Saksela K, Cheng GH, Baltimore D. Proline-Rich (Pxxp) Motifs in Hiv-1 Nef Bind
to Sh3 Domains of a Subset of Src Kinases and Are Required for the Enhanced
Growth of Nef(+) Viruses but Not for down-Regulation of Cd4. EMBO J.
1995;14(3):484-91.
40. Lee CH, Saksela K, Mirza UA, Chait BT, Kuriyan J. Crystal structure of the
conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell.
1996;85(6):931-42.
41. Haberg K, Lundmark R, Carlsson SR. SNX18 is an SNX9 paralog that acts as a
membrane tubulator in AP-1-positive endosomal trafficking. J Cell Sci.
2008;121(Pt 9):1495-505.
42. Noviello CM, Benichou S, Guatelli JC. Cooperative binding of the class I major
histocompatibility complex cytoplasmic domain and human immunodeficiency
virus type 1 Nef to the endosomal AP-1 complex via its mu subunit. J Virol.
2008;82(3):1249-58.
43. Iijima S, Lee YJ, Ode H, Arold ST, Kimura N, Yokoyama M, et al. A noncanonical
mu-1A-binding motif in the N terminus of HIV-1 Nef determines its ability to
downregulate major histocompatibility complex class I in T lymphocytes. J Virol.
2012;86(7):3944-51.
44. Dikeakos JD, Atkins KM, Thomas L, Emert-Sedlak L, Byeon IJL, Jung JW, et al.
Small Molecule Inhibition of HIV-1-Induced MHC-I Down-Regulation Identifies
a Temporally Regulated Switch in Nef Action. Mol Biol Cell. 2010;21(19):3279-
92.
45. Hung CH, Thomas L, Ruby CE, Atkins KM, MorriS NP, Knight ZA, et al. HIV-1
nef assembles a src family Kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-
surface MHC-I. Cell Host Microbe. 2007;1(2):121-33.
46. Atkins KM, Thomas L, Youker RT, Harriff MJ, Pissani F, You HH, et al. HIV-1
Nef binds PACS-2 to assemble a multikinase cascade that triggers major
histocompatibility complex class I (MHC-I) down-regulation - Analysis using short
interfering RNA and knock-out mice. J Biol Chem. 2008;283(17):11772-84.
47. Dirk BS, Pawlak EN, Johnson AL, Van Nynatten LR, Jacob RA, Heit B, et al. HIV-
1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and
recycling. Sci Rep. 2016;6:37021.
48. Dikeakos JD, Thomas L, Kwon G, Elferich J, Shinde U, Thomas G. An interdomain
binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to
down-regulate MHC-I. Mol Biol Cell. 2012;23(11):2184-97.
49. Ali A, Ng HL, Dagarag MD, Yang OO. Evasion of cytotoxic T lymphocytes is a
functional constraint maintaining HIV-1 Nef expression. Eur J Immunol.
2005;35(11):3221-8.
207
50. Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D. HIV-1 Nef protein
protects infected primary cells against killing by cytotoxic T lymphocytes. Nature.
1998;391(6665):397-401.
51. Wonderlich ER, Leonard JA, Kulpa DA, Leopold KE, Norman JM, Collins KL.
ADP ribosylation factor 1 activity is required to recruit AP-1 to the major
histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I
trafficking in HIV-1-infected primary T cells. J Virol. 2011;85(23):12216-26.
52. Yi L, Rosales T, Rose JJ, Chowdhury B, Knutson JR, Venkatesan S. HIV-1 Nef
binds a subpopulation of MHC-I throughout its trafficking itinerary and down-
regulates MHC-I by perturbing both anterograde and retrograde trafficking. J Biol
Chem. 2010;285(40):30884-905.
53. Roeth JF, Williams M, Kasper MR, Filzen TM, Collins KL. HIV-1 Nef disrupts
MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J Cell Biol.
2004;167(5):903-13.
54. Piguet V, Wan L, Borel C, Mangasarian A, Demaurex N, Thomas G, et al. HIV-1
Nef protein binds to the cellular protein PACS-1 to downregulate class I major
histocompatibility complexes. Nat Cell Biol. 2000;2(3):163-7.
55. Yang OO, Nguyen PT, Kalams SA, Dorfman T, Gottlinger HG, Stewart S, et al.
Nef-mediated resistance of human immunodeficiency virus type 1 to antiviral
cytotoxic T lymphocytes. J Virol. 2002;76(4):1626-31.
56. Kuang XT, Li X, Anmole G, Mwimanzi P, Shahid A, Le AQ, et al. Impaired Nef
function is associated with early control of HIV-1 viremia. J Virol.
2014;88(17):10200-13.
57. Gupta P, Husain M, Hans C, Ram H, Verma SS, Misbah M, et al. Sequence
heterogeneity in human immunodeficiency virus type 1 nef in patients presenting
with rapid progression and delayed progression to AIDS. Arch Virol.
2014;159(9):2303-20.
58. Pereira LA, Bentley K, Peeters A, Churchill MJ, Deacon NJ. A compilation of
cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic
Acids Res. 2000;28(3):663-8.
59. Arts EJ, Hazuda DJ. HIV-1 antiretroviral drug therapy. Cold Spring Harb Perspect
Med. 2012;2(4):a007161.
60. Kirchhoff F, Easterbrook PJ, Douglas N, Troop M, Greenough TC, Weber J, et al.
Sequence variations in human immunodeficiency virus type 1 Nef are associated
with different stages of disease. J Virol. 1999;73(7):5497-508.
61. Casartelli N, Di Matteo G, Argentini C, Cancrini C, Bernardi S, Castelli G, et al.
Structural defects and variations in the HIV-1 nef gene from rapid, slow and non-
progressor children. AIDS. 2003;17(9):1291-301.
62. Shugars DC, Smith MS, Glueck DH, Nantermet PV, Seillier-Moiseiwitsch F,
Swanstrom R. Analysis of human immunodeficiency virus type 1 nef gene
sequences present in vivo. J Virol. 1993;67(8):4639-50.
208
63. Verma S, Ronsard L, Kapoor R, Banerjea AC. Genetic characterization of natural
variants of Vpu from HIV-1 infected individuals from Northern India and their
impact on virus release and cell death. PLoS One. 2013;8(3):e59283.
64. Zhang L, Huang Y, Yuan H, Tuttleton S, Ho DD. Genetic characterization of vif,
vpr, and vpu sequences from long-term survivors of human immunodeficiency
virus type 1 infection. Virology. 1997;228(2):340-9.
65. Nakagawa F, Miners A, Smith CJ, Simmons R, Lodwick RK, Cambiano V, et al.
Projected Lifetime Healthcare Costs Associated with HIV Infection. PLoS One.
2015;10(4):e0125018.
66. Sloan CE, Champenois K, Choisy P, Losina E, Walensky RP, Schackman BR, et
al. Newer drugs and earlier treatment: impact on lifetime cost of care for HIV-
infected adults. AIDS. 2012;26(1):45-56.
67. Martin AR, Siliciano RF. Progress Toward HIV Eradication: Case Reports, Current
Efforts, and the Challenges Associated with Cure. Annu Rev Med. 2016;67:215-
28.
68. Deeks SG. HIV: Shock and kill. Nature. 2012;487(7408):439-40.
69. Dekaban GA, Dikeakos JD. HIV-I Nef inhibitors: a novel class of HIV-specific
immune adjuvants in support of a cure. AIDS Res Ther. 2017;14(1):53.
70. Kerkau T, Bacik I, Bennink JR, Yewdell JW, Hunig T, Schimpl A, et al. The human
immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step
in the biosynthesis of major histocompatibility complex (MHC) class I molecules.
J Exp Med. 1997;185(7):1295-305.
71. Apps R, Del Prete GQ, Chatterjee P, Lara A, Brumme ZL, Brockman MA, et al.
HIV-1 Vpu Mediates HLA-C Downregulation. Cell Host Microbe.
2016;19(5):686-95.
72. Veillette M, Desormeaux A, Medjahed H, Gharsallah NE, Coutu M, Baalwa J, et
al. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by
antibody-dependent cell-mediated cytotoxicity. J Virol. 2014;88(5):2633-44.
73. Veillette M, Coutu M, Richard J, Batraville LA, Dagher O, Bernard N, et al. The
HIV-1 gp120 CD4-bound conformation is preferentially targeted by antibody-
dependent cellular cytotoxicity-mediating antibodies in sera from HIV-1-infected
individuals. J Virol. 2015;89(1):545-51.
74. Richard J, Veillette M, Brassard N, Iyer SS, Roger M, Martin L, et al. CD4
mimetics sensitize HIV-1-infected cells to ADCC. Proc Natl Acad Sci U S A.
2015;112(20):E2687-94.
209
Appendix 1 Copyright permission for published work
Appendix 2 Ethics statement
Informed consent was obtained from all subjects according to an ethics protocol
approved by The University of Western Ontario Research Ethics Board for Health
Sciences Research Involving Human Subjects (HSREB; project ID: 103782; project title:
Nef is an HIV-1 protein at the forefront of pathogenesis).
210
Curriculum Vitae
Emily N. Pawlak
Education
Doctorate of Philosophy
May/2015 – current
Supervisor: Dr. JD Dikeakos
Department of Microbiology and Immunology
Schulich School of Medicine and Dentistry
The University of Western Ontario, London, Ontario, Canada
Master’s of Science
September/2013 – April/2015
Supervisor: Dr. JD. Dikeakos
Department of Microbiology and Immunology
Schulich School of Medicine and Dentistry
The University of Western Ontario, London, Ontario, Canada
Completed PhD candidacy examination with distinction
Bachelor of Medical Sciences (Honors)
September/2009 – April/2013
Honors Specialization in Biochemistry of Infection and Immunity
Schulich School of Medicine and Dentistry
The University of Western Ontario, London, Ontario, Canada
Graduation with Distinction
Western Scholars Designation
Western University Gold Medal Recipient
Dean’s honor list – 2010, 2011, 2012, 2013
Peer-Reviewed Publications
Van Nynatten L.R., Johnson A.L., Dirk B.S., Pawlak E.N., Jacob R.A., Haeryfar S.M. &
Dikeakos J.D. 2018. Identification of Novel Subcellular Localization and Trafficking of
HIV-1 Nef Variants from Reference Strains G (F1.93.HH8793) and H (BE.93.VI997).
Viruses 10(9)
Pawlak E.N., Dirk B.S., Jacob R.A., Johnson A.L., & Dikeakos J.D. 2018. The HIV-1
accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells.
Retrovirology 15:6
211
Jacob R.A., Johnson A.L., Pawlak E.N., Dirk B.S., Van Nynatten L.R., Haeryfar S.M. &
Dikeakos J.D. 2017. The interaction between HIV-1 Nef and adaptor protein-2 reduces
Nef-mediated CD4+ T cell apoptosis. Virology 509:1-10
Dirk B.S., Pawlak E.N., Johnson AL, Van Nynatten LR, Jacob RA, Heit B & Dikeakos JD.
2016. HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis
and recycling. Scientific Reports 6:37021
Dirk B.S., Jacob R.A., Johnson A.L., Pawlak E.N., Cavanagh P.C., Van Nynatten L.,
Haeryfar S.M. & Dikeakos J.D. 2015. Viral bimolecular fluorescence complementation: a
novel tool to study intracellular vesicular trafficking pathways. PLoS One 10(4):e0125619
Pawlak E.N. & Dikeakos J.D. 2015. HIV-1 Nef: A Master Manipulator of the Membrane
Trafficking Machinery. Biochimica et Biophysica Acta General Subjects 1850: 733-741.
Conference Presentations
Pawlak E.N., Dirk B.S., Jacob R.A., Johnson A.L., & Dikeakos J.D. The immune cell
surface receptor CD28 is hijacked by the HIV-1 accessory proteins Nef and Vpu. (10 May
2018) London Health Research Day, London ON (poster presentation)
Pawlak E.N., Dirk B.S., Jacob R.A., Johnson A.L., & Dikeakos J.D. The HIV-1 accessory
proteins Nef and Vpu downregulate the immune cell surface receptor CD28 in CD4+ T
cells. (26-29 April 2018) Canadian Conference on HIV/AIDS Research, Vancouver BC
(selected oral presentation)
Pawlak E.N. & Dikeakos J.D. (24-28 June 2017) The HIV-1 accessory proteins Nef and
Vpu downregulate the cell surface receptor CD28. American Society for Virology Annual
Meeting, Madison WI (selected oral presentation)
Pawlak E.N. & Dikeakos J.D. (6-9 June 2017) The HIV-1 Accessory Proteins Nef and Vpu
Hijack Membrane Trafficking to Downregulate the Immune Cell Surface Receptor CD28.
Canadian Student Health Research Forum, Winnipeg MB (poster presentation, nominated
for participation by Schulich School of Medicine and Dentistry)
Pawlak E.N., Van Nynatten L.R. & Dikeakos J.D. (6-9 April 2017) Investigating the Role
of Membrane Trafficking Regulator Sorting Nexin Proteins in HIV-1 Nef-mediated
Extracellular Vesicle Release. Annual Canadian Conference on HIV/AIDS Research,
Montreal QC (poster presentation)
Pawlak E.N. & Dikeakos J.D. (28 March 2017) The HIV-1 accessory proteins Nef and Vpu
hijack membrane trafficking to downregulate the immune cell surface receptor CD28.
London Health Research Day, London ON (selected oral presentation, *awarded prize)
212
Pawlak E.N. & Dikeakos J.D. (23 September 2016) The HIV-1 accessory protein Nef
hijacks cell surface receptor trafficking. Infection and Immunity Research Forum, London
ON (poster presentation)
Pawlak E.N. & Dikeakos J.D. (29 March 2016) The HIV-1 Nef protein hijacks cell surface
receptor trafficking. London Health Research Day, London ON (poster presentation)
Pawlak E.N. & Dikeakos J.D. (12-16 December 2015) The role of membrane trafficking
regulator sorting nexin proteins in cell surface receptor downregulation mediated by the
HIV-1 accessory protein Nef. American Society for Cell Biology Annual Meeting, San
Diego CA (poster presentation)
Pawlak E.N. & Dikeakos J.D. (6 November 2015) Investigating the role of membrane
trafficking regulator sorting nexin proteins in HIV-1 Nef-mediated cell surface receptor
downregulation. Infection and Immunity Research Forum, London ON (poster
presentation *awarded prize)
Pawlak E.N. & Dikeakos J.D. (11-15 July 2015) Investigating the role of membrane
trafficking regulator sorting nexin proteins in HIV-1 Nef-mediated cell surface receptor
downregulation. American Society for Virology Annual Meeting, London ON (selected
oral presentation)
Pawlak E.N. & Dikeakos J.D. (3 May 2015) Exploring the Role of Membrane Trafficking
Regulator Sorting Nexin Proteins in HIV-1 Nef MHC-I Downregulation. Annual Canadian
Conference on HIV/AIDS Research, Toronto ON (poster presentation)
Pawlak E.N. & Dikeakos J.D. (1 April 2015) A novel protein interaction between the
membrane trafficking regulator SNX18 and the HIV-1 accessory protein Nef. London
Health Research Day, London ON (selected oral presentation)
Pawlak E.N. & Dikeakos J.D. (6 November 2014) Exploring a novel protein interaction
between HIV-1 Nef and the membrane trafficking regulator SNX18 and HIV-1 Nef.
Infection and Immunity Research Forum, London, ON (poster presentation)
Pawlak E.N. & Dikeakos J.D. (18 March 2014) Examining the interaction between the
membrane trafficking regulator SNX18 and HIV-1 Nef. London Health Research Day,
London, ON (poster presentation)
Pawlak E.N. & Dikeakos J.D. (1 November 2013) Molecular dissection of the interaction
between HIV-1 Nef and the membrane trafficking regulator SNX18, Infection and
Immunity Research Forum, London, ON (poster presentation)
Teaching Experience
Microbiology and Immunology 4300A – Clinical Immunology
September – December / 2014; 2015; 2016; 2017
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The University of Western Ontario, London, Ontario Canada
*nominated for a graduate teaching award, 2016 & 2017
Scholarships and Awards
➢ Academic scholarship, Canadian Conference on HIV/AIDS Research, April 2018
➢ Travel award, American Society for Virology, June 2017 ($500)
➢ Oral Presentation Award, London Health Research Day, April 2017 ($600)
➢ Doctoral Excellence Research Award, The University of Western Ontario -
$10,000/year, September/2016- current
➢ Alexander Graham Bell Canada Graduate Scholarship, NSERC - $35,000/year
for three years, May/2016- current
➢ Queen Elizabeth II Scholarship, Ministry of Training, Colleges and Universities
– May/2016 – April/2017 ($15,000)- Declined
➢ Poster prize, Infection and Immunity Research Forum, graduate student over 6
months category, 2015 $100
➢ 2015 Dr. Frederick W. Luney Graduate Entrance Fellowship, $3000
(September 2015)
➢ 2014 Dr. John Robinson Graduate Scholarship, $1300 (September 2015)
➢ Institute Community Support (Spring 2015) Travel Award – Canadian Institutes
of Health Research Institute Community Support (Spring 2015) ($2500)
➢ Ontario Graduate Scholarship (OGS), Ministry of Training, Colleges and
Universities – May/2015 – April/2016 ($15000)
➢ Microbiology and Immunology Graduate Travel Award, Department of
Microbiology and Immunology, Western University – May/2015 ($1000)
➢ Ontario Graduate Scholarship (OGS), Ministry of Training, Colleges and
Universities – May/2014 – April/2015 ($15000 Declined)
➢ Frederick Banting and Charles Best Canada Graduate Scholarship, Canadian
Institutes of Health Research Masters Award – May/2014 – April/2015 ($17,500)
➢ Dr. Frederick W. Luney Graduate Entrance Fellowship, Department of
Microbiology and Immunology graduate entrance scholarship - 2013 ($2000)