identification of putative functional motifs in - UNSWorks

IDENTIFICATION OF PUTATIVE FUNCTIONAL MOTIFS IN

VIRAL PROTEINS ESSENTIAL FOR HUMAN

CYTOMEGALOVIRUS (HCMV) DNA REPLICATION

Heng Giap Woon

Master of Science (Research)

2008

I

ABSTRACT

Human cytomegalovirus (HCMV) is a ubiquitous virus that causes significant morbidity

and mortality in immunocompromised individuals. Although there are prophylactic

treatments available, all current antiviral drugs ultimately target the DNA polymerase,

resulting in the increasing emergence of antiviral resistant strains in the clinical setting.

There is a fundamental need for understanding the role of other essential genes in DNA

replication as a foundation for developing new antiviral treatments that are safe and

which utilize a mechanism of action different to existing therapies. In this study we

looked at six HCMV replication genes encoding for the DNA polymerase accessory

protein (UL44), single stranded DNA binding protein (UL57), primase (UL70), helicase

(UL105), primase-helicase associated protein (UL102), and the putative initiator protein

(UL84) in order to increase our understanding of their role in DNA replication. The aim

of this project was to identify variation within these genes as well as to predict putative

domains and motifs in order to ultimately express and study the functional properties of

the HCMV primase (UL70) through the use of recombinant mutants. Sequencing of

these genes revealed a high degree of conservation between the isolates with amino acid

sequence identity of >97% for all genes. Using ScanProsite software from the Expert

Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs

throughout these HCMV replication genes. In particular, highly conserved putative N-

linked glycosylation sites were identified in UL105 that were also conserved across 33

homologues as well as several unique motifs including casein kinase II phosphorylation

sites (CKII) in UL105 and UL84, a microbodies signal motif in UL57 and an integrin

binding site in the UL102 helicase-primase associated protein. Our investigations have

also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein,

mapped functionally important domains of the UL105 helicase and identified cysteine

motifs that have implications for folding of the UL70 primase. Taken together, these

findings provide insights to regions of these HCMV replication proteins that are

important for post-translation modification, activation and overall function, and this

information can be utilized to target further research into these proteins and advance the

development of novel antiviral agents that target these processes.

II

ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr Gillian Scott, for her guidance and

encouragement throughout the project, and to my co-supervisor, Professor Bill

Rawlinson, for his advice and for giving me the opportunity to work in research. I

would also like to extend my thanks to Professor Andrew Lloyd for his advice and

guidance.

I would also like to thank my colleagues at the Virology Research Lab for their

company, friendship, encouragement, advice and humor. In particular, thanks to Sharon

and Min for the countless conversations over lunch.

Thanks to the people at the UNSW Ramaciotti Centre for their technical aid in this

project.

I would like to give a special thanks to Professor Patrick Tam for his insights and advice

on science research in general and to my parents who have supported me throughout.

Finally I would like to give a very special thanks to Ying, who kept me company on the

long nights in the lab and while writing this thesis.

III

TABLE OF CONTENTS

ABSTRACT………………………………………………………………………….....I

ACKNOWLEDGEMENTS………………………………………………………......II

TABLE OF CONTENTS…………………………………………………………….III

LIST OF TABLES…………………………………………………………………....VI

LIST OF FIGURES……………………………………………………………….....VII

COMMONLY USED ABBREVIATIONS………………………………………..VIII

1 INTRODUCTION............................................................................................... 1

1.1 BIOLOGY....................................................................................................... 1

1.1.1 The herpesviridae .................................................................................. 1

1.1.2 The Cytomegaloviruses.......................................................................... 2

1.2 HCMV EPIDEMIOLOGY................................................................................ 3

1.2.1 Transmission ......................................................................................... 4

1.2.2 Congenital infection............................................................................... 5

1.2.3 Immunocompromised infection and disease outcomes............................ 6

1.2.4 Antiviral treatment................................................................................. 6

1.2.5 Antiviral resistance................................................................................ 8

1.3 HCMV GENOME STRUCTURE AND ORGANIZATION ..................................... 13

1.3.1 Open reading frame organization ........................................................ 13

1.4 CMV GROWTH CYCLE ................................................................................ 14

1.4.1 Cell attachment and viral entry............................................................ 15

1.4.2 HCMV replication genes...................................................................... 16

1.4.3 DNA replication................................................................................... 24

1.4.4 Capsid assembly, maturation and egress.............................................. 25

1.4.5 Latency and reactivation...................................................................... 26

1.5 AIMS AND OBJECTIVES ............................................................................... 26

IV

2 REAGENTS AND EQUIPMENT .................................................................... 28

2.1 VIRAL STOCKS ............................................................................................ 28

2.2 DNA EXTRACTION ...................................................................................... 28

2.2.1 Buffers ................................................................................................. 28

2.2.2 Chenicals............................................................................................. 28

2.2.3 Kits...................................................................................................... 29

2.3 POLYMERASE CHAIN REACTION (PCR) ...................................................... 29

2.3.1 Buffers ................................................................................................. 29

2.3.2 Enzymes............................................................................................... 29

2.3.3 Equipment ........................................................................................... 30

2.3.4 Reagents .............................................................................................. 30

2.4 ELECTROPHORESIS ..................................................................................... 30

2.4.1 Buffers ................................................................................................. 30

2.4.2 DNA Markers ...................................................................................... 31

2.4.3 Equipment ........................................................................................... 31

2.4.4 Reagents .............................................................................................. 31

2.5 DNA SEQUENCING ...................................................................................... 32

2.5.1 Equipment ........................................................................................... 32

2.5.2 Reagents .............................................................................................. 32

2.6 RESTRICTION ENZYMES .............................................................................. 32

2.6.1 Buffers ................................................................................................. 32

2.6.2 Enzymes............................................................................................... 33

2.6.3 Reagents .............................................................................................. 33

2.7 CLONING REAGENTS ................................................................................... 33

2.7.1 Antibiotics ........................................................................................... 33

2.7.2 Cells .................................................................................................... 34

2.7.3 Equipment ........................................................................................... 34

2.7.4 Kits...................................................................................................... 35

2.7.5 Media .................................................................................................. 36

2.7.6 Reagents .............................................................................................. 36

2.7.7 Vectors ................................................................................................ 36

2.8 PROTEIN EXPRESSION AND DETECTION ....................................................... 37

2.8.1 Antibodies............................................................................................ 37

V

2.8.2 Buffers ................................................................................................. 37

2.8.3 Equipment ........................................................................................... 38

2.8.4 Kits...................................................................................................... 38

2.8.5 Protein Markers................................................................................... 38

2.8.6 Reagents .............................................................................................. 39

2.8.7 Solutions.............................................................................................. 39

3 IDENTIFICATION OF PUTATIVE FUNCTIONAL MOTIFS IN VIRAL

PROTEINS ESSENTIAL FOR HUMAN CYTOMEGALOVIRUS DNA

REPLICATION ........................................................................................................ 40

3.1 INTRODUCTION ........................................................................................... 40

3.2 METHODS ................................................................................................... 41

3.2.1 Viral stocks and DNA extraction.......................................................... 41

3.2.2 Polymerase chain reaction (PCR) amplification .................................. 41

3.2.3 DNA sequencing and analysis.............................................................. 44

3.2.4 Phylogenetic analysis .......................................................................... 44

3.2.5 Genbank Accession Numbers ............................................................... 44

3.3 RESULTS ..................................................................................................... 45

3.3.1 Strain variation amongst HCMV isolates ............................................. 45

3.3.2 Prediction of potential motifs ............................................................... 45

3.3.3 Strain variation and identification of protein motifs in UL44 ............... 46



3.3.6 Strain variation and identification of protein motifs in UL102 ............. 67

3.3.7 Strain variation and identification of protein motifs in UL105 ............. 73


3.4 DISCUSSION................................................................................................. 85

4 HCMV PRIMASE EXPRESSION ................................................................... 93

4.1 INTRODUCTION ........................................................................................... 93

4.2 METHODS ................................................................................................... 95

4.2.1 Cloning of UL70 into high-copy pBluescript II primase vector............. 95

4.2.2 Construction of baculovirus entry clone for HCMV primase expression

99

VI

4.2.3 In vitro UL70 primase expression using TNT Quick Coupled

Transcription/Translation ................................................................................. 102

4.3 RESULTS ................................................................................................... 108

4.3.1 UL70 was cloned into pBluescript II high-copy vector ....................... 108

4.3.2 Generating Baculovirus entry clones via BP recombination............... 110

4.3.3 UL70 was cloned into pCITE expression vector ................................. 114

4.3.4 UL70 in vitro expression using the TNT system.................................. 115

4.4 DISCUSSION............................................................................................... 117

5 CONCLUDING DISCUSSION AND FUTURE DIRECTIONS................... 119

6 REFERENCES................................................................................................ 121

VII

LIST OF TABLES

Table 1 Classification of Human herpesviruses and pathological outcomes................... 2

Table 2 Sequenced cytomegalovirus (CMV) genomes................................................... 3

Table 3 Epidemiological seroprevalence of HCMV infection........................................ 4

Table 4 Mutations of UL97 and UL27 antiviral resistance .......................................... 11

Table 5 Mutations of UL54 associated with antiviral resistance .................................. 12

Table 6 Essential DNA replication proteins in HCMV and HSV-1.............................. 40

Table 7 Primers used for PCR amplification of HCMV DNA fragments ..................... 43

Table 8 Sequence variation within HCMV replication genes....................................... 45

Table 9 Summary of the putative motifs identified in the HCMV replication genes..... 46

Table 10 Nucleotide primers used for UL70/pBluescript cloning ................................ 96

Table 11 Nucleotides primers used for UL70/pDONR cloning.................................. 100

Table 12 Nucleotide primers used for UL70/pCITE cloning...................................... 103

VIII

LIST OF FIGURES

Figure 1 Arrangement of the HCMV genome.............................................................. 13

Figure 2 CMV growth cycle........................................................................................ 15

Figure 3 Relative positions of genes investigated in this study .................................... 27

Figure 4 HCMV strain alignments of UL44 ................................................................ 50


Figure 6 Putative zinc finger domain of UL57............................................................. 59


Figure 8 Conserved domains between UL70 homologues. .......................................... 66

Figure 9 HCMV strain alignments for UL102 ............................................................. 72

Figure 10 HCMV strain alignments for UL105 ........................................................... 78

Figure 11 Putative motifs conserved across UL105 homologues ................................. 79

Figure 12 HCMV strain alignments for UL84 ............................................................. 83

Figure 13 Overlapping putative casein kinase-2 (CKII) phosphorylation sites of UL8484

Figure 14 Isolate variation in the HCMV primase-helicase complex. .......................... 91

Figure 15 Insert to vector ratio formula ....................................................................... 96

Figure 16 Overview of cloning UL70 into pBluescript ................................................ 98

Figure 17 pDONR/Zeocin Vector ............................................................................. 100

Figure 18 converting femtomoles to nanograms ........................................................ 101

Figure 19 Overview of UL70/pDONR recombination ............................................... 102

Figure 20 Overview of cloning UL70 into pCITE expression vector ......................... 105

Figure 21 Overview of UL70 in vitro protein expression........................................... 107

Figure 22 pBluescript UL70a .................................................................................... 108

Figure 23 pBluescript UL70b.................................................................................... 109

Figure 24 Screening of entry clone BPR4 ................................................................. 111

Figure 25 The BPR4 truncation removes key motifs from UL70 ............................... 111

Figure 26 Colony PCR screening for entry clone BPR5 ............................................ 112

Figure 27 The BPR5 truncation of UL70................................................................... 113

Figure 28 The BPR8 truncation removes the putative zinc finger from UL70............ 113

Figure 29 UL70/pCITE expression vector................................................................. 114

Figure 30 Expression of UL70 in TNT system .......................................................... 115

Figure 31 Time course expression of UL70 with TNT system................................... 116

Figure 32 Sequence analysis of BPR4 and BPR8 ...................................................... 118

IX

COMMONLY USED ABBREVIATIONS

AIDS Acquired immunodeficiency syndrome

ANGIS Australian National Genomic Information Service

ATP Adenosine triphosphate

bp Base pairs

CDV Cidofovir

CMV Cytomegalovirus

DNA Deoxyribonucleic acid

dNTP Deoxyribonucleoside triphosphate

EBV Epstein-Barr virus

FOS Foscarnet

GCV Ganciclovir

HCMV Human cytomegalovirus

HHV Human Herpesvirus

HIV Human immunodeficiency virus

HSV-1/2 Herpes simplex virus type 1/2

IE Immediate early gene

kbp Kilo-base pairs

MIE Major immediate early gene

mRNA Messenger RNA

NLS Nuclear localization signal

X

OBP Origin binding protein

ORF Open reading frame

PCR Polymerase chain reaction

PEG Polyethylene glycol

RNA Ribonucleic acid

UL

Long unique genome region

US Short unique genome region

vGCV Valganciclovir

VZV Varicella-zoster virus

1

1 Introduction

1.1 Biology

1.1.1 The herpesviridae

The herpesviridae family consists of a large group of host-specific animal viruses that

are ubiquitous to the vertebrate species and in at least one invertebrate (Davison, 2002).

Herpesviruses are defined by their virion morphology, which consists of the core, capsid,

tegument, and envelope (Arvin, 2007). The core consists of a single, linear double-

stranded DNA genome of 125–240 kilo-base pairs (kbp) packaged within an icosahedral

capsid that is imbedded in an inner proteinaceous tegument matrix and an outer

glycoprotein-rich lipid membrane (Davison, 2002). Initial phylogenetic analysis of fish

and mammalian herpesviruses dated the divergence of the Alpha-, Beta- and

Gammaherpesvirinae subfamilies to approximately 180-200 million years ago

(McGeoch, 1995), but the inclusion of avian and reptilian herpesviruses have pushed

back the common ancestor of the herpesvirinae subfamilies to about 400 million years

ago (McGeoch, 2005). Classification into Alpha-, Beta- and Gammaherpesvirinae

subfamilies were based on biological properties such as host range and ability to

establish latent infection in certain cell types. The Alphaherpesvirinae include four

genera (Simplexvirus, Varicellovirus, Mardivirus, and Iltovirus) as well as the reptilian

herpesviruses and are characterized by their relatively rapid replication and ability to

maintain latent infections in sensory ganglia as well as productively infect mucous

membranes and skin tissue (Arvin, 2007). The Betaherpesvirinae include the

Cytomegalovirus, Muromegalovirus, and Roseolovirus lineages and are characterized by

their slower replication rate in vitro relative to other herpesviruses (Arvin, 2007). The

Gammaherpesvirinae are divided into two genera, the Lymphocryptoviridae and

Rhadinoviridae, which primarily infect mammals and are known to induce

lymphoproliferation and tumors in endothelial cells (Arvin, 2007). In addition, there is a

genus (Ictalurivirus) that is unattached to any subfamily as well as a large number of

species not assigned to genera (Arvin, 2007). Currently, there are eight herpesviruses

known to infect humans with pathological consequences (Table 1).

2

The herpesviridae subfamilies share many homologous ‘core genes’ which were

presumably inherited from a common ancestor and are associated with DNA replication,

processing and packaging, capsid assembly and egress, as well as a host of tegument

proteins and surface glycoproteins (Davison, 2002).

Table 1 Classification of Human herpesviruses and pathological outcomes

Common Name Genome Subfamily Pathological Outcomes

(Classification) size (kbp)*

Herpes Simplex Virus 1 (HHV-1) 152 α Predominantly oro-facial lesions,

keratoconjunctivitis

Herpes Simplex Virus 2 (HHV-2) 155 α Predominantly genital lesions

Varicella Zoster Virus (HHV-3) 125 α Chickenpox, shingles

Epstein-Barr Virus (HHV-4) 184 γ Burkitt’s and Hodgkin’s lymphoma

infectious mononucleosis, tumors

Human Cytomegalovirus (HHV-5) ~230 β Vasculature and end-organ disease,

congenital abnormalities, retinitis,

Human Herpesvirus 6 (HHV-6A/B) 159/162 β Exanthem subitum (HHV6A)

Human Herpesvirus 7 (HHV-7) ~149 β Exanthem subitum

Human Herpesvirus 8 (HHV-8) ~141 γ Kaposi’s sarcoma

*in kilobase pairs (Arvin, 2007; Davison, 2002)

1.1.2 The Cytomegaloviruses

The cytomegalovirus genus infects a wide range of mammalian species including

rodents (Rawlinson, 1996; Vink, 2000), primates (Davison, 2003; Hansen, 2003), and

humans (Chee, 1990; Dolan, 2004; Dunn, 2003; Murphy, 2003) with several sequenced

genomes available on public databases (Table 2). The human cytomegalovirus (HCMV)

is the prototype of the betaherpesvirinae subfamily and is the most complex of the eight

human herpesvirus species (Davison, 2003). The first HCMV strains were isolated 50

years ago (Craig, 1957; Rowe, 1956; Smith, 1956) while the genetic content of the virus

has been available for almost two decades (Chee, 1990). However, the mechanism of

HCMV DNA replication and its regulation is not completely understood, with the

function of several genes inferred from studies on the herpes simplex virus (HSV). In

addition, current anti-viral treatments exclusively target only one aspect of the CMV

replication cycle, by competitive inhibition of the DNA polymerase, and with the advent

3

of anti-viral resistance strains, the efficacy of treatment is greatly reduced. Hence other

aspects of the HCMV replication machinery need to be studied in greater detail as a

basis of identifying a broader range of potential anti-viral targets in the future.

Table 2 Sequenced cytomegalovirus (CMV) genomes

Common Name Strain Accession size (kbp) Reference

Human CMV AD169 X17403 229 Chee et al. (1990)

Towne AY315197 231 Dunn et al. (2003)

Merlin AY446894 236 Dolan et al. (2004)

Toledo AC146905 227 Murphy et al. (2003)

Chimpanzee CMV - AF480884 241 Davison et al. (2003)

Rhesus CMV 68-1 AY186194 221 Hansen et al. (2003)

Mouse CMV Smith U68299 230 Rawlinson et al. (1996)

Rat CMV Maastricht AF232689 230 Vink et al. (2000)

1.2 HCMV Epidemiology

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is acquired early in life

in most populations and generally increases in seroprevalence with age. However, the

patterns of acquisition vary greatly based on geographic and socioeconomic

backgrounds such that in developing nations, acquisition of HCMV is nearly universal

in early childhood (Kubo, 1991; Miles, 2007; Prabhakar, 1992). HCMV infection is

endemic in the human population (Table 3) and is not influenced by seasonal variations

(Malm, 2007; Pass, 2005). The seroprevalence of HCMV infection within the adult

population is very common ranging from 60% in developed countries, to 100% in

developing countries (Munro, 2005). In particular, countries in Africa (Adjei, 2006;

Pultoo, 2001), Asia (Kangro, 1994; Kothari, 2002; Liu, 1990; Shen, 1992; Urwijitaroon,

1993; Wong, 2000), and the Eurasian border (Hizel, 1999) tend to have a higher

incidence of HCMV infection with less significant differences between age groups than

the European countries (Alanen, 2005; Andersson-Ellström, 1995; de Ory, 2004;

Hecker, 2004; Kangro, 1994; Knowles, 2005; Natali, 1997), Australia (Seale, 2006),

New Zealand (Beresford, 1988), and the United States (Staras, 2007).

4

Table 3 Epidemiological seroprevalence of HCMV infection

Location Population Age Seropositivity Reference

Study (years) (%)

Europe

Germany Blood donor 18+ 46 Hecker et al. (2004)

Parma, Italy General 2-54 72 Natali et al. (1997)

Finland Women 16-45 56 Alanen et al. (2005)

Madrid, Spain Women 2-40 66 de Ory et al. (2004)

United Kingdom General 12-21 19 Kangro et al. (1993)

Ankara, Turkey Women 15-49 99 Hizel et al. (1999)

Ireland Women 15-46 30 Knowles et al. (2005)

Sweden Women 16 45 Andersson-Ellström et al. (1995)

Asia

Chengdu, China General 4-7 60 Liu et al. (1990)

Taipei, Taiwan General 4-12 58 Shen et al. (1992)

Delhi, India Blood donor 95 Kothari et al. (2002)

Thailand Blood donor 17-59 93 Urwijitaroon et al. (1993)

Hong Kong General 12-21 80 Kangro et al. (1993)

Nepal General 1+ 100 Kubo et al. (1991)

Singapore Pregnant women ≤30 77 Wong et al. (2000)

North America

USA General 6+ 59 Staras et al. (2006)

Cuernavaca, Mexico Women 13-44 92 Echániz-Avilés et al. (1993)

South America

Jamaica General 15-25 90+ Prabhakar et al. (1992)

Santiago, Chile General <30 60 Abarca et al. (1997)

Buenos Aires, Argentina Socioeconomic* <15 46 Damilano et al. (1992)

Africa

Ghana Blood donor 20-69 93 Adjei et al. (2006)

Gambia General 1 85 Miles et al. (2007)

Mauritius Blood donor 18-60 94 Pultoo et al. (2001)

Australia General 1-59 57 Seale et al. (2006)

New Zealand Blood donor 56-65 65 Beresford et al. (1988)

*Population study based on middle-socioeconomic classes

1.2.1 Transmission

HCMV infection is acquired through direct contact with body fluids from an infected

person and is present in saliva, urine, breast milk, blood products, semen, cervical and

vaginal secretions, as well as allograft tissues (Arvin, 2007). In a hospital environment,

allograft recipients are among the highest at risk to HCMV infection, with over 75% of

5

solid organ transplant patients contracting or reactivating latent CMV after

transplantation (Fishman, 1998). Transfusion associated HCMV infections were first

described 40 years ago (Kääriäinen, 1966) and is attributed to the virus’s ability to

establish latency in cells of the myeloid lineage (Cervia, 2007; Miller, 1991). In a child-

care environment, horizontal transmission of HCMV is common amongst young

children, parents and other care-givers from the community (Adler, 1988; Adler, 1989;

Bale, 1999; de Mello, 1996; Kiss, 2002; Pass, 1990; Pass, 1984) while in young adult

populations, sexual activity is the major mode of transmission for CMV (Handsfield,

1985). The presence of infectious virus is common in salivary glands and cervical

secretions (Britt, 1996; Shen, 1994). HCMV has also been cultured from semen despite

being urine, blood, and saliva negative for infectious virus (Biggar, 1983; Lang, 1975).

In addition, artificial insemination therapy has been suggested as another route of

transmission (Mansat, 1997; Prior, 1994), with HCMV detected in 0.02% to 34% of

donor semen depending on the population study (Mansat, 1997; McGowan, 1983; Shen,

1994a; Tjiam, 1987; Yang, 1995).

1.2.2 Congenital infection

HCMV is the leading cause of congenital viral infection, occurring in 0.15-3.0% of

newborns worldwide (Hassan, 2007; Malm, 2007). Infection can occur via viral

transmission through the placenta, during delivery via cervical secretions and blood or

from the mother via breast milk (Malm, 2007). The risk for viral transmission is higher

in primary infected mothers than in mothers with reactivated disease (Gaytant, 2002).

Primary CMV infections are reported in 1-4% of seronegative women during pregnancy

and the risk for viral transmission to the fetus is 30-40% (Stagno, 1986; Stagno, 1982)

while reactivation of a CMV infection during pregnancy is reported in 10-30% of

seropositive women but the risk of transmitting the virus is only 1-3% (Stagno, 1982).

Approximately 30% of those infected will develop symptomatic disease (Ahlfors, 1999),

although only 10-15% of children with congenital CMV are symptomatic during the

neonatal period (Malm, 2007). Hearing loss is the most common sequela of congenital

CMV infection, occurring in 10–15% of infected children (Dahle, 2000; Dollard, 2007;

Pass, 2005; Ross, 2006) while other neurological deficits such as mental retardation,

autism, learning disabilities, cerebral palsy, epilepsy, visual impairment, microcephaly,

encephalitis, seizures, and upper motor neuron disorders may also develop (Gandhi,

6

2004; Ross, 2005). The most severely affected infants have a mortality rate of about

30% with death occurring as a result of hepatic dysfunction, bleeding, disseminated

intravascular coagulation or secondary bacterial infections (Malm, 2007).

1.2.3 Immunocompromised infection and disease outcomes

HCMV infection often remains asymptomatic within an immunocompetent host,

whereas it may cause significant morbidity and mortality in immunocompromised

individuals such as transplant patients and HIV/AIDS patients (Arvin, 2007). Over 75%

of solid organ transplant patients are newly infected or reactivate latent CMV after

transplantation (Fishman, 1998), often within the first 3 months when

immunosuppression is most intense (Dummer, 1983). CMV disease manifests in the

vast majority of transplant recipients as a viral syndrome that includes fever, malaise,

muscle pain or headache (Steininger, 2007) and has been associated with diminished

graft survival and allograft rejection (Grattan, 1989; Pouteil-Noble, 1993; Ricart, 2005).

In HIV-infected patients, retinitis is the single most common manifestation of CMV

disease, accounting for 85% of all cases (Yust, 2004). CMV infection has also been

linked to the onset of diabetes mellitus in kidney transplant recipients (Hjelmesaeth,

2004) as well as accelerated cardiac allograft vasculopathy in heart transplant recipients

(Lemström, 1995).

1.2.4 Antiviral treatment

Currently, only five compounds have been licensed to treat established HCMV

infections: ganciclovir (GCV), its oral pro-drug, valganciclovir (vGCV), Foscarnet

(FOS), cidofovir (CDV) and fomivirsen (Mercorelli, 2007). GCV is a nucleoside analog

commonly used to treat CMV disease and in particular, CMV retinitis in the

immunocompromised (De Clercq, 2001). GCV requires intracellular phosphorylation,

firstly by the UL97 viral kinase and then by cellular kinases to create a triphosphate

active form that acts as a competitive inhibitor of deoxyguanosine triphosphate,

incorporating itself into the viral DNA via the DNA polymerase during elongation to

disrupt DNA synthesis (Abdel-Haq, 2006). Valganciclovir is an inactive pro-drug form

of GCV that is rapidly converted to active GCV in the plasma (Abdel-Haq, 2006) and

has a ten-fold higher bioavailability than GCV (Cocohoba, 2002; Pescovitz, 2000),

7

which is normally administered intravenously. Hematologic toxicities such as

neutropenia, anemia, and thrombocytopenia are among the most frequently reported

adverse side effects (Abdel-Haq, 2006; Mercorelli, 2007) while nausea, diarrhea,

vomiting, fever, and abdominal pain have also been reported during GCV and

valganciclovir treatment (Cocohoba, 2002). CDV is an acyclic nucleoside phosphonate

with a mechanism of action similar to that of other nucleoside analogs, but unlike GCV,

is not dependent on activation by a viral encoded enzyme (Mercorelli, 2007). Instead,

host kinases convert CDV to the active diphosphoryl form, and CDV disphosphate then

acts as a competitive inhibitor of the viral DNA polymerase, causing premature chain

termination in viral DNA synthesis (Biron, 2006). Although CDV is approved for use to

treat CMV retinitis in AIDS patients, severe nephrotoxicity is a major limitation of its

use (Biron, 2006; Ho, 2000; Mercorelli, 2007) and thus remains a second-line therapy

option (Biron, 2006). In addition, it has low oral bioavailability and is only available as

an intravenous treatment (Mercorelli, 2007). Neutropenia is another toxicity associated

with CDV, and CDV was shown to be both carcinogenic and teratogenic in preclinical

toxicological studies (Biron, 2006). FOS is a pyrophosphate analogue that inhibits the

viral DNA polymerase by binding to the pyrophosphate binding site and blocking

cleavage of pyrophosphate from the terminal nucleoside triphosphate added to the

growing DNA chain (Biron, 2006; Wagstaff, 1994). FOS is also considered a second-

line therapy due to its nephrotoxic potential (Biron, 2006; Mercorelli, 2007), but is the

preferred drug for patients who are failing GCV therapy due to viral resistance, or those

who cannot be treated with GCV due to dose-limiting neutropenia or leucopenia

(Razonable, 2004). Fomivirsen is a 21-nucleotide anti-sense RNA (5’-GCG TTT GCT

CTT CTT CTT GCG-3‘) (De Clercq, 2001), specifically targeted against the major

immediate-early (MIE) transactivator gene of CMV (Geary, 2002). As it is administered

only as an intraocular injection, it is an end-organ treatment that does not affect

systemic CMV (Arvin, 2007). The most commonly reported adverse effect associated

with fomivirsen is ocular inflammation, which occurs in 25% patients (Group., 2002),

with other less common side-effects including abnormal vision, cataracts, bleeding in

and around the eye, reduced color vision, eye pain, retinal detachment, stomach pain,

low blood count, weakness, dehydration, cough, flu-like symptoms, and chest pain

(Group., 2002).

8

In addition to these licensed drugs, several anti-CMV drugs are also currently in clinical

development. Maribavir (MBV), (1-(β-L-ribofuranosyl)-2-isopropylamino-5, 6-

dichlorobenzimidazole), is a riboside analog with a novel mechanism of action that is

being developed to inhibit viral DNA synthesis in CMV and EBV (Biron, 2002; Zacny,

1999). In CMV-infected cells, Maribavir targets the HCMV protein kinase gene (UL97)

that has been shown to play a role in viral nucleocapsid egress from the nucleus, thereby

reducing the yield of infectious CMV (Evers, 2004; Krosky, 2003a). Its mechanism of

action is not fully understood although it has been hypothesized that inhibition of viral

DNA synthesis is a consequence of preventing the phosphorylation of the DNA

polymerase accessory protein (pUL44)by the protein kinase (Krosky, 2003; Marschall,

2003). In addition, laboratory-generated resistant mutations have been mapped to the

UL27 gene, a protein of unknown function (Chou, 2004; Komazin, 2003). Maribavir

pre-clinically shows advantages over existing anti-CMV drugs in its in vitro potency,

bioavailability, safety profile in acute, chronic and genetic toxicology testing, and the

lack of cross-resistance with GCV, FOS, and CDV resistant strains (Biron, 2006; Drew,

2006).

BAY 38-4766 represents a novel class of non-nucleoside antiviral agents that are highly

selective inhibitors of CMV in vitro (Reefschlaeger, 2001). The compound

demonstrates antiviral activity similar to that of GCV (Weber, 2001) and showed a

favorable safety profile in healthy male volunteers at single oral doses up to 2000 mg

(Biron, 2006). The mechanism of action is largely unknown although resistance

mutations have been mapped to the UL89 and UL56 genes (Krosky, 1998;

Reefschlaeger, 2001), which encode the two terminase subunit proteins involved in

DNA cleavage and packaging (Bogner, 2001). It is also active against strains that are

resistant to GCV (McSharry, 2001) and does not have cross-resistance with other

current antiviral drugs (Reefschlaeger, 2001).

1.2.5 Antiviral resistance

Resistance to antiviral treatment mainly occurs under selective pressure from lengthy

periods of antiviral treatment, although other risk factors including host immune

competence and levels of ongoing viral replication are also involved (Chou, 2001). In

addition, currently approved drugs for treatment share a similar mechanism of action by

9

targeting the DNA polymerase, hence increasing the risk of mutations that confer

resistance to multiple anti-viral drugs. Antiviral resistant CMV strains may emerge

between 6 weeks to 2 months of treatment depending on the patient group (Boivin, 2001;

Eckle, 2002; Limaye, 2000; Springer, 2005) although low levels of resistant genotypes

are present in patients even before antiviral treatment commences (Emery, 2000).

Antiviral resistance can be determined by genotypic or phenotypic assays. Phenotypic

assays measure the concentration of an antiviral agent necessary to inhibit viral

replication while genotypic assays, screen for key resistance mutations in the UL97 and

UL54 genes (Springer, 2005). These two gene products encode for the CMV protein

kinase and DNA polymerase respectively. Point mutations or deletions of portions of

UL97 can lead to GCV resistance (Baldanti, 1995; Baldanti, 1998; Chou, 1995; Chou,

1997; Chou, 2000a; Chou, 2005; Chou, 2002; Faizi-Khan, 1998; Hanson, 1995; Hantz,

2005; Ijichi, 2002; Lurain, 1994; Marfori, 2007; Sullivan, 1992; Wolf, 1995) (Table 4),

while UL54 mutations, depending on their locations, can confer resistance to one or

more anti-viral drugs (Chou, 2003; Chou, 1997; Chou, 1998; Chou, 2007a; Cihlar, 1998;

Cihlar, 1998a; Ducancelle, 2006; Gilbert, 2005a; Marfori, 2007; Mousavi-Jazi, 2001;

Mousavi-Jazi, 2003; Scott, 2007; Springer, 2005; Weinberg, 2003) (Table 5).

UL97 GCV resistance mutations are clustered at codons 460, 520, and 590-607 (Chou,

1999), but the degree of resistance varies depending on the mutation or combination of

mutations present. For example, the most commonly encountered mutations in UL97,

A594V, L595S, and M460V, confer a 5-10 fold increase resistance to GCV while

C592G and A594T confer only a 2-3 fold increase resistance to GCV (Chou, 1999;

Chou, 2005). Maribavir resistance mutations have also been mapped to the UL97 gene

(Biron, 2002; Chou, 2007), although the affected codons are clustered upstream of the

known GCV resistance mutations and share no cross-resistance with GCV (Chou, 2007).

In addition to GCV, UL54 mutations are known to confer varying degrees of resistance

to FOS and/or CDV as well. Among the most frequent mutations associated with

resistances to these drugs are V715M, V781I, and L802M, which confer resistance to

FOS (Cihlar, 1998), F412C, L501I, and P522S, which confer resistance to GCV and

CDV (Chou, 1997; Cihlar, 1998), and A809V, which confer resistance to GCV and

FOS (Chou, 1998). In addition, an increasing number of GCV and FOS cross-resistant

strains have been observed in several laboratory and clinical isolates (Biron, 2006;

Mercorelli, 2007) while there have also been cases of mutations that confer resistance to

10

all three anti-CMV drugs (Chou, 2000; Scott, 2007). The issue of cross-resistance

arising from the above mentioned factors poses a need to develop drugs with a novel

mechanism of action to use with patients not responding to current treatment or in

conjunction with existing anti-viral drugs to reduce the incidence of resistance.

11

Table 4 Mutations of UL97 and UL27 antiviral resistance

Gene Mutation Domain Resistance* Reference

UL97 V353A --- MBV Biron 2002, Chou 2007

L397R --- MBV Biron 2002

T409M --- MBV Biron 2002, Chou 2007

H411 --- MBV Chou 2008

M460V VI GCV Chou 1995

M460I VI GCV Lurain 1994

H520Q --- GCV Hanson 1995

590-93 deletion IX GCV Sullivan 1992

A591V IX GCV Chou 2002

591-607 deletion IX GCV Chou 2002

C592G IX GCV Chou 2005

A594V IX GCV Chou 1995

A594P IX GCV Ijichi 2002

A594T IX GCV Chou 2002

L595S IX GCV Chou 1995

L595F IX GCV Wolf 1995

L595W IX GCV Chou 2002

L595 deletion IX GCV Baldanti 1995

595-603 deletion IX GCV Chou 2000a

E596G --- GCV Chou 2002

K599T --- GCV Faizi-Khan 1998

601 deletion --- GCV Hantz 2005

601-03 deletion --- GCV Marfori 2007

C603W/R/S --- GCV Chou 1997

C607Y --- GCV Baldanti 1998

C607F --- GCV Chou 2002

UL27 L355P --- MBV Komazin 2003

R233S --- MBV Chou 2004

W362R --- MBV Chou 2004

A406V/415Stop --- MBV Chou 2004

*GCV = Gancyclovir, MBV = Maribavir

12

Table 5 Mutations of UL54 associated with antiviral resistance

Gene Mutation Domain Resistance* Reference

UL54 D301N EXOI GCV Chou 2003

N408D IV GCV, CDV Cihlar 1998

N408K IV GCV, CDV Scott 2007

N410K IV GCV, CDV Chou 2003

F412C/V IV GCV, CDV Chou 1997, Cihlar 1998

D413E or A IV GCV, CDV Chou 2003

D413A IV GCV, CDV Marfori 2007

T419M IV FOS Mousavi 2003

N495K δ-C FOS Ducancelle 2006

L501I δ-C GCV, CDV Cihlar 1998

T503I δ-C GCV, CDV Chou 2003

K513N or E δ-C GCV, CDV Cihlar 1998a

L516R δ-C GCV, CDV Chou 2003

P522S δ-C GCV, CDV Cihlar 1998

L545S δ-C GCV, CDV Cihlar 1998

Q578H δ-C FOS Mousavi 2003

D588N or E δ-C FOS Mousavi 2001, Cihlar 1998

A692S --- FOS Chou 2003

T700A II FOS Cihlar 1998

V715M II FOS Cihlar 1998

E756K --- GCV, CDV, FOS Mousavi 2001, Chou 2003

E756D or Q --- FOS Chou 2003, Weinberg 2003

L773V VI FOS Mousavi 2003

V781I VI GCV, FOS Cihlar 1998, Mousavi 2001

V787L VI FOS Weinberg 2003

L802M III FOS Cihlar 1998, Chou 1997

K805Q III CDV Cihlar 1998

A809V III GCV, FOS Chou 1998

V812L III GCV, CDV, FOS Chou 1997, Cihlar 1998a

T813S III GCV, FOS Chou 2007a

T821I III GCV, CDV, FOS Cihlar 1998

P829S III FOS Gilbert 2005a

A834P III GCV, CDV, FOS Scott 2007

T838A III FOS Springer 2005

G841A III GCV, FOS Chou 2007a

981-82 deletion V GCV, CDV, FOS Chou 2000

A987G V GCV, CDV Cihlar 1998

*GCV = Ganciclovir, CDV = Cidofovir, FOS = Foscarnet

13

1.3 HCMV genome structure and organization

Human cytomegalovirus has the largest genome of all the herpesviruses with a linear

double stranded DNA genome of 230-240kbp. It is also the only known betaherpesvirus

to have a class E genome structure that consists of a long unique (UL) and short unique

(US) sequence capable of inverting to give four sequence isomers (Gibson, 1999). Each

unique region is flanked by inverted repeat sequences (TRL and IRL, TRS and IRS), such

that the overall structure of the HCMV genome is TRL-UL-IRL-IRS-US-TRS (Mocarski,

2001) (Figure 1). Large scale sequencing analysis has revealed the existence of seven

conserved sequence blocks present in all herpesviruses, but arranged in different orders

(Chee, 1990). These conserved regions were found to have a functional role in DNA

replication, DNA repair, nucleotide metabolism, and virion structure (Mocarski, 2001).

Figure 1 Arrangement of the HCMV genome

1.3.1 Open reading frame organization

Initial analysis of the fully sequenced AD169 laboratory strain predicted the existence

of 208 open reading frames (ORFs) (Chee, 1990), including 33 ORFs that have

substantial sequence similarity to HSV-1, varicella zoster virus (VZV), and Epstein-

Barr virus (EBV) (Chee, 1990), as well as 41 ORFs that are dispensable for replication

(Spaete, 1987). Several studies have proposed their models for a consensus genome

(Chee, 1990; Davison, 2003; Murphy, 2003), while various interpretations to the coding

potential of AD169 (Chee, 1990) has led to several revisions of its genomic content.

Sequence comparisons with the closely related chimpanzee cytomegalovirus (CCMV)

discounted 51 previously identified ORFs, re-interpreted 24 ORFs, as well as predicting

the existence of 10 novel genes, including five in AD169 (UL15A, UL21A, UL128,

UL131A, and US34A) (Davison, 2003). Another study, using the Bio-Dictionary-based

Gene Finder (BDGF) analysis tool, found that 37 ORFs from the original AD169

sequence can be discarded as non-polypeptide encoding as well as the addition of up to

14

12 ORFs that had not been previously identified (Murphy, 2003). In addition, sequence

differences have been pointed out in ORF UL102 (Smith, 1995a), US28 (Neote, 1993),

and UL15 (Davison, 2003). Certain stocks of AD169 were also found to contain an

additional 929 base pairs that were not in the original sequence of AD169 and which

resulted in alterations to the UL42 and UL43 genes (Dargan, 1997; Mocarski, 1997) as

well as a reinterpretation of the UL41 gene (Dargan, 1997). Several ORFs have also

been modified to account for splicing events, including UL111A, UL118, UL119, UL22,

US3 (Rawlinson, 1993), and UL33 (Davis-Poynter, 1997). Furthermore, various regions

of the genome are known to be hypervariable among clinical isolates (Prichard, 2001).

These include: three major genotype variations in UL11 (Hitomi, 1997), UL144 (Lurain,

1999), four major genotype variations in US9, US28 (Rasmussen, 2003), UL4 (Bar,

2001), UL73 (Dal Monte, 2004; Pignatelli, 2001), UL55 (Chou, 1991; Meyer-König,

1998), and five major genotype variations in UL37 (Hayajneh, 2001), as well as several

associated with UL146 (Hassan-Walker, 2004). Nevertheless, laboratory strain AD169

is still widely regarded as the consensus strain for interpretation of coding.

1.4 CMV growth cycle

HCMV generally replicates slowly in cell culture, taking days for cytopathic effects to

appear (Britt, 1996). However, in vivo, CMV has been observed to have a doubling time

of approximately 1-2 days, depending on the methods used (Emery, 1999). The HCMV

growth cycle consists of cell attachment and viral entry, transport to the nucleus,

transcription and translation of viral proteins, DNA replication, capsid assembly, and

maturation and egress from the cell (Figure 2). The CMV replication cycle has not been

completely elucidated, although it shares numerous homologies to HSV-1 replication

(Boehmer, 2003).

15

Figure 2 CMV growth cycle

1. Cell attachment and viral entry; 2. Transport to the nucleus; 3. Transcription/translation of viral

proteins; 4. DNA replication; 5. Capsid assembly; and 6. Maturation and egress

1.4.1 Cell attachment and viral entry

HCMV cell attachment and penetration of the host cell occurs by fusion with the plasma

membrane (Compton, 2004) and is mainly mediated by envelope glycoproteins (Arvin,

2007; Mach, 2005). Entry of HCMV involves the interaction of these envelope

glycoproteins with a number of distinct receptors, initiated by the use of the cell surface

proteoglycan heparan sulfate by glycoprotein B (gB) (Compton, 1993). HCMV gB

(UL55) and the gH:gL (UL75:UL115) complex have been the most extensively studied

(Baldwin, 2000; Feire, 2004; Lopper, 2004; Navarro, 1993) while recent evidence

suggests core envelope glycoproteins gM (UL100) and gN (UL73) may also be

important for viral entry (Mach, 2005). HCMV and other herpesviruses exploit normal

cytoplasmic transport systems of the host (Dohner, 2005; Ogawa-Goto, 2003) to control

nucleocapsid transit through the cytoplasm. It relies on microtubules to gain access to

the nucleus and nuclear pores where un-coating is completed and the viral genome is

released into the nucleoplasm (Dohner, 2005). The importance of essential protein-

16

protein interactions in receptor recognition and signaling for viral attachment and entry

underscores the potential for developing drugs which target other aspects of the CMV

replication cycle.

1.4.2 HCMV replication genes

HCMV replication is associated with eleven loci that have been identified as necessary

for transient complementation of oriLyt-dependent DNA replication (Pari, 1993). Six of

these were found to encode homologs of herpes simplex virus (HSV-1) DNA replication

proteins (Anders, 1996; Pari, 1993), including: a two-subunit DNA polymerase (UL54

and UL44), a single-stranded DNA binding protein (UL57), a primase (UL70), a

helicase (UL105), and a primase-associated factor (UL102). In addition to these six core

replication genes, five other loci supplemented in regulatory and transactivation roles.

The UL36-38 locus is involved in the up-regulation of viral transcription, inhibition of

apoptosis, and growth in human cells (Colberg-Poley, 1996; Goldmacher, 1999; Pari,

1993a; Smith, 1995; Terhune, 2007), while the four spliced variants of UL112-113

cooperate with each other to relocate UL44 and possibly other core replication proteins

to the pre-replication foci (Iskenderian, 1996; Park, 2006). The TRS1/IRS1 locus is

involved in the disruption of host cell antiviral response pathways, the production of

viable virus, as well as having a regulatory function for the other immediate early genes

(Adamo, 2004; Blankenship, 2002; Child, 2004; Hakki, 2005; Romanowski, 1997). The

major immediate-early region (IE1/IE2) plays a role in regulating the cell cycle,

metabolism and apoptosis (Chiou, 2006; Lukac, 1999; Yu, 2002; Zhu, 1995). Although

not considered to be part of the core set of replication machinery proteins, UL84 has

been shown to have an essential role in oriLyt-dependent DNA replication (Sarisky,

1996; Xu, 2004). It is also involved in promoting the formation of replication

compartments (Sarisky, 1996; Xu, 2002) as well as interacting with IE2 to regulate

transactivation activity in the UL112-113 locus (Gebert, 1997; Spector, 1994).

1.4.2.1 DNA polymerase (UL54)

HCMV encodes a two-subunit DNA polymerase which comprises a catalytic subunit,

UL54 (Heilbronn, 1987) and an accessory protein, UL44, which acts as a processivity

factor (Ertl, 1992). Initial studies revealed that UL54 possesses DNA-dependent DNA

17

polymerase activity (Mar, 1981) as well as 3’-5’ exonuclease activity (Nishiyama, 1983)

and was highly homologous to the UL30 protein of HSV-1 (Gottlieb, 1990; Hernandez,

1990). UL54 shares a series of conserved regions and structural domains with the α-

family of DNA polymerases. These conserved regions are located within amino acid

residues 379–1100 and include conserved domains I–VII, which are involved in the

DNA polymerisation reaction (Hwang, 1992; Wong, 1988), the δ-region C (Zhang,

1991) and three motifs designated Exo I–III which are located within conserved

domains IV and δ-region C (Bernad, 1989). Mutations conferring resistance to GCV,

CDV, and FOS map to several of these conserved motifs (Lurain, 1992; Sullivan, 1993)

while mutations induced in these regions leading to a loss of function (Ye, 1993)

demonstrate the importance of these conserved domains. In particular, domains I, II and

III have been shown to directly participate in binding to deoxynucleoside triphosphates,

in chelating Mg2+

ions and in interacting with primer and template (Ye, 1993) while it

has been suggested that domains III, V, and δ -region C can also form part of the dNTP-

binding site (Ye, 1993). The region involved with interacting with the UL44 subunit has

been mapped to C terminal residues 1221-1242 of UL54 and appears to be dependent on

hydrophobic interactions (Loregian, 2003).

1.4.2.2 DNA polymerase accessory protein (UL44)

Initial studies on the HCMV DNA polymerase (UL54) and its accessory subunit (UL44)

were based on the HSV-1 equivalent, UL30 and UL42. UL54 is a functional homolog of

the HSV-1 UL30 DNA polymerase, both sharing polymerase activity (Mar, 1981) as

well as 3’-5’ exonuclease activity (Nishiyama, 1983) that are dependent on salt

concentration (Cihlar, 1997; Weiland, 1994). UL44 is analogous to the HSV-1

processivity factor UL42 (Gottlieb, 1990), functioning to bind double-stranded DNA

and stimulating DNA synthesis by the main catalytic subunit UL54 (Appleton, 2004;

Ertl, 1992; Loregian, 2004a; Weiland, 1994). The observation that both UL54 and UL44

are required for DNA synthesis and that disruption to either subunit severely inhibits

viral DNA replication (Digard, 1993; Pari, 1993a; Ripalti, 1995) provides a strong basis

to study the UL54/UL44 interaction. The UL44 binding site on UL54 had previously

been elucidated to the C-terminal 22 amino-acids of UL54 with two cysteine residues

suggested to play a key role in binding or stabilizing the UL54/UL44 interaction

(Loregian, 2004; Loregian, 2004a; Loregian, 2003). Alanine mutants of the C-terminal

18

region in UL54 further identified Leu1227 and Phe1231 as key residues for subunit

interaction (Loregian, 2004). Although the HSV-1 homolog interaction between

UL30/UL42 had been mapped to an analogous region earlier (Monahan, 1993; Zuccola,

2000), neither UL30 nor UL54 shared any sequence similarity with each other

(Loregian, 2005).

Analysis of residues 1-290 from the UL44 crystal structure revealed an overall fold that

was similar to other processivity factors including UL42 of HSV-1 and the proliferating

cell nuclear antigen (PCNA) of eukaryotic DNA polymerases, despite sharing no

obvious sequence similarity (Appleton, 2004). Crystallization of the N-terminal two-

thirds of UL44 revealed a C-clamp-shaped head-to-head homodimer (Appleton, 2004)

in contrast to UL42, which carries out its functions as a monomer (Gottlieb, 1990;

Randell, 2004; Zuccola, 2000). Analysis of the dimer binding region revealed specific

residue interactions involving six main-chain-to-main-chain hydrogen bonds and

extensive packaging of hydrophobic side chains at the interface (Appleton, 2004). At

the binding interface, F121 of each monomer is buried against a hydrophobic loop

composed of P85, L86 and L87 of the other monomer. L86 and L87 also pack against

M123 and L93 of the opposite monomer (Appleton, 2004). Furthermore, L86 and L87

are conserved as hydrophobic residues in UL44 homologs of other β-herpesvirus

(Appleton, 2004), suggesting an importance in specific binding mechanisms. Alanine

substitutions of the two lysine residues in the hydrophobic loop disrupted DNA binding

by up to 100 fold (Appleton, 2004; Loregian, 2005).

In addition, UL44 shares a ‘connector loop’ structure similar to UL42 which was shown

to connect two topologically similar domains of UL42 (Zuccola, 2000) as well as

playing a crucial role in interacting with UL30 (Bridges, 2001). Indeed, recent studies

have shown that the connector loop in UL44 has functional similarities to its homolog

in HSV-1 (Appleton, 2004; Loregian, 2004). Constructed alanine mutants of the

connector loop region showed that substitutions that affected residues 133-136 severely

reduced physical and functional interaction with UL54 while a substitution at residue

I135 disrupted UL54/UL44 binding and long-chain DNA synthesis completely

(Appleton, 2004; Loregian, 2004). Similar studies done with the UL42 connector loop

identified the glutamine residue at position 171 as crucial for subunit interaction

(Bridges, 2001). However, subtle differences between the two systems have also been

19

described, suggesting that despite the similarities in function and analogous positioning,

the mechanism of binding is probably different. Firstly, the residues important for

binding the two HCMV subunits are hydrophobic while their counterparts in the HSV-1

interaction are hydrophilic (Bridges, 2001; Loregian, 2004; Zuccola, 2000). This is

further supported by findings that the accessory subunit of HSV-1 cannot stimulate the

catalytic subunit of HCMV, and vice-versa (Loregian, 2005). Secondly, despite the

substitution of residue Q171 in UL42, weak binding could still be detected in maltose

binding protein (MBP)-pulldown assays (Bridges, 2001), whereas substitution of the

I135 residue in UL44 abolished binding completely (Loregian, 2004). The observation

that even a single substitution can disrupt key interactions to such an extent has

provided some insight into possible anti-viral treatments such as the use of small

inhibitory molecules (Loregian, 2004; Pilger, 2004).

Although the crucial functions of UL44 had been mapped to the N-terminal two-thirds

of the gene, the region beyond residue 290 has been less well defined functionally.

Earlier studies had determined that the C-terminus of the gene was dispensable for

UL44 activity (Loregian, 2004a; Weiland, 1994), despite the presence of salient

glycine-rich strings (Ertl, 1992). However, more recent studies suggest the C-terminus

to be involved in nuclear transport with the key nuclear localization site (NLS) located

at residues 425-431 of the C-terminus (Alvisi, 2005). This is consistent with similar

studies on nuclear localization sites flanked by protein kinase phosphorylation sites of

other homologous herpesviruses (Loh, 1999; Takeda, 2000). Apart from its function as

the polymerase processivity factor, UL44 has also been associated with mediating cell

adhesion via a putative integrin binding RGD motif that has been studied in murine

CMV (Loh, 2000).

1.4.2.3 Single-stranded DNA binding protein (UL57)

HCMV UL57 encodes an early single-stranded DNA-binding protein that is

homologous to the HSV-1 major DNA-binding protein, ICP8 (Anders, 1996; Pari,

1993). ICP8 has been studied more extensively than UL57 and as such, much of our

understanding of UL57 is based on observations derived from studies on ICP8. Studies

in the HSV-1 major DNA-binding protein ICP8 have been linked to multiple functions

within the HSV replication cycle. Its ability to modulate activity of the HSV-1 DNA

20

polymerase (Hernandez, 1990), affect genome recombination and processing (Bortner,

1993; Dutch, 1993; Nimonkar, 2003), regulate late viral gene expression (Chen, 1996;

Gao, 1989; Gao, 1991), organize DNA replication enzymes into nuclear replication

compartments (Bush, 1991; de Bruyn Kops, 1988) as well as interactions with the

helicase-primase complex (Boehmer, 1998; Boehmer, 1993; Crute, 1991; Falkenberg,

1998; Hamatake, 1997; He, 2001; Lee, 1997; Makhov, 1996) provides an insight into

potential functional parallels that may be found with its HCMV counterpart. However, it

has already been established that like ICP8, UL57 serves as an essential component for

transient complementation in HCMV oriLyt-mediated DNA replication (Pari, 1993;

Sarisky, 1996). Physical aspects of the ICP8 protein have also been characterized to a

certain extent. Cooperative DNA binding has been mapped to the C-terminus of ICP8

(Dudas, 1998; Mapelli, 2000) with the possible involvement of two cysteine residues at

245 and 455 playing a role in modulation (Dudas, 1998). The DNA binding region has

been harder to elucidate with studies mapping regions which range from residues 564-

1160 (Gao, 1989), 300-849 (Wang, 1990), and 368-902 (White, 1999) as potential

active sites. Several other functional regions have been mapped including: a nuclear

localization signal at the C-terminal 28 amino acid residues (Gao, 1992), an intranuclear

localization site located in the region between residues 1080-1135 (Taylor, 2003), and a

putative zinc binding motif between residues 499-512 (Gao, 1988; Gupte, 1991).

UL57 is located about 1kb downstream of oriLyt, raising the possibility that UL57

promoter elements contribute to oriLyt function (Kiehl, 2003). Indeed, mapping the 5’

ends of the UL57 transcripts elucidated a 42 base-pair sequence spanning the oriLyt-

proximal start site for UL57 that was found to play a role for both UL57 transcription

and oriLyt function, albeit not an essential one for the latter (Kiehl, 2003). Furthermore,

the 1kb non-coding region between oriLyt and UL57 consists of numerous potential

transcriptional control signals, including transcription factor binding sites and

polyadenylation signals, all of which have been shown to participate in replicator

functions in other herpesvirus systems (Nguyen-Huynh, 1998; Schepers, 1993).

1.4.2.4 Primase-helicase complex (UL70, UL102, and UL105)

Despite several studies detailing the functionality of the helicase-primase complex, most

of them were done in the context of the herpes simplex virus model (Barnard, 1997;

21

Biswas, 1999; Dracheva, 1995; Graves-Woodward, 1996; Graves-Woodward, 1997;

Klinedinst, 1994). Apart from the initial characterization of the HCMV helicase-primase

proteins (Pari, 1993; Smith, 1995a; Smith, 1996), there have been relatively few

publications on the specific interactions of these proteins. Although studies have shown

that the UL70, UL102, and UL105 proteins interact with each other to form a

heterotrimeric complex (McMahon, 2002), the specific regions that bind the complex

together have not been studied. Based on sequence and positional similarities with the

herpes simplex virus counterparts, the helicase protein (UL105) is predicted to be

involved in unwinding the DNA helix, while the HCMV primase protein (UL70) is

predicted to be involved in initiating DNA synthesis. However, the primase-helicase

associated protein (UL102) is not specifically involved in the activity of either, yet it

interacts with both UL70 and UL105 to enhance their activities. By elucidating the

binding relationship of these three proteins, we hope to understand more about the role

of UL102.

The HCMV primase ORF (UL70) encodes a 947 amino acid protein that shares 27%

homology with the HSV-1 primase, UL52 (Chee, 1990). Similar to the HSV-1 primase,

pUL70 contains several conserved regions including a putative DXD motif that is

associated with primase catalysis (Dracheva, 1995; Klinedinst, 1994). Furthermore,

substitutions of the aspartate residues in the motif have been shown to abolish primase

but not helicase or ATPase activity in vitro (Dracheva, 1995; Klinedinst, 1994). The

HSV-1 UL52 protein contains a putative zinc-finger motif at its C terminus that is

highly conserved among herpesviruses as well as other prokaryotic, and eukaryotic

primases (Ilyina, 1992; Mendelman, 1994). Although the role of the zinc finger is not

well defined, alanine substitutions of the third and fourth conserved cysteines resulted in

the loss of primase, helicase, ATPase, and DNA-binding activities of the HSV helicase-

primase complex (Biswas, 1999). UL70 is predicted to engage in primer synthesis

during DNA replication on the basis of its relative homology to the HSV-1 primase,

which has been shown to produce short oligoribonucleotide primers up to 10-13

nucleotides long on ssDNA (Crute, 1991; Ramirez-Aguilar, 1995), allowing the DNA

polymerase to begin replicating DNA via dNTP polymerization (Gottlieb, 1994;

Nimonkar, 2003). In addition, the HSV-1 primase was found to initiate primer synthesis

with a purine, at the second nucleotide of a 3'-deoxyguanylate-pyrimidine-pyrimidine

22

(3'-G-pyr-pyr) template sequence (Ramirez-Aguilar, 1995), which may be similar in the

HCMV primase.

HCMV also encodes a helicase-primase associated protein (UL102) that shares little

sequence homology to its HSV counterpart, UL8 (Chee, 1990), but instead is

recognized as a positional homolog (McGeoch, 1988; Pari, 1993). Initial identification

of the UL102 ORF revealed a 798 amino acid protein that was essential for origin-

dependent DNA replication (Chee, 1990; Pari, 1993). In addition, the UL101 ORF

located upstream of UL102 was also shown to be an essential component in replication

(Pari, 1993). However, comparison against cDNA and subsequent re-sequencing of

UL102 in Towne and AD169 (Smith, 1995a) revealed that the genomic stop codon for

UL101 was an error in the original published sequence (Chee, 1990). As a result,

UL102 has been redefined as a 2.7kb un-spliced transcript encoding an 873 amino acid

protein that has the capacity to encode several smaller proteins, all within frame of the

UL102 stop codon (Smith, 1995a). Although further elucidations of the biochemical

activities of UL102 have yet to be published, its positional and functional homology to

HSV UL8 may provide an insight into potential functional parallels derived from

studies on UL8. Firstly, studies looking at primase activity have demonstrated that UL8

may serve a key role in stimulating the synthesis of RNA primers (Tenney, 1994) as

well as increasing the efficiency of primer utilization by DNA polymerase (Sherman,

1992). A central segment of UL8 has been attributed to interact with both the HSV

helicase (UL5) and primase (UL52) subunits (Barnard, 1997). However, in the absence

of UL8, the HSV primase and helicase protein form a sub-complex that is enzymatically

indistinguishable from a complex formed by all three proteins (Calder, 1992; Crute,

1991), suggesting that UL8 may play a role in promoting nuclear localization of the

complex (Calder, 1992). In addition, UL8 also interacts directly with the origin binding

protein (UL9) and at least functionally with the single-stranded DNA binding protein

(ICP8) (Hamatake, 1997; McLean, 1994). UL8 has also been hypothesized to help

direct the polymerase to the initiation complex, where it coordinates polymerase and

helicase-primase activities (Marsden, 1997).

The HCMV helicase ORF (UL105) encodes a 982 amino acid protein that shares 34%

sequence homology with the HSV-1 helicase, UL5 (Chee, 1990), and is present in

infected cells as early as 24 hrs post-infection (Smith, 1996). Like the HSV-1 helicase,

23

the HCMV helicase protein (pUL105) contains six conserved helicase motifs common

to superfamily-1 DNA helicases (Gorbalenya, 1989; Zhu, 1992). Motif I consists of a

GxxGxGKT/S Walker A motif associated with binding the di- or triphosphate moiety of

the nucleotide cofactor (Walker, 1982) while Motif II consists of a Walker B motif

containing a group of hydrophobic residues terminated by an Asp and a Glu residue and

is associated with the stabilization of the coordinated Mg2+

ion (Walker, 1982). In HSV,

motif I has been shown to be directly involved in ATP binding and/or hydrolysis while

motif II appears to be required for coupling of DNA binding to ATP hydrolysis

(Graves-Woodward, 1997). The functional significance of motifs III, IV, V, and VI are

yet to be fully elucidated but the strong conservation of these six motifs suggests that

they may be important for helicase activity. However, mutations generated in motifs III,

IV, V, and VI did not eliminate ATP hydrolysis nor affect DNA binding and therefore is

postulated to be involved in the coupling of these two activities to the process of DNA

unwinding (Graves-Woodward, 1997). Other studies on the HSV helicase have shown

that single amino acid substitutions in the most conserved residues of these motifs

abolish the ability of the HSV helicase to support DNA replication in vivo suggesting

that these conserved residues are essential to DNA replication (Zhu, 1992).

1.4.2.5 Putative Initiator Protein (UL84)

HCMV UL84 encodes a 586 amino acid protein that is detected in infected cells as early

as 2.5 hrs post-infection (He, 1992). Currently, unlike the other replication genes, UL84

shares no functional or positional homology to any HSV replication gene. Analysis of

the UL84 amino acid sequence has revealed the presence of two potential leucine zipper

domain in the N-terminal half of the protein, at amino acids 114–135 and at 325-373

(He, 1992). The leucine zipper domain between residues 114-135 has been implicated in

the interaction of UL84 with the IE2 protein (Colletti, 2004), an immediate-early protein,

that has been identified as one of the auxiliary components required for origin-

dependent DNA replication in human fibroblasts (Pari, 1993). Substitutions of the

leucine residues in this domain rendered UL84 incapable of complementing oriLyt-

dependent DNA replication (Colletti, 2004) while further studies on the functional role

of the UL84–IE2 interaction revealed that an over-expression of UL84 interfered with

the IE2-mediated transactivation of the UL112/113 promoter, decreasing IE2-mediated

transient transactivation (Gebert, 1997). UL84 has also been shown to undergo

24

oligomerization with key interaction domain mapped to residues 151-201 (Colletti,

2004). Initial studies identified UL84 as utilizing a nuclear localization signal similar to

that of the simian virus 40 large T antigen and IE2 nuclear localization signals (Xu,

2002), although subsequent studies report that UL84 interacts with at least four

members of the α importin protein family in vitro and in vivo to utilize a classic

importin-mediated pathway for nuclear import (Lischka, 2003). The region of UL84

responsible for the interaction with α importin has been identified as a complex domain

spanning residues 226-508 and containing a cluster of basic amino acids similar to that

of the classical nuclear localization signals (NLS), albeit lacking classical NLS activity

(Lischka, 2003). This domain also contains two motifs homologous to leucine-rich

nuclear export signals (NES) which have been shown to enable UL84 to shuttle between

the nucleus and the cytoplasm (Lischka, 2006). There are also evidence to suggest that

UL84 may have enzymatic properties that is similar to that of a helicase (Colletti, 2005).

Studies have demonstrated UTPase activity in UL84 that has been suggested to be part

of an energy-generating system for helicase activity (Colletti, 2005; Davison, 2005). In

addition, the amino acid sequence of UL84 displays some structural homology to the

DExD/H box family of RNA helicases (Colletti, 2005). However, most importantly are

recent implications that UL84 provides the role as an initiator of DNA replication, with

several studies demonstrating that UL84 interacts with DNA replication genes UL57

and UL44 (Gao, 2007).

1.4.3 DNA replication

Herpesvirus DNA replication is thought to begin with genome circularization and theta

form replication which then proceeds to a rolling circle form which has been well

documented in HSV (Boehmer, 2003). Initiation of CMV DNA synthesis has yet to be

elucidated. However, in general, initiation of DNA synthesis involves targeted

unwinding of particular DNA sequences to enable assembly of a replisome. It has been

proposed that DNA synthesis in herpesvirus initially proceeds by a theta type

mechanism in which DNA replication proceeds bi-directionally from the lytic origin of

replication, although this has yet to be seen in vitro (Boehmer, 2003). HCMV has a

highly complex oriLyt region that spans over a kilo-base of DNA upstream of the UL57

ORF (Kiehl, 2003). However, unlike HSV, it is the only lytic-phase replicator identified

in HCMV (Anders, 1992). Herpesviruses encode a virion-associated transcript that

25

associates with oriLyt to form a three-stranded structure whose precise role in DNA

synthesis is yet to be elucidated (Prichard, 1998). In general, binding of the Origin

Binding Protein (OBP) with specific sites in OriLyt is followed by an interaction with

single stranded DNA binding proteins which lead to localized unwinding and access of

replication fork proteins. Here, the replication fork machinery consisting of the DNA

polymerase catalytic and accessory subunits, and the hetero-trimeric helicase-primase

complex direct continuous, leading strand viral DNA replication in a rolling circle

mechanism (Boehmer, 2003).

1.4.4 Capsid assembly, maturation and egress

Rolling circle replication generates long head-to-tail concatemers consisting of multiple

unit-length genomes that accumulate in the nuclei. These concatemers are cleaved at

regular intervals and packaged into individual, pre-formed capsids by a mechanism that

is conserved throughout the entire herpesviridae family (Homa, 1997). Structural studies

of the HSV capsid have indicated that structural and functional protein homologues of

HSV capsid proteins are present in the capsid of HCMV (Gibson, 1996). In addition,

cryo-electron microscopic analysis of the capsids has revealed that HCMV and HSV

have near identical structures (Butcher, 1998; Chen, 1999) suggesting that the assembly

of the HCMV capsid follows a very similar assembly pathway as that of HSV

(Grunewald, 2003). The HCMV capsid is composed of 162 capsomeres consisting of

150 hexons and 12 pentons (Butcher, 1998; Chen, 1999) of which the most abundant

protein components consist of the major capsid protein (MCP, UL86) and the smallest

capsid protein (SCP, UL48–49). Two copies of the minor capsid protein (MnCP, UL85)

combined with a single copy of the minor capsid binding protein (MnCP-BP, UL46)

form the triplexes that are located between adjacent pentons and hexons (Butcher, 1998;

Chen, 1999). The process of capsid assembly involves UL86 and UL48-49 interacting

in the cytoplasm in the presence of assembly protein precursor gene UL80a before

translocating to the nucleus. UL85 and UL46 are also translocated to the nucleus, albeit

separately from the UL86/UL48-49 complex. Once in the nucleus, self-interaction

domains in the products of the UL80a lead to formation of pentons and hexons and the

generation of the capsid scaffold. The UL86/UL48-49 complex then interacts with

UL85 and UL46 to form the immature shell of the capsid (Gibson, 1996). In HCMV,

the packaging of viral DNA involves a portal protein, proposed to be encoded by

26

UL104 (Komazin, 2004), and is mediated through virus encoded protein recognition of

two conserved sequence motifs, the pac-1 and pac-2 sequences, that are located at each

end of the viral genome (Mocarski, 2001). In HSV, this process is also coupled with

capsid maturation (Heymann, 2003), which involves the proteolytic cleavage of the

carboxyl terminal of the MCP binding domain (Gibson, 1996). The process of HCMV

egress is not well known, but is thought to involve an initial envelope obtained at the

inner nuclear membrane that is lost by a fusion event at the outer nuclear membrane,

releasing free nucleocapsids into the cytoplasm, where egress continues through a

second and final envelopment step as the virion leaves the host cell (Mettenleiter, 2004).

1.4.5 Latency and reactivation

A unique biological property common to all herpesviruses, including HCMV, is the

ability of the virus to establish lifelong persistence within the host following initial

infection. In general, HCMV reactivation from latency is observed in 13% of healthy

adults (Ling, 2003), and though episodes of sporadic reactivation may occur, they are

generally well-controlled by cell-mediated immuno-surveillance. However, as with

primary infection, reactivation within an immunocompromised individual can lead to

high levels of morbidity and mortality. Recent studies have implicated endothelial cells

and specific cell types of the myeloid lineage as sites of HCMV persistence and latency

(Jarvis, 2002). The factors leading to reactivation from latency are not completely

understood, although studies have shown that myeloid differentiation can stimulate the

production of viral gene expression and in some cases, the production of infectious virus

(Reeves, 2005; Soderberg-Naucler, 2001; Taylor-Wiedeman, 1994). In addition, the

production of the stress hormones cortisol, adrenocorticotrophic hormone, epinephrine

and norepinephrine has been implicated in the reactivation and shedding of CMV in

urine (Mehta, 2000).

1.5 Aims and Objectives

There is a fundamental need for understanding the role of other HCMV genes essential

for DNA replication as a foundation for developing new antiviral treatments. In this

study we looked at six HCMV replication genes encoding for the DNA polymerase

accessory protein (UL44), single stranded DNA binding protein (UL57), primase

27

(UL70), helicase (UL105), primase-helicase associated protein (UL102), and the

putative initiator protein (UL84) in order to increase our understanding of their role in

DNA replication (Figure 3). In particular, UL70 is an essential component of the

primase-helicase complex and elucidation of its functional domains is essential for

understanding its role in DNA replication and subsequently, ways to inhibit its activity

as a potential drug target. Hence, the aims of this project are to:

1. Identify strain variation within the UL44, UL57, UL70, UL102, UL105,

and UL84 genes.

2. Predict putative functional domains and motifs for these genes using the

ScanProsite software, and

3. Express and study the UL70 (Primase) gene through the use of

recombinant mutants.

Figure 3 Relative positions of genes investigated in this study

28

2 Reagents and equipment

All reagents and equipment used in the study are detailed in this section. Individual

experimental methods are outlined in their corresponding chapters. In general, most

chemicals used were molecular biology grade and solutions sterilized by autoclaving or

filtration. Processing of biological specimens was carried out in Class II cabinets using

sterile techniques. In addition all purchased reagents were tested sterile and pyrogen-

free by the manufacturers. Biological waste was sterilized by autoclaving prior to

disposal.

2.1 Viral stocks

HCMV low-

passage strains

Clinical isolates 44A, 70A, 77A, 90A, 91A, 16B, 21B, 80B, 4E,

and 30E were obtained from the Prince of Wales Hospital.

2.2 DNA extraction

2.2.1 Buffers

T10E50

buffer

T10E50 buffer consisted of 10mM Tris-HCl (pH 8.0), 50mM EDTA in

MilliQ water and autoclaved before use.

TE buffer TE buffer consisted of 10mM Tris-HCl (pH 8.0), 1mM EDTA in MilliQ

water and autoclaved before use.

2.2.2 Chenicals

Chloroform Chloroform was obtained from Sigma-Aldrich (USA).

Ethanol 99.5% ethanol was obtained from Sigma-Aldrich (USA).

NaCl >98% (titration) sodium chloride for molecular biology was obtained

from Sigma-Aldrich (USA).

Phenol Phenol was obtained from Sigma-Aldrich (USA).

29

2.2.3 Kits

QIAamp DNA

Mini Kit

QIAamp DNA mini kits (Qiagen, USA) were used to extract DNA

from frozen cell-associated viral stock using a QIAamp spin-column

procedure.

2.3 Polymerase chain reaction (PCR)

2.3.1 Buffers

ExpandLong

template reaction

buffer 1

10X Expand Long Template Buffer 1 was obtained from Roche

(Germany) and is supplemented with 17.5mM MgCl2.

GoTaq green reaction

buffer

5X GoTaq green reaction buffer was obtained from

Promega (USA) and is supplemented with 7.5mM MgCl2.

Platinum Taq High

Fidelity PCR buffer

10X Platinum Taq high fidelity PCR buffer was obtained from

Invitrogen (USA) and consisted of 600mM Tris-SO4 buffer (pH

8.9), 180mM ammonium sulfate.

Red Hot reaction

buffer

10X Red Hot reaction buffer was obtained from AB gene (UK)

and consisted of 750mM Tris-HCl (pH 8.8), 200mM

(NH4)2SO4, 0.1% (v/v) Tween 20.

2.3.2 Enzymes

ExpandLong template

enzyme

Expand Long Template enzyme mix was obtained from

Roche (Germany) and stored at -20°C before use.

GoTaq Flexi DNA

polymerase

GoTaq Flexi DNA polymerase was obtained from

Promega (USA) and stored at -20°C before use.

Platinum Taq High Fidelity

DNA Polymerase

Platinum Taq DNA polymerase was obtained from

Invitrogen (USA) and stored at -20°C before use.

Red Hot DNA polymerase Red Hot DNA polymerase was obtained from ABgene

(UK) and stored at -20°C before use.

30

2.3.3 Equipment

Thermal

cycler

PTC 200 Peltier Thermal cyclers (GMI, USA) were used for

amplification of DNA fragments.

2.3.4 Reagents

dNTP Deoxynucleoside triphosphates, dATP, dCTP, dGTP and dTTP, each at stock

concentrations of 100mM (Promega, USA) were diluted 1:10 and 1:100 with

sterile water (Baxter, Australia) to a final concentration of 10mM and 1mM

respectively. Diluted dNTP stocks were stored at -20°C.

MgCl2 25mM magnesium chloride was obtained from AB gene (UK) or Promega

(USA) and stored at -20°C before use.

MgSO4 50mM magnesium sulfate was obtained from Invitrogen (USA) and stored at -

20°C before use.

Primers Primers were synthesized by Sigma Genosys (Australia) as 400µM stock

solutions. These were further diluted to 10µM with sterile water (Baxter,

Australia) and stored at -20°C before use.

2.4 Electrophoresis

2.4.1 Buffers

TBE

buffer

10X TBE consisted of Trizma base (Sigma-Aldrich, USA), boric acid (Sigma-

Aldrich, USA) and EDTA (Sigma-Aldrich, USA) in MilliQ water and

autoclaved. 0.5X working stock made by diluting 1:20 in MilliQ water.

31

2.4.2 DNA Markers

1kb step-ladder

marker

0.3µg/µl 1kb step-ladder markers were obtained from Promega

(USA), diluted 1:10 with sterile water (Baxter, Australia) and

stored at -20°C before use.

BenchTop pGEM

DNA markers

BenchTop pGEM markers were obtained from Promega (USA)

and stored at room temperature.

Lambda/HindIII

marker

Lambda/HindIII markers were obtained from Promega (USA),

diluted 1:10 with sterile water (Baxter, Australia) and stored at -

20°C before use.

2.4.3 Equipment

Electrophoresis

Equipment

The Mini-Sub Cell GT System (Biorad, USA) was used to

run 2D agarose gels.

2.4.4 Reagents

Agarose Agarose (analytical grade) was obtained from Promega (USA) and used

for gels at concentrations of 0.75% to 1.5% depending on PCR product

size.

Ethidium

bromide

10mg/ml ethidium bromide was obtained from Sigma-Aldrich (USA)

and diluted in sterile water (Baxter, Australia) to a final concentration of

5mg/ml. This was further diluted to 0.5mg/ml in agarose gels and TBE

running buffer.

SYBR safe

DNA gel

stain

10,000X SYBR Safe gel stain was obtained from Invitrogen (USA) and

stored at room temperature.

32

2.5 DNA sequencing

2.5.1 Equipment

DNA

sequencer

A 3730 DNA Analyzer (Applied Biosystems, USA) was used for

analysis of sequencing products at the Ramaciotti Centre, University of

New South Wales.

2.5.2 Reagents

PEG PCR

mix

Polyethylene glycol PCR mix consisted of 26.7% (w/v) polyethylene

glycol (PEG) 8000 (Sigma-Aldrich, USA), 0.6M NaOAc (pH 5.2),

6.5mM MgCl2.

Primers Primers were synthesized by Sigma Genosys (Australia) as 400µM

stock solutions. These were further diluted to 10µM with sterile water

(Baxter, Australia) and stored at -20°C before use.

Sequencing

mix

BigDye Terminator v3.1 was obtained from Applied Biosystems (USA)

and stored at -20°C before use.

Sequencing

buffer

5X sequencing buffer was obtained from Applied Biosystems (USA)


2.6 Restriction enzymes

2.6.1 Buffers

CIAP stop

buffer

CIAP stop buffer consisted of 10mM Tris-HCl (pH 7.5), 1mM EDTA

(pH 7.5), 200mM NaCl, and 0.5% (w/v) SDS.

Multi-core

buffer

10X multi-core buffer was obtained from Promega (USA) and consisted

of 10mM Tris-HCl (pH 7.5), 100mM NaCl, 50% glycerol.

33

2.6.2 Enzymes

BamHI BamHI was obtained from New England BioLabs (USA) with NEBuffer 3


HindIII HindIII was obtained from Promega (USA) with buffer B and stored at -20°C

before use.

SalI SalI was obtained from New England BioLabs (USA) with NEBuffer 3 and


SpeI SpeI was obtained from Promega (USA) with buffer B and stored at -20°C

before use.

2.6.3 Reagents

BSA 1µg/µl Bovine serum albumin (BSA) was obtained from Promega (USA) and


CIAP Calf intestinal alkaline phosphatase (CIAP) was obtained from Promega (USA)


TSAP Thermosensitive alkaline phosphatase (TSAP) was obtained from Promega


2.7 Cloning reagents

2.7.1 Antibiotics

Ampicillin 91.0-100.5% Ampicillin was obtained from Sigma-Aldrich (USA),

diluted with sterile water (Baxter, USA) to a working concentration of

10mg/ml, filtered and stored at -20°C before use.

Tetracycline 100mg/ml Tetracycline was obtained from Invitrogen (USA) and stored

at -20°C before use.

Zeocin 100mg/ml Zeocin was obtained from Invitrogen (USA) and stored at -

20°C before use.

34

2.7.2 Cells

Library efficiency DH5α

competent cells

DH5α competent cells were obtained from Invitrogen


XL10 Gold Ultracompetent

cells

XL10 Gold ultracompetent cells were obtained from

Stratagene (USA) and stored at -80°C before use.

2.7.3 Equipment

Low-salt LB

zeocin plates

Low salt LB consisted of 1% (w/v) tryptone (Oxoid, Australia), 0.5%

NaCl (Sigma-Aldrich, USA), 0.5% yeast (Oxoid, Australia), 1.5%

(w/v) agar (Promega, USA), adjusted to pH 7.5 and autoclaved.

Allowed to cool to 55°C and zeocin (Invitrogen, USA) added to final

concentration of 50µg/ml before pouring.

LB

Ampicillin

plates

2% (w/v) agar (Promega, USA) added to LB broth and autoclaved.

Allowed to cool to 55°C and Ampicillin (Sigma-Aldrich, USA) added

to final concentration of 100µg/ml before pouring.

LB

tetracycline

plates

2% (w/v) agar (Promega, USA) added to LB broth and autoclaved.

Allowed to cool to 55°C and tetracycline (Sigma-Aldrich, USA) added

to final concentration of 125µg/ml before pouring.

35

2.7.4 Kits

DNA ligation kit DNA ligation kit was obtained from Stratagene (USA) and stored at

-20°C before use. Includes pUC18 (BamH I digested) and cI857

wild-type lambda (Hind III digested) control DNA, 10mM rATP

(pH 7.5), T4 DNA ligase, and 10X ligase buffer consisting of

500mM Tris-HCl (pH 7.5), 70mM MgCl2, 10mM dithiothreitol

(DTT).

Gateway BP

Clonase II

enzyme mix

BP Clonase II kit was obtained from Invitrogen (USA) and stored

at -20°C before use.

Wizard Plus

Midipreps DNA

purification

system

Wizard Plus midiprep kits (Promega, USA) were used to extract

plasmid DNA using a column and vacuum procedure. Reagents

were stored at room temperature.

Wizard Plus

Minipreps DNA

purification

system

Wizard Plus miniprep kits (Promega, USA) were used to extract

plasmid DNA using a column and vacuum procedure. Reagents

were stored at room temperature.

Wizard SV

Gel/PCR Clean-

up System

Wizard PCR clean-up kits (Promega, USA) were used to purify

restriction digested DNA. Reagents were stored at room

temperature.

36

2.7.5 Media

Luria

broth

Luria broth (LB) consisted of 1% (w/v) NaCl (Sigma-Aldrich, USA), 1%

(w/v) tryptone (Oxoid, Australia), 0.5% (w/v) yeast (Oxoid, Australia),

adjusted to pH 7.0, and autoclaved prior to use.

NZY

broth

NZY broth consisted of 1% (w/v) casein hydrolysate (Merck, USA), 0.5%

(w/v) yeast (Oxoid, Australia), and 0.5% (w/v) NaCl (Sigma-Aldrich, USA),

adjusted to pH 7.5 and autoclaved. Add 12.5ml of 1M MgCl2 (Sigma, USA),

12.5ml of 1M MgSO4 (BDH, UK), and 10ml of 2M glucose (Gibco BRL,

USA) per liter of broth prior to use.

S.O.C

media

S.O.C media consisted of 2% (w/v) tryptone (Oxoid, Australia), 0.5% (w/v)

yeast (Oxoid, Australia), 10mM NaCl (Sigma-Aldrich, USA), 2.5mM KCl

(BDH, UK), 10mM MgCl2 (Sigma, USA), 10mM MgSO4 (BDH, UK), 20mM

glucose (Gibco BRL, USA), and autoclaved before use.

2.7.6 Reagents

IPTG 100mM Isopropyl B-D-Thiogalactopyranoside was obtained from

Promega (USA).

Proteinase

K

2µg/µl Proteinase K was obtained from Invitrogen (USA) and stored at -

20°C before use.

X-gal 50mg/ml 5-bromo-4-chloro-3-indolyl-B-D-galactosidase was obtained

from Promega (USA).

2.7.7 Vectors

pBluescript II

SK(+)

pBluescript II SK(+) vectors were obtained from Stratagene (USA)


pCITE-4A(+) pCITE-4A(+) vectors were obtained from Novagen (Germany) and


pDONR/Zeo pDONR/Zeo vectors were obtained from Invitrogen (USA) and


37

2.8 Protein expression and detection

2.8.1 Antibodies

Anti-His G Antibody Anti-His G antibodies (Invitrogen, USA) were used for

protein detection and stored at -20°C before use.

S-Protein alkaline

phosphatase conjugate

S-Protein alkaline phosphatase conjugate (S-Tag) was

obtained from Novagen (Germany) and stored at -20°C

before use.

2.8.2 Buffers

Loading

buffer

4X loading buffer consisted of 0.25M Tris-Cl (Sigma-Aldrich, USA), 40%

(w/v) glycerol, 8% (w/v) SDS, 0.4% (w/v) Bromophenol Blue (BDH, UK),

8% (v/v) β-mercaptoethanol (Sigma-Aldrich, USA)

PBS 10X phosphate buffered solution (PBS) were obtained from Gibco (USA).

Running

buffer

1X running buffer consisted of 25mM Trizma base (Sigma-Aldrich, USA),

250mM (pH 8.3) glycine (Sigma-Aldrich, USA), and 0.1% (w/v) SDS in

MilliQ water.

Transfer

buffer

1X transfer buffer consisted of 25mM Trisma base (Sigma-Aldrich, USA),

250mM (pH 8.3) glycine (Sigma-Aldrich, USA), and 20% methanol

(Mallinckrodt Chemical, UK) in MilliQ water.

38

2.8.3 Equipment

2-D gel

electrophoresis

equipment

The Mini-PROTEAN 3 Cell electrophoresis system (Biorad,

USA) was used to run 2D poly-acrylamide gels.

Nitrocellulose Hybond-ECL nitrocellulose was obtained from Amersham

Biosciences.

Resolving gel 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE) resolving gels consisted of 34% (v/v) 30%

Bis/Acrylamide, 0.39M Tris (pH 8.8), 0.1% SDS, 0.1% APS, and

0.04% TEMED.

Stacking gel 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE) stacking gels consisted of 16.5% (v/v) 30%

Bis/Acrylamide, 0.125M Tris (pH 6.8), 0.1% SDS, 0.1% APS,

and 0.1% TEMED.

2.8.4 Kits

TNT Quick Coupled

Transcription/Translation

System

TNT Quick Coupled Transcription/Translation

System (Promega, USA) was used to express protein

utilizing the pCITE expression vector.

2.8.5 Protein Markers

Kaleidoscope marker Precision Plus Protein Kaleidoscope Standards were obtained

from Biorad (USA) and stored at -20°C before use.

Perfect Protein

Western markers

Perfect protein western markers were obtained from Novagen

(Germany) and stored at -20°C before use.

39

2.8.6 Reagents

APS >98% ammonium persulfate (APS) was obtained from

Sigma-Aldrich (USA) and stored at room temperature.

Bis/Acrylamide 30% Acrylamide/Bis Solution (37.5:1) was obtained from

Biorad (USA) and stored at 4°C before use.

Gelcode blue stain Gelcode blue stain (Pierce, USA) was used to stain

PAGE gels for total protein.

Glycerol >99% glycerol was obtained from Sigma-Aldrich (USA)

Ponceau S 0.1% (w/v) Ponceau S in 5% acetic acid (Sigma-Aldrich,

USA) was used to stain nitrocellulose membranes for

total protein.

SDS >98.5% Sodium dodecyl sulfate (for molecular

biology, .98.5%) was obtained from Sigma-Adrich

(USA).

SuperSignal West Pico

chemiluminescent substrate

SuperSignal West Pico Chemiluminescent Substrate kit

was obtained from Pierce (USA) and stored at 4°C before

use.

TEMED >99% N, N, N’, N’-Tetramethylethylenediamine was

obtained from Sigma-Aldrich (USA) and stored at room

temperature.

2.8.7 Solutions

Blocking

solution

Blocking solution consisted of 5% skim milk powder (Diploma,

Australia) in PBST.

PBST

solution

Phosphate buffered solution with Tween (PBST) consisted of 1X PBS,

and 0.1% Tween (Sigma-Aldrich, USA)

40

3 Identification of putative functional motifs in viral proteins

essential for human cytomegalovirus DNA replication

3.1 Introduction

HCMV DNA replication is associated with eleven loci necessary for transient

complementation of oriLyt-dependent DNA replication (Pari, 1993). These include six

core genes homologous to six of the seven essential replication genes in Herpes Simplex

Virus (HSV) (Anders, 1996) (Table 6). In HCMV, they consist of a two-subunit DNA

polymerase (UL54 and UL44), single-stranded DNA binding protein (UL57), primase

(UL70), helicase (UL105), and the primase-helicase associated factor (UL102). In

addition, five other loci (TRS1/IRS1, IE1/IE2, UL112-113, UL84, and UL36-38) have

supporting regulatory and trans-activation roles in HCMV replication (Iskenderian,

1996; Pari, 1993). DNA replication in HCMV is considered to be analogous to that in

HSV, with the function of several HCMV proteins being predicted on the basis of

sequence and functional homology with their HSV counterparts (Anders, 1996; Chee,

1990).

Table 6 Essential DNA replication proteins in HCMV and HSV-1

Function HCMV HSV1

DNA Polymerase UL54 UL30

DNA Polymerase accessory protein UL44 UL42

Single-Stranded DNA Binding Protein UL57 UL29

Primase UL70 UL52

Helicase UL105 UL5

Primase-Helicase Associated Protein UL102 UL8

Initiator Protein UL84 UL9

The role of the HCMV DNA polymerase accessory protein (UL44) has been well

documented over the years with the elucidation of key functional motifs and residues

(Appleton, 2004; Loregian, 2004) as well as a partial crystal structure of the functional

N terminal half of the protein (Appleton, 2006; Appleton, 2004). In contrast, the HCMV

single stranded DNA binding protein (UL57) has been less well elucidated although

early studies established biochemical properties that were comparable to that of the

41

HSV major DNA-binding protein, ICP8 (Anders, 1987). The HCMV primase (UL70),

helicase (UL105) and primase-helicase associated factor (UL102) form a trimeric

complex (McMahon, 2002) that shares biologically important motifs with the HSV

primase-helicase complex (Dracheva, 1995; Klinedinst, 1994; Zhu, 1992). The UL84

ORF encodes the only non-core replication protein that is required for origin-dependent

DNA replication in human fibroblasts (Sarisky, 1996). Although initially classed as a

regulatory protein for HCMV replication (Pari, 1993), recent functional studies (Colletti,

2004; Xu, 2004) have suggested that the UL84 ORF encodes the initiator protein for

HCMV DNA synthesis (Xu, 2004a).

The residues comprising a protein are not all equally important. Some serve as

indispensable mediators of protein function while others can be readily replaced without

impacting on functionality. As the sequences of biologically significant residues are

evolutionarily conserved, conservation analysis has become one of the most widely used

techniques for predicting protein function. In this study we report our findings on the

variation of the HCMV replication proteins amongst isolate strains.

3.2 Methods

3.2.1 Viral stocks and DNA extraction

Viral DNA from 10 low-passage isolates (44A, 70A, 77A, 90A, 91A, 16B, 21B, 80B,

4E, and 30E) were extracted from frozen cell-associated viral stock using the QIAamp

DNA Mini Kit (Qiagen, USA) as per manufacturer’s instructions, with DNA eluted in

50µl of buffer AE after 5 min incubation instead of the recommended 200µl.

3.2.2 Polymerase chain reaction (PCR) amplification

Full-length ORFs encoding the accessory protein (UL44), helicase-primase complex,

(UL70, UL102, and UL105), single-stranded DNA-binding protein (UL57), and

putative initiator protein (UL84) were amplified in overlapping segments from viral

DNA by single rounds of PCR. PCR reaction mixes consisted of 1x buffer, 2mM MgCl2,

0.25mM deoxynucleoside triphosphates (dNTP), 0.2µM forward and reverse primers

(Table 7), 1 U/µl Red Hot DNA polymerase (AB Gene), and 5µl of extracted DNA

template (approximately 100ng). Single round cycling conditions were 94°C for 5 min

42

followed by 30 cycles of 94°C for 1 min, 55°C for 30 sec, and 72°C for 2 min. PCR

products were visualized following electrophoresis on 1.5% agarose gels.

43

Table 7 Primers used for PCR amplification of HCMV DNA fragments

Gene Primer** Region Amplified* Sequence (5' to 3')

UL44 UL44.1 [F] 172843 - 173547 ATGGATCGCAAGACGCGCCT

UL44.2 [R] GACGGCGCAATTGAGCAGCG

UL44.3 [F] 173438 - 174199 GCGAGCTGGAATTCACGGCC

UL44.4 [R] TTTCTCCATCACGGGACCGCG

UL57 UL57.1 [F] 138815 - 140062 CATCACGCTATTTTCGCGGGC

UL57.2 [R] CGGTGATCGGTTGCGTTGGTC

UL57.3 [F] 139960 - 141135 TGGCCTTCAGTTTTGCCTCGG

UL57.4 [R] AATAGGCGTAGGCTAGCAGCG

UL57.5 [F] 141020 - 142123 TTCGGAGGTGCGCCTCAAGAG

UL57.6 [R] CCGTTGCCCGAAAAGTAGCCG

UL57.7 [F] 141978 - 142993 ACTGTGGCGCGTGAATCGTTG

UL57.8 [R] TTCGGGGCTGTTGCTGCTGTT

UL70 UL70.1 [F] 125951 - 126606 GCTGGGCCAGCTGCATCGTGCCGGCGCGACG

UL70.2 [R] GCGAGCCCAGTAGCAGACGCGCGAA

UL70.3 [F] 126497 - 128309 CATCTCCACCGTGGAGGAGTACGTG

UL70.4 [R] GGGGCAGATTTTGTCGCGTACGCTG

UL70.5 [F] 128221 - 128853 CCCAACTGGATCTGCGTAACCTGCT

UL70.6 [R] GGCGGGTCACCGGCGCCGTGGAAAGTGAGGC

UL102 UL102.1 [F] 147750 - 148466 GCTCACGACGCGGTTTGAGCA

UL102.2 [R] ATACTTGTGAGTGCCGACGCG

UL102.3 [F] 148341 - 148966 CGTGTGGTCACGCATGCCGCGGAAC

UL102.4 [R] GCCGTGGCATTGAGACGCACGGCGC

UL102.5 [F] 148896 - 149960 GTGCCCGAGGATGAGTGGCAGGTCT

UL102.6 [R] GAGGATCTCGCGATACACGGCTTCC

UL102.7 [F] 149870 - 150480 CGCGGCCGGTGGCGACTGGCTCTCG

UL102.8 [R] TACCCACGTAGTTCCCCTACGTGACTCG

UL105 UL105.1 [F] 153111 - 153909 AGAGCAGCTGCTTGCGCAGCA

UL105.2 [R] CATTTGTCCACGATGTCGGCG

UL105.3 [F] 153748 - 155593 CTTCGTCAGCAAGCACGTGCCGCTG

UL105.4 [R] CTGCGAGCTGATCTGACAGTTGGCC

UL105.5 [F] 155478 - 156130 TCAAGCGCTACCAGCTCATGCAGCG

UL105.6 [R] GTCACGTTTTCCTTACACGGTGTTGTG

UL84 UL84.1 [F] 108076 - 109175 AGAAGGGCGACGCGCTATGCG

UL84.2 [R] TCTTCTTGCGACGTCGCGGGG

UL84.3 [F] 109101 - 110188 TCTGCTCTCTACGCCGCTGCA

UL84.4 [R] ACGTGGCGCCATTCTCGTCGC

UL84.5 [F] 110115 - 111151 GCCCGTTACGGTCTGGCTACC

UL84.6 [R] CCGACGGGTAGTGGTGCACGT

*Given in relation to AD169 for UL44, UL57, and UL70, Merlin for UL102 and UL105,

and Towne for UL84.

**[F] denotes forward primer and [R] denotes reverse primer

44

3.2.3 DNA sequencing and analysis

All PCR products were precipitated from solution with polyethylene glycol as

previously described (Craxton, 1991). Sequencing was carried out using a reaction mix

consisting of Big Dye Terminator (Applied Biosystems, USA), premix buffer, 0.25µM

forward or reverse primer (Table 7), and approximately 50ng/300bp of DNA. Cycling

conditions consisted of 25 cycles of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min.

Sequencing products were purified and analyzed at the University of New South Wales

DNA analysis facility. Forward and reverse sequences were compared for sequencing

accuracy using GAP (GCG) (Accelrys).

3.2.4 Phylogenetic analysis

Full length nucleotide sequences for UL44, UL70, UL102, UL105, UL57, and UL84

were compiled for each individual strain, translated to protein using Translate (GCG)

(Accelrys) and aligned with three laboratory strains (AD169, Towne, and Merlin) using

ClustalW (Thompson, 1994). Genetic distances between pairs of nucleotide and amino

acid sequences were calculated with DNAdist and Protdist programs (Felsenstein, 1989)

respectively. Homologous proteins from other virus or cellular systems were also

identified using BLASTP (Altschul, 1997) and the results of the searches aligned to

their respective CMV counterpart using ClustalW (fast) (Thompson, 1994). All of these

programs were accessed through the Australian National Genomic Information Service

(ANGIS). The PROSITE database of biologically meaningful motif descriptors derived

from multiple alignments and ProRule (Hulo, 2006; Sigrist, 2005) were used to scan the

sequences of each HCMV replication protein. Potential motifs were identified using the

ScanProsite program (de Castro, 2006) from the Expert Protein Analysis System

(ExPASy) proteomics server of the Institute of Bioinformatics.

3.2.5 Genbank Accession Numbers

Sequences derived from each of the HCMV clinical isolates were submitted to Genbank

and have the accession numbers: UL44 (EU294431 - EU294440), UL57 (EU294441 -

EU294450), UL70 (EU294451 - EU294460), UL84 (EU294461 - EU294469), UL102

(EU294470 - EU294479), and UL105 (EU294480 - EU294489).

45

3.3 Results

3.3.1 Strain variation amongst HCMV isolates

The DNA polymerase accessory protein (UL44), single-stranded DNA binding protein

(UL57), primase (UL70), helicase (UL105), primase-helicase associated factor (UL102),

and putative initiator protein (UL84) are highly conserved among the ten HCMV

clinical isolates and three reference strains (AD169, Towne, and Merlin). All six genes

studied were found to be highly conserved with nucleotide (nt) identity ranging from

97.9% to 100% and amino acid (aa) identity ranging from 97.46% to 100% (Table 8).

Major alterations of the coding sequence, such as frame-shifts or premature stop codons,

were not observed. Amino acid polymorphisms in UL44, UL57, UL70, UL102, UL105,

and UL84 are summarized in Table 8. Polymorphisms existed outside of known

functional domains with the exceptions of a K430R substitution in the nuclear

localization signal (NLS) of UL44 as well as various polymorphisms in the NLS of

UL84. Results of phylogenetic analysis showed no preferential clustering of isolates

although certain amino acid changes were linked in UL84 and UL44. In particular, a

S296G substitution was consistently associated with an I319L substitution in UL44

(AD169, Towne, 44A, 70A, 77A, and 4E) while three isolates (16B, 21B, and 30E) had

a S27T substitution in UL84 which correlated with A390T substitutions further towards

the carboxy terminal end of the protein.

Table 8 Sequence variation within HCMV replication genes

Gene Protein Nucleotide Amino Acid Amino Acid

Length Identity (%) Identity (%) Variations

UL44 433 98.1 - 100 98.6 - 100 18

UL57 1235 99.1 - 100 99.7 - 100 16

UL70 946 98.6 - 100 99.1 - 100 36

UL105 956 99.0 - 100 99.0 - 100 31

UL102 875 98.6 - 100 99.2 - 100 39

UL84 587 97.9 - 100 97.5 - 100 73

3.3.2 Prediction of potential motifs

No motifs were detected in the HCMV replication genes when ScanProsite was

designated to exclude motifs with a high probability of occurrence. However, this also

tended to omit documented motifs which had previously been shown to be biologically

significant (Alvisi, 2005; Colletti, 2004; Graves-Woodward, 1997; Lischka, 2006). By

46

contrast, the inclusion of motifs with a high probability of occurrence generated a range

of motifs associated with post-translational modifications as well as several domains

unique to specific genes (Table 9). In addition, several compositionally biased regions

(regions consisting of a high proportion of one amino acid type) that are often abundant

in certain motifs were also observed. For example, glycine-rich domains were evident

for UL44, UL57 and UL102. A number of conserved functionally significant motifs

were identified by comparison of the HCMV proteins with other Herpesvirus

homologues, and related viral and cellular proteins discovered by the BLASTP search.

Excluding HCMV, we were able to identify 12 homologues for UL44, 34 for UL57, 29

for UL70, 12 for UL102, 32 for UL105, and 4 for UL84.

Table 9 Summary of the putative motifs identified in the HCMV replication genes

Gene

Motif UL44 UL57 UL70 UL102 UL105 UL84

N-linked glycosylation site 4 3 3 1 9 4

cAMP-dependent protein kinase phosphorylation site 1 0 1 0 2 1

Casein kinase II phosphorylation site 5 14 20 8 15 12

Protein kinase C phosphorylation site 10 15 10 7 10 12

Amidation site 1 1 1 0 0 0

N-myristoylation site 27 42 5 22 11 9

Tyrosine kinase phosphorylation site 0 1 2 2 2 0

Cell attachment sequence 1 0 0 1 0 0

Leucine zipper 0 0 0 0 0 1

Microbodies targeting signal 0 1 0 0 0 0

ATP/GTP-binding site motif A (P-loop) 0 0 0 0 1 0

Total 49 77 42 41 50 39

3.3.3 Strain variation and identification of protein motifs in UL44

Sequence alignments of the ten HCMV strains show a highly conserved protein with

variations outside of essential UL44 domains such as the DNA polymerase connector

loop (Loregian, 2004) and dimer binding domains (Appleton, 2004). A lysine to

arginine substitution was observed at residue 430 of 4E in a region that was previously

identified as a nuclear localization signal (NLS) (Alvisi, 2005). However, the

significance of the substitution is unknown although both amino acids share similar

basic properties. Interestingly, a serine to glycine substitution at residue 296 of AD169,

Towne, 44A, 70A, 77A, and 4E correlated with an isoleucine to leucine substitution in

the same isolates (Figure 4). An unusual integrin binding motif was identified at residue

47

274 for HCMV, but was not present in other homologues except pongine Herpesvirus 4.

More than half of the potential motifs identified for UL44 by ScanProsite (Table 9)

were found in the carboxy-terminal third of the gene (codons 290-433), including a

putative casein kinase-2 (CKII) signal thought to enhance the carboxy-terminal NLS of

UL44 (Alvisi, 2005). In addition, half of the motifs identified by ScanProsite also

occurred in the glycine rich region of UL44 (codons 284-397).

48

α δ α δ α δ γ UL44 1 11 21 31 41 51 61 71 81 91 Consensus MDRKTRLSEPPTLALRLKPYKTAIQQLRSVIRALKENTTVTFLPTPSLILQTVRSHCVSKITFNSSCLYITDKSFQPKTINNSTPLLGNFMYLTSSKDLT

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ............V.......................................................................................

77A ....................................................................................................

90A .................................................................................................H..

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ...Q................................................................................................

30E ....................................................................................................

δ α 101 111 121 131 141 151 161 171 181 191

Consensus KFYVQDISDLSAKISMCAPDFNMEFSSACVHGQDIVRESENSAVHVDLDFGVVADLLKWIGPHTRVKRNVKKAPCPTGTVQILVHAGPPAIKFILTNGSE

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

49

γ δ γ ε δ η 201 211 221 231 241 251 261 271 281 291

Consensus LEFTANNRVSFHGVKNMRINVQLKNFYQTLLNCAVTKLPCTLRIVTEHDTLLYVASRNGLFAVENFLTEEPFQRGDPFDKNYVGNSGKSRGGGGGSGSLS

AD169 ...............................................................................................G....

Merlin ....................................................................................................

Towne ...............................................................................................G....

44A ................I..............................................................................G....

70A ...............................................................................................G....

77A ...............................................................................................G....

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ...............................................................................................G....

30E ....................................................................................................

η η η ζ η δ β η δ γ δ η η η 301 311 321 331 341 351 361 371 381 391

Consensus SLANAGGLHDDGPGLDNDIMNEPMGLGGLGGGGGGGGKKHDRGGGGGSGTRKMSSGGGGGDHDHGLSSKEKYEQHKITSYLTSKGGSGGGGGGGGGGLDR

AD169 ..................L.................................................................................

Merlin ....................................................................................................

Towne ..................L.................................................................................

44A ..................L.................................................................................

70A ..................L.................................................................................

77A ..................L.................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ..................L......................................S..........................................

30E ....................................................................................................

50

η γ δ 401 411 421 431

Consensus NSGNYFNDAKEESDSEDSVTFEFVPNTKKQKCG

AD169 .................................

Merlin .................................

Towne .................................

44A .................................

70A .................................

77A .................................

90A .................................

91A .................................

16B .................................

21B .................................

80B .................................

4E ..............................R..

30E .................................

Figure 4 HCMV strain alignments of UL44

Residues highlighted in yellow indicate functionally significant amino acids (Alvisi, 2005; Appleton, 2004; Loregian, 2004) while underlined sequences represent putative

motifs identified with ScanProsite. α = N-linked glycosylation site (N-x-T/S-x), β = cAMP-dependent protein kinase phosphorylation site (RR/KK/RK-x-S/T), γ = Casein

kinase II phosphorylation site (S/T-xx-D/E), δ = Protein kinase C phosphorylation site (S/T-x-R/K), ε = Cell attachment sequence (R-G-D), ζ = Amidation site (x-G-

RR/KK/RK), η = N-myristoylation site (G-A/N/C/Q/G/I/L/M/S/T/V-xx-all except P).

51


Although no well defined functional domains exist in UL57, sequence alignments

indicate a highly conserved protein with very little variation between strains (Figure 5).

An unusual micro-bodies targeting signal was identified at the extreme C-terminus of

UL57 (residues 1233-1235) while two short glycine-rich domains spanning residues

536-591 (score = 18.78) and 1167–1226 (score =13.29) were also observed. An

alignment of the UL57 protein and its 34 homologues identified several highly

conserved cysteine residues at amino acid positions Cys107, Cys483, Cys472, and

Cys475, with the latter two forming a putative canonical disulfide bond motif that was

conserved in 97% of the homologues. Three of the conserved cysteine residues (the

disulfide bond residues Cys472 and Cys475, as well as Cys483) are homologous to a

putative zinc binding motif identified in the HSV1 homolog, ICP8 (Wang, 1990). In this

study, we observed that this domain was retained in 16 of the 35 (46%) UL57

homologues (Figure 6).

52

γ γ δ η UL57 1 11 21 31 41 51 61 71 81 91

Consensus MSHEELTALAPVGPAAFLYFSRLNAETQEILATLSLCDRSSSVVIAPLLAGLTVEADFGVSVRTPVLCYDGGVLTKVTSFCPFALYFHHTQGIVAFTEDH

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ...................................................................................T................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

η δ 101 111 121 131 141 151 161 171 181 191

Consensus GDVHRLCEDARQKYALEAYTPEADRVPTDLAALCAAVGCQASETTVHVVVGNGLKEFLFAGQLIPCVEEATTVRLHGGEAVRVPLYPPTLFNSLQLDAEA

AD169 ...................M................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ..............................V.....................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

53

δ δ δ γ 201 211 221 231 241 251 261 271 281 291

Consensus DEVSLDARSAFVEARGLYVPAVSETLFYYVYTSWCQSLRFSEPRVLIEAALRQFVHDSQQSVKLAPHKRYLGYMSQRLSSLEKDHLMLSDAVVCELAFSF

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ................F...................................................................................

70A ................F...................................................................................

77A ....................................................................................................

90A ................F...................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ................F...................................................................................

4E ................F...................................................................................

30E ....................................................................................................

α δ η η 301 311 321 331 341 351 361 371 381 391

Consensus ASVFFDSAYQPAESMLFSEWPLVTNATDHRDLIRALTELKLHLSTHVAALVFSANSVLYQHRLVYLQSSARHPSAGGTASQETLLKAIQFTNGLSAACED

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

54

η η η 401 411 421 431 441 451 461 471 481 491

Consensus VYNDARKVLKFQGAPLKDERYGPQHLALVCGTCPQLVSGFVWYLNRVSVYNTGLSGSSTLTNHLVGCAAGLCEACGGTCCHTCYQTAFVRVRTRLPVVPK

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

ζ η η η η η η δ 501 511 521 531 541 551 561 571 581 591

Consensus QPKKEPCVITVQSRFLNDVDILGSFGRRYNVDAKDGGLDGKGDDGVPGGGAGGGGGRDVSGGPSDGLGGGRGGGGGGDSGGMMGRGGRMLGASVDRTYRL

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

55

γ δ γ η 601 611 621 631 641 651 661 671 681 691

Consensus NRILDYCRKMRLIDPVTGEDTFSAHGKSDFVAVFSALNKFVDDEALGFVSEVRLKSSRDEVAGATQAFNLDLNPYAVAFQPLLAYAYFRSVFYVIQNVAL

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

η δ δ γ β 701 711 721 731 741 751 761 771 781 791

Consensus ITATSYIVDNPLTTNLVSKWMTQHFQSIHGAFSTTSSRKGFLFTKQIKSSKNSDHDRLLDFRLYAQGTYAVVPMEIKLSRLSVPTLIMVRVKNRPIYRAG

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....A...............................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

56

η γ 801 811 821 831 841 851 861 871 881 891

Consensus KGNAGSVFFRRDHVPRRNPAKGCLGFLLYRHHERLFPECGLPCLQFWQKVCSNALPKNVPIGDMGEFNAFVKFLVAVTADYQEHDLLDVAPDCVLSYVES

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

η δ δ γ η γ η α 901 911 921 931 941 951 961 971 981 991

Consensus RFHNKFLCYYGFKDYIGSLHGLTTRLTTQNHAQFPHVLGASPRFSSPAEFALHVKGLKTAGVPAPMAATVARESLVRSVFEHRSLVTVPVSVEKYAGINN

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A .......................................................S............................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

57

η α γ γ δ δ γ 1001 1011 1021 1031 1041 1051 1061 1071 1081 1091

Consensus SKEIYQFGQIGYFSGNGVERSLNVSSMSGQDYRFMRQRYLLATRLADVLIKRSRRENVLFDADLIKNRVMLALDAENLDCDPEVMAVYEILSVREEIPAS

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

γ η η η 1101 1111 1121 1131 1141 1151 1161 1171 1181 1191

Consensus DDVLFFVDGCEALAASLMDKFAALQEQGVEDFSLENLRRVLDADAQRLTDAAGGEVHDLSALFAPSGVGAASGVGGGGLLLGESVAGNSICFGVPGETGG

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne .....................................................................................T..............

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ..................................D.................................................................

4E ....................................................................................................

30E ....................................................................................................

58

η η ε 1201 1211 1221 1231

Consensus GCFLVNAGEDEAGGVGGSSGGGGGSGLLPAKRSRL

AD169 ...................................

Merlin ...................................

Towne ...................................

44A ...................................

70A .......S...........................

77A ...............S...................

90A .......S...........................

91A ...................................

16B ...................................

21B ...................................

80B ...................................

4E .......S...........................

30E ...................................


Underlined sequences represent putative motifs identified with ScanProsite. α = N-linked glycosylation site (N-x-T/S-x), β = Tyrosine kinase phosphorylation site (R/K-xx-

D/E-xxx-Y/R/K-xxx-D/E-xxx-Y), γ = Casein kinase II phosphorylation site (S/T-xx-D/E), δ = Protein kinase C phosphorylation site (S/T-x-R/K), ε = Microbodies C-terminal

targeting signal (S/T/A/G/C/N-R/K/H-L/I/V/M/A/F/Y) ζ = Amidation site (x-G-RR/KK/RK), η = N-myristoylation site (G-A/N/C/Q/G/I/L/M/S/T/V-xx-all except P).

59

AHV1 (O36360) YNITEYVGSAANSP-VCSLCSGQCPCVCINTLF

AtHV3 YSVAQYVGTVAVSE-LCELCQGKCPAACIHTLF

BHV1 (Q89549) DALKYVASTLEGDV-PCGLCSRDDRHACAHTTL

BHV4 YNISNYVGTAASSD-MCNLCQGKKPAVCINTLF

BHV5 (Q6X241) DALKYVASTLEGDV-PCGLCSRDDRHACAHTTL

CHV3 (Q993K9) HEIGSYVAGAATSS-VCSLCEGSTPAVCLNTLF

EBV (U75698) PETGSYVAGAAASP-MCSLCEGRAPAVCLNTLF

EHV1 DALRYLANTLESDV-PCGLCNQATRPACAHTTL

EHV2 (Q66611) YNVPTYVGTAANTP-MCELCRGSCPASCVNTLF

GHV1 (Q9QH63) SISNSGKAPCTGAVPECRWCNDESRNHCIRYTM

GTH (Q6VQA1) TLLQYLNAKTEEGA--CDLCDVETRHVCPATTF

HGTH (Q5ZR67) TLLQYLNAKTEEGA--CDLCDVETRHVCPATTF

HSV1 (X14112) DVFRYVADSNQTDV-PCNLCTFDTRHACVHTTL

HSV2 DVFRYVADSGQTDV-PCNLCTFETRHACAHTTL

HHV6 TEIYNHLVNCSAN--LCEFCDGKCCQSCIGTAM

HHV7 SEIYNHIVNCSSN--LCEFCEGKCCHSCIGTAL

HHV8 (P88904) YNVVDYVGTAAPSQ-MCDLCQGQCPAVCINTLF

HCMV STLTNHLVGCAAG--LCEACGGTCCHTCYQTAF

LHV1 (Q82172) YNIAQYISTA-----------------------

MCMV DTVFSHIVNAGSK--LCGACGGRCCHTCYATSF

PorcHV4 (Q8QS31) STLSNHIVGCASS--LCEACGGTCCHTCYNTAF

PorcHV1 (Q8JYD5) YNISQYVGSAAVSN-VCQQCKGSYPCVCINTLF

PorcHV2 (Q8B424) YNICQYVGSAAVSN-VCQQCKGNYPCVCINTLF

PorcHV3 (Q8B408) YSISQYVGSVAGSN-VCQLCHGTCPCTCLNTLF

PHV1 (Q6UDK2) NTLKFVAAETTMAA-ECRWCTETTRQYCVRHTL

RCMV HAVYQHVVHSVGN--LCEACGGRCCHTCYATPF

Rhadinovirus (Q9WRU1) FNMVHYVGTAANSE-MCTLCHGNTPATCLNTLF

RhCMV STLSNHLIGCSSS--LCGACGGTCCHTCYNTAF

SaimHV2 YSVPQYVGTAAASD-LCELCQGTCPASCIHTLF

SCMV STLSNHLIGCSSS--LCGACGGTCCHTCYNTAF

SuidHV1 (Q5PPC5) DAVRYVAGSLDAEV-PCSLCDRASRPACAHTTL

THV1 SALYEHLVHCAVN--LCPACRGRCCQSCYQTAF

THV2 SALYEHLVHCAVN--LCPACRGRCCQSCYQTAF

Turtle HV (Q5Y969) TLLQYLNAKTEEGA--CDLCDVETRHVCPATTF

VZV DALKYVTGTFDSEI-PCSLCEKHTRPVCAHTTV

Figure 6 Putative zinc finger domain of UL57.

The DNA binding protein from 35 homologues are aligned to show conservation of the cysteines residues

(grey) in a region that is otherwise poorly conserved. Accession numbers (where available) are denoted

in brackets. Species abbreviations are as follows: AHV1: Alcelaphine herpesvirus 1 (wildebeest

herpesvirus); AtHV3: Ateline herpesvirus 3; BHV1/4/5: Bovine herpesvirus 1/4/5; CHV3: Callitrichine

herpesvirus 3 (marmoset lymphocryptovirus); EBV: Epstein-Barr virus; EHV1/2: Equine herpesvirus 1/2;

FATP: Fibropapilloma-associated turtle herpesvirus; GHV1: Gallid herpesvirus 1; GTH: Green turtle

herpesvirus; HGTH: Hawaiian green turtle herpesvirus; HCMV: Human cytomegalovirus; HHV6/7/8:

Human herpesvirus 6/7/8; HSV1/2: Herpes simplex virus 1/2; LHV1: Leporid herpesvirus 1; MCMV:

Murine cytomegalovirus; MMR: Macca Mulatta rhadinovirus; PongHV4: Pongine herpesvirus 4;

PorcHV1/2/3: Porcine herpesvirus 1/2/3; PHV1: Psittacid herpesvirus 1; RCMV: Rat cytomegalovirus;

RhCMV: Rhesus cytomegalovirus; SaimHV2: Saimiriine herpesvirus 2; SCMV: Simian cytomegalovirus;

SuidHV1: Pseudorabies virus; THV1/2: Tupaiid herpesvirus 1/2; VZV: Varicella-Zoster virus

60


Sequence alignments of the ten HCMV strains show that most variations occur in the C-

terminal third of the protein (Figure 7) and that key functional motifs predicted in HSV

are also highly conserved in HCMV (Biswas, 1999; Dracheva, 1995) (Figure 8A). In

addition, alignment of the UL70 homologues reaffirms the importance of the highly

conserved DxD catalytic motif (Dracheva, 1995) in a region that is otherwise poorly

conserved. The HCMV primase also retains a putative zinc finger motif originally

identified in HSV (Biswas, 1999). The zinc finger motif, spanning residues 881-920,

remained highly conserved amongst the HCMV isolates studied and was present in 90%

of the aligned homologues. In addition, a key phenylalanine residue involved in zinc

finger structural stability (Michael, 1992) was present in all HCMV strains at position

896 and conserved in 22 of the 30 (73%) homologues (Figure 8D). Further analysis also

revealed a pair of cysteines conserved across all 30 homologues (Figure 8C) while a

previously uncharacterized domain spanning amino acids 566-572 contained several

highly conserved aromatic residues (Figure 8B).

61

γ γ δ δ δ α γ UL70 1 11 21 31 41 51 61 71 81 91

Consensus MTLVLFATEYDSAHIVANVLSQTPTDHCVFPLLVKHQVSRRVYFCLQTQKCSDSRRVAPVFAVNNETLQLSRYLAARQPIPLSALIASLDEAETQPLYRH

AD169 ....................................................................................................

Merlin ..............................................................................................R.....

Towne ....................................................................................................

44A ..............................................................................................R.....

70A ..............................................................................................R.....

77A ....................................................................................................

90A ....................................................................................................

91A ..............................................................................................R.....

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

η γ γ 101 111 121 131 141 151 161 171 181 191

Consensus LFRTPVLSPEHGGEVREFKHLVYFHHAAVLRHLNQVFLCPTSPSWFISVFGHTEGQVLLTMAYYLFEGQYSTISTVEEYVRSFCTRDLGTIIPTHASMGE

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

62

γ ζ 201 211 221 231 241 251 261 271 281 291

Consensus FARLLLGSPFRQRVSAFVAYAVARNRRDYTELEQVDTQINAFRERARLPDTVCVHYVYLAYRTALARARLLEYRRVVAYDADAAPEAQCTREPGFLGRRL

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

β η η γ γ δ 301 311 321 331 341 351 361 371 381 391

Consensus STELLDVMQKYFSLDNFLHDYVETHLLRLDESPHSATSPHGLGLAGYGGRIDGTHLAGFFGTSTQLARQLERINTLSESVFSPLERSLSGLLRLCASLRT

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

63

δ γ 401 411 421 431 441 451 461 471 481 491

Consensus AQTYTTGTLTRYSQRRYLLPEPALAPLLERPLPVYRVHLPNDQHVFCAVASETWHRSLFPRDLLRHVPDSRFSDEALTETVWLHDDDVASTSPETQFYYT

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

γ γ γ γ 501 511 521 531 541 551 561 571 581 591

Consensus RHEVFNERLPVFNFVADFDLRLRDGVSGLARHTVFELCRGLRRVWMTVWASLFGYTHPDRHPVYFFKSACPPNSVPVDAAGAPFDDDDYLDYRDERDTEE

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A .................................I................................................S.................

70A ....................................................................................................

77A ....................................................................................................

90A ..................................................................................S.................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ..................................................................................S.................

30E ....................................................................................................

64

γ γ γ γ δ 601 611 621 631 641 651 661 671 681 691

Consensus DEDGKEDKNNVPDNGVFQKTTSSVDTSPPYCRCKGKLGLRIITPFPACTVAVHPSVLRAVAQVLNHAVCLDAELHTLLDPISHPESSLDTGIYHHGRSVR

AD169 ....................................................................................................

Merlin ............G....................................I..................................................

Towne ......N.............................................................................................

44A ....................................................................................................

70A ............G....................................I..................................................

77A .......................I............................................................................

90A ...................P..T..........................I..................................................

91A ............G....................................I..................................................

16B ..........................L.........................................................................

21B ..........................L.........................................................................

80B ............G.......................................................................................

4E ...................P..T.............................................................................

30E ..........................L.........................................................................

ε γ δ α ε 701 711 721 731 741 751 761 771 781 791

Consensus LPYMYKMDQDDGYFMHRRLLPLFIVPDAYREHPLGFVRAQLDLRNLLHHHPPHDLPALPLSPPPRVILSVRDKICPSTEANFIETRSLNVTRYRRRGLTE

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ......................................................................H.............................

21B ......................................................................H.............................

80B .............................................................................S......................

4E .............................................................................................Q......

30E ......................................................................H.............................

65

η γ α δ 801 811 821 831 841 851 861 871 881 891

Consensus VLAYHLYGGDGATAAAISDTDLQRLVVTRVWPPLLEHLTQHYEPHVSEQFTAPHVLLFQPHGACCVAVKRRDGARTRDFRCLNYTHRNPQETVQVFIDLR

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

δ δ 901 911 921 931 941

Consensus TEHSYALWASLWSRCFTKKCHSNAKNVHISIKIRPPDAPMPPATAV

AD169 .......................................V......

Merlin ..............................................

Towne ..............................................

44A ..............................................

70A .......................................V......

77A .......................................V......

90A ..............................................

91A ..............................................

16B .......................................V......

21B .......................................V......

80B ..............................................

4E ..............................................

30E .......................................V......


Residues highlighted in yellow indicate functionally significant amino acids (Biswas, 1999; Dracheva, 1995) while underlined sequences represent putative motifs identified

with ScanProsite. α = N-linked glycosylation site (N-x-T/S-x), β = cAMP-dependent protein kinase phosphorylation site (RR/KK/RK-x-S/T), γ = Casein kinase II

phosphorylation site (S/T-xx-D/E), δ = Protein kinase C phosphorylation site (S/T-x-R/K), ε = Tyrosine kinase phosphorylation site (R/K-xx-D/E-xxx-Y/R/K-xxx-D/E-xxx-

Y), ζ = Amidation site (x-G-RR/KK/RK), η = N-myristoylation site (G-A/N/C/Q/G/I/L/M/S/T/V-xx-all except P).

66

518 A 526 562 B 566 633 C 644 883 D 924

HCMV FVADFDLRL PVYFFKSACP PYCRCKGKLGLRI FRCLNYTHRN.24.SRCFTKKCHS

AHV1 WVLDLDLPI EVFFFKSACI TFCTCTEKLGMRV FKCLRYNHRG.24.SQCFANKCQS

AtHV3 LVLDLDLHI PVYFFKSSCE KFCYCTKKLGFRI FSCLNFKHKL.24.SQCFASKCNN

BHV1 LVMDFDLKV PVYFFKSAC- RFCNCNSKIGLRI FRCLTFNHRG.24.SQCFANKCNS

BHV4 IVLDVDFRL PCFFYKSACP AACGCEDKMGFRV FGCLRAAHGR.25.QQCFATKCGS

BHV5 IVLDVDFRL PCFFYKSACP TACGCDDKMGFRV FGCLRAAHGR.25.QQCFATKCGS

CercoHV1 IILDLDVAL PCYFFKSACS PRCTCAKKIGLRV LSCLRFKHGR.25.QQCFASKCDS

CercoHV9 IILDVDFHV PCYFYKSSC- NTCSCNKNLGFRV FTCLRYPH-R.25.QQCFATKCDS

CHV3 LVLDMDVKI PVYFFKSACP PFCICREKLGLRV FKCLRYQHRN.24.SQCFSGRCGS

EBV LVLDLDLKI PVYFFKSACP PFCICTGKLGFRV FACLRHTHRA.24.SQCFAGRCGA

EHV1 IILDVDFGI PCYFYKTSCP DACECTEKMGFRV FSCLRAKHLR.25.QQCFATKCGN

EHV2 LVLDFDLPL PVYFFKSACP PFCTCDAKLGLRI FVCISSNHRN.24.SQCFAHKCNS

EHV4 IILDVDFDI PCYFYKTQCP NACQCTEKMGFRI FSCLRAKHLR.25.QQCFATKCGN

GHV1 LILDIDIPL PVYFYKSHCS TLCKCKEKLGFRV FTCLKHSHRS.25.D---------

HHV6 YIGDLDLPL PIFFFKTQCD QFCVCRKKIGLRI FSCLNRQHRG.24.STCFATKCQS

HHV7 YIGDLDLPL PIFFFKTTCS AFCVCKKKIGLRI FSCLTRNHKG.24.SKCFTTKCKS

HHV8 WVLDFDLPV PVYFFKSACP SFCRCHDKLGMRI FLCINHNHKN.24.SQCFASKCNN

HSV1 IILDLDIAL PCYFFKSACR PVCSCTDKIGLRV LSCLRFKHGR.25.QQCFAAKCDS

HSV2 IILDLDIAL PCYFFKSACR VVCSCADKIGLRV LSCLRFKHGR.25.QQCFATKCDN

MCMV FVGDVDLKL PVFFFKSAC- PFCVCRRKLGLRV FSCLARETYT.24.SRCFTRRCNS

MDHV1 IVLDVDIHL PCYFYKSSCK KPCGCHDKIGLRV FTCVRFKHAR.25.QQCFAAKCGN

MMR WVLDFDLKV PVYFFKSACP AFCHCDAKIGMRI FFCINHKHRN.24.SQCFAAKCNH

PhoHV2 LVLDFDLPL QVYFFKSSCP MFCNCSEKLGLRV ------------------------

PHV1 LPLDLDITL PVYFYKTQCD RFCRCERKIGFRI FTCLKYQHRG.25.TRCFATKCGS

PongHV4 FVADMDLRL PVYFFKSACR LYCRCTEKLGLRI FRCLNYVHRN.24.SRCFTKKCHS

RCMV FVGDLDLKL PVYFFKSACD -FCTCRRKIGMRI FRCLTTEHHF.25.SRCFANKCQS

RhCMV FIADFDLRL PVYFFKSACK DYCKCSEKLGLRI FRCLNYTHRN.24.SRCFTKKCHS

SaimHV2 LVLDLDLHI HVYFFKSACE KFCYCTKKLGFRI FSCLNFKHKL.24.SQCFASKCNS

THV1 LALDVDLAL PVYFFKSACP DFCACRAKLGFRV FRCLSRAHRI.24.SRCFATKCRS

VZV LILDVDFHI PCYFYKTACP LPCNCKEKIGFRV FTCLRYPH-R.25.QQCFATKCDS

* * * * * * * * * * * * ** *

Figure 8 Conserved domains between UL70 homologues.

(A) The DxD catalytic motif. (B) Domain consisting of several conserved aromatic residues. (C)

Conserved cysteines. (D) Putative zinc finger domain. Species abbreviations are as follows: AHV1:

Alcelaphine herpesvirus 1 (wildebeest herpesvirus); AtHV3: Ateline herpesvirus 3; BHV1/4/5: Bovine

herpesvirus 1/4/5; CercoHV1: Cercopothecine herpesvirus 1 (Simian herpes B virus); CercoHV9:

Cercopothecine herpesvirus 9 (Simian varicella virus); CHV3: Callitrichine herpesvirus 3 (marmoset

lymphocryptovirus); EBV: Epstein-Barr virus; EHV1/2/4: Equine herpesvirus 1/2/4; GHV1: Gallid

herpesvirus 1; HCMV: Human cytomegalovirus; HHV6/7/8: Human herpesvirus 6/7/8; HSV1/2: Herpes

simplex virus 1/2; MCMV: Murine cytomegalovirus; MDHV1: Meleagrid herpesvirus 1; MMR: Macca

Mulatta rhadinovirus; PongHV4: Pongine herpesvirus 4; PorcHV1/2/3: Porince herpesvirus 1/2/3;

PhoHV2: Phocid herpesvirus 2; PHV1: Psittacid herpesvirus 1; RCMV: Rat cytomegalovirus; RhCMV:

Rhesus cytomegalovirus; SaimHV2: Saimiriine herpesvirus 2; THV1: Tupaiid herpesvirus 1; VZV:

Varicella-Zoster virus. * denotes highly conserved residues

67


Sequence alignments of the UL102 isolates show a highly conserved protein, although

alignments with homologues yielded a low degree of conservation for any region. As

with UL44, a putative integrin binding motif was also detected in UL102 at amino acid

residue 58. An alanine to glycine substitution at residue 76 in AD169 allowed the

formation of another N-myristoylation site while the insertion of additional glycines in

isolates 44A and 80B also increased the number of N-myristoylation sites formed

(Figure 9). ScanProsite detected a glycine-rich domain (score = 12.88) between residues

553-687 and a short serine-rich domain spanning residues 795-836 (score = 12.8;

normative score = 7.22). Similar to UL44, the glycine-rich domain in UL102 accounted

for a significant number of the motifs identified. Isolate variation within the glycine-rich

region spanning residues 629-633 was observed, with some isolates containing

additional and deleted glycine residues. Similar variations were not observed in the

glycine rich regions of UL44 or UL57.

68

η η ζ η γ UL102 1 11 21 31 41 51 61 71 81 91

Consensus MTAQPPLHHRHHPYTLFGTSCHLSWYGLLEASVPIVQCLFLDLGGGRAEPRLHTFVVRGDRLPPAEVRAVHRASYAALASAVTTDADERRRGLEQRSAVL

AD169 ..........................................................................-G........................

Merlin ....................................................................................................

Towne ..............A.....................................................................................

44A .....S..............................................................................................

70A .............C......................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................V...............................

30E ....................................................................................................

γ η γ 101 111 121 131 141 151 161 171 181 191

Consensus ARVLLEGSALIRVLARTFTPVQIQTDASGVEILEAAPALGVETAALSNALSLFHVAKLVVIGSYPEVHEPRVVTHAAERVSEEYGTHAHKKLRRGYYAYD

AD169 ...........................................................................T........................

Merlin ...........................................T........................................................

Towne .....................................................................S..............................

44A ............................................................................T.......................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ...........................................T........................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

69

δ δ η 201 211 221 231 241 251 261 271 281 291

Consensus LAMSFRVGTHKYVLERDDEAVLARLFEVREVCFLRTCLRLVTPVGFVAVAVTDEQCCLLLQSAWTHLYDVLFRGFAGQPPLRDYLGPDLFETGAARSFFF

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

γ η α 301 311 321 331 341 351 361 371 381 391

Consensus PGFPPVPVYAVHGLHTLMRETALDAAAEVLSWCGLPDIVGSAGKLEVEPCALSLGVPEDEWQVFGTEAGGGAVRLNATAFRERPAGGDRRWLLPPLPRDD

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ......................................................................................S.............

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

70

β δ δ 401 411 421 431 441 451 461 471 481 491

Consensus GDGENNVVEVSSSTGGAHPPSDDATFTVHVRDATLHRVLIVDLVERVLAKCVRARDFNPYVRYSHRLHTYAVCEKFIENLRFRSRRAFWQIQSLLGYISE

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ...........................................L........................................................

21B ...........................................L........................................................

80B ....................................................................................................

4E ....................................................................................................

30E ...........................................L........................................................

β α δ η 501 511 521 531 541 551 561 571 581 591

Consensus HVTSACASAGLLWVLSRGHREFYVYDGYSGHGPVSAEVCVRTVVDCYWRKLFGGDDPGPTCRVQESAPGVLLVWGDERLVGPFNFFYGNGGAGGSPLHGV

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ..........................................................L.........................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ................................................................................................F...

30E ....................................................................................................

71

η η η δ η η η δ η 601 611 621 631 641 651 661 671 681 691

Consensus VGGFAAGHCGGACCAGCVVTHRHSSGGGG--SGVGDADHASGGGLDAAAGSGHNGGSDRVSPSTPPAALGGCCCAAGGDWLSAVGHVLGRLPALLRERVS

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A .............................GG............D......G.................................................

70A ....................................................................................................

77A .............................G......................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ............................-..--.......................G...........................................

80B .............................GG.....................................................................

4E ............................-.......................................................................

30E ............................-..--.......................G...........................................

γ η 701 711 721 731 741 751 761 771 781 791

Consensus VSELEAVYREILFRFVARRNDVDFWLLRFQPGENEVRPHAGVIDCAPFHGVWAEQGQIIVQSRDTALAADIGYGVYVDKAFAMLTACVEVWARELLSSST

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

72

γ 801 811 821 831 841 851 861 871

Consensus ASTTACSSSSVLSSALPSVTSSSSGTATVSPPSCSSSSATWLEERDEWVRSLAVDAQHAAKRVASEGLRFFRLNA

AD169 ...........................................................................

Merlin ....T......................................................................

Towne ...........................................................................

44A ...........................................................................

70A .........-.................................................................

77A ...........................................................................

90A ...........................................................................

91A ....T......................................................................

16B ...........................................................................

21B ...........................................................................

80B ....T......................................................................

4E ....T....-.................................................................

30E ...........................................................................

Figure 9 HCMV strain alignments for UL102

Underlined sequences represent putative motifs identified with ScanProsite. α = N-linked glycosylation site (N-x-T/S-x), β = Tyrosine kinase phosphorylation site (R/K-xx-

D/E-xxx-Y/R/K-xxx-D/E-xxx-Y), γ = Casein kinase II phosphorylation site (S/T-xx-D/E), δ = Protein kinase C phosphorylation site (S/T-x-R/K), ζ = Cell attachment site (R-

G-D), η = N-myristoylation site (G-A/N/C/Q/G/I/L/M/S/T/V-xx-all except P).

73


Sequence alignments of the UL105 isolates show a highly conserved protein with sparse

variation occurring outside of predicted functional domains (Figure 10). UL105 is

postulated to contain six motifs typical of superfamily-1 class of helicase proteins (Zhu,

1992) based on studies of the HSV helicase, UL5 (Graves-Woodward, 1997).

ScanProsite detected an ATP/GTP-binding site in motif I as well as a putative

cAMP/cGMP-dependent protein kinase phosphorylation site in Motif IV. Protein

alignments of the UL105 homologues predicted 2 potential N-linked glycosylation sites

as well as a potential CKII phosphorylation site based on conservation across 33

homologues (Figure 11).

74

δ δ α γ UL105 1 11 21 31 41 51 61 71 81 91

Consensus MSMTASSSTPRPTPKYDDALILNLSSAAKIERIVDKVKSLSRERFAPEDFSFQWFRSISRVERTTDNNPSAATTAAATTTVHSSASSSAAAAASSEAGGT

AD169 ....................................................................................................

Merlin ..............................................................................A.....V...............

Towne ....................................................................................................

44A ....................................................................................................

70A ..I..........S......................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ..............................................................................A.....V...............

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ..............................................................................A.....................

30E ....................................................................................................

ζ α α 101 111 121 131 141 151 161 171 181 191

Consensus RVPCVDRWPFFPFRALLVTGTAGAGKTSSIQVLAANLDCVITGTTVIAAQNLSAILNRTRSAQVKTIYRVFGFVSKHVPLADSAVSHETLERYRVCEPHE

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

75

201 211 221 231 241 251 261 271 281 291

Consensus ETTIQRLQINDLLAYWPVIADIVDKCLNMWERKAASASAAAAAAACEDLSELCESNIIVIDECGLMLRYMLQVVVFFYYFYNALGDTRLYRERRVPCIIC

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B .............................G.......................................VVG..L.........................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

γ γ α δ β δ 301 311 321 331 341 351 361 371 381 391

Consensus VGSPTQTEALESRYDHYTQNKSVRKGVDVLSALIQNEVLINYCDIADNWVMFIHNKRCTDLDFGDLLKYMEFGIPLKEEHVAYVDRFVRPPSSIRNPSYA

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

76

γ δ ε α α 401 411 421 431 441 451 461 471 481 491

Consensus AEMTRLFLSHVEVQAYFKRLHEQIRLSERHRLFDLPVYCVVNNRAYQELCELADPLGDSPQPVELWFRQNLARIINYSQFVDHNLSSEITKEALRPAADV

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B D.............................C.....................................................................

80B ................................................................F...................................

4E ....................................................................................................

30E ....................................................................................................

α η η δ δ η γ γ ε 501 511 521 531 541 551 561 571 581 591

Consensus VATNNSSVQAHGGGGSVIGSTGGNDETAFFQDDDTTTAPDSRETLLTLRITYIKGSSVGVNSKVRACVIGYQGTVERFVDILQKDTFIERTPCEQAAYAY

AD169 ....................................................................................................

Merlin .....P..............................................................................................

Towne ....................................................................................................

44A .........T..........................................................................................

70A ....................................................................................................

77A .....P..............................................................................................

90A ..................................I.................................................................

91A .....P..............................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ..N.-P..K...........................................................................................

4E .....P..............................................................................................

30E ....................................................................................................

77

η γ γ δ η 601 611 621 631 641 651 661 671 681 691

Consensus SLVSGLLFSAMYYFYVSPYTTEEMLRELARVELPDVSSLCAAAAATAAAPAWSGGENPINNHVDADSSQGGQSVPVSQRMEHGQEETHDIPCLSNHHDDS

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ...............................................................................I....................

70A ...................................................................................................L

77A ....................................................................................................

90A ................................................................................K...................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E .........................................................L..........................................

30E ....................................................................................................

γ γ δ γ η η 701 711 721 731 741 751 761 771 781 791

Consensus DAITDAELMDHTSLYADPFFLKYVKPPSLALLSFEETVHMYTTFRDIFLKRYQLMQRLTGGRFATLPLVTYNRRNVVFKANCQISSQTGSFVGMLSHVSP

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ..........Y.........................................................................................

44A ....................................................................................................

70A .V..................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

78

δ α γ δ 801 811 821 831 841 851 861 871 881 891

Consensus AQTYTLEGYTSDNVLSLPSDRHRIHPEVVQRGLSRLVLRDALGFLFVLDVNVSRFVESAQGKSLHVCTTVDYGLTSRTAMTIAKSQGLSLEKVAVDFGDH

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ....................................................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

80B ....................................................................................................

4E ....................................................................................................

30E ....................................................................................................

γ β 901 911 921 931 941 951

Consensus PKNLKMSHIYVAMSRVTDPEHLMMNVNPLRLPYEKNTAITPYICRALKDKRTTLIF

AD169 ........................................................

Merlin ........................................................

Towne ........................................................

44A ........................................................

70A ........................................................

77A ........................................................

90A ........................................................

91A ........................................................

16B ........................................................

21B ........................................................

80B ........................................................

4E ........................................................

30E ........................................................


Residues highlighted in yellow indicate functionally significant motifs (Graves-Woodward, 1996; Graves-Woodward, 1997) while underlined sequences represent putative

motifs identified with ScanProsite. α = N-linked glycosylation site (N-x-T/S-x), β = cAMP-dependent protein kinase phosphorylation site (RR/KK/RK-x-S/T), γ = Casein

kinase II phosphorylation site (S/T-xx-D/E), δ = Protein kinase C phosphorylation site (S/T-x-R/K), ε = Tyrosine kinase phosphorylation site (R/K-xx-D/E-xxx-Y/R/K-xxx-

D/E-xxx-Y), ζ = ATP/GTP-binding site motif A (A/G-xxxx-G-K-S/T), η = N-myristoylation site (G-A/N/C/Q/G/I/L/M/S/T/V-xx-all except P).

79

20 A 27 409 B 415 473 C 481

HCMV LILNLSSAA MTRLFLSHVEVQA RIINYSQFV

AHV1 FILNMTSEA WTRLFLSHSEVKQ RLSNYSQFI

AtHV3 FILNMTSDA WTRLFLSHAEVKS RLSNYSQFI

BHV1 FILNMTSDA WTRLFLSHNEVKA RLSNYSQFI

BHV4 VFLNFTSMH WTRLYSSHREVSA RITNYSQSR

BHV5 VFLNFTSMH WTRLYSSHREVSA RITNYSQSR

CHV3 FMLNMTSDA WTRLFLSHAEVKQ RLGNYSQFA

CercoHV1 VYLNFTSMH WTRLYSSHKEVSA RLNNYSQSR

CercoHV9 AFLNFTSMH WTRLFSSHKEVSA RITNYSQSQ

EBV FMLNMTSDA WTRLFLSHAEVKT RLGNYSQFA

EHV1 VYLNFTSMH WTRLYSSHKEVSA RVSNYSQSR

EHV2 FILNMTSDS WTRLFVSHREVKA RLSNYSQFI

EHV4 VYLNFTSMH WTRLYSSHKEVSA RVSNYSQSR

GHV1 TYLNFTAMH WTRLYSSHKEVSA RLSNWSQSR

GHV3 VYLNFSAMQ WTRLFSSHEEVKE RLGNYSQSR

HSV1 AFLNFTSMH WTRLFSSHKEVSA RITNYSQSQ

HSV2 AFLNFTSMH WTRLFSSHKEVSA RITNYSQSQ

HHV6 FLLNMSSAP TTRLFLSHNEVKN RLNTYSQFA

HHV7 FLLNMSSAA MTRLFLSHYEVKS RLNTYSQFA

HHV8 FILNMTSDA WTRLFISHQEVKS RLSNYSQFA

MCMV FVLNMSSAS VTRLFISHAEVKR RISNYSQFT

MDHV1 TYLNFTAMH WTRLYSSHKEVST RLSNWSQSR

MD TYLNFTAMH WTRLYSSHKEVSA RLSNWSQSR

MMR FILNMTSDA WTRLFLSHSEVKA KLSNYSQFV

PorcHV1 FFLNMTSDS WTRLFLSHIEVKN RLSNYSQFV

PorcHV2 FFLNMTSDA WTRLFLSHAEVKN RLSNYSQFV

PongHV4 LILNLSSAA MTRLFLSHAEVQL RIINYSQFV

Pseudorabies TYLNFTSMH WTRLYSSHREVSA RITNYSQSR

RCMV FVLNTSSAL ATRLFVSHREVKD RIGNYSQFT

RhCMV LILNLSSAA MTRLFLSHVEVQA RIINYSQFV

SuidHV1 TYLNFTSMH WTRLYSSHREVSA RITNYSQSR

THV1 FILNMSSAV MTRLFISHAEVKR RIINYSQFV

VZV VYLNFTSMH WTRLYSSHKEVSA RLHNYSQSR

*** * * *

Figure 11 Putative motifs conserved across UL105 homologues

(A) and (C) Conserved putative N-linked glycosylation sites. (B) Conserved putative CK2

phosphorylation site. Species abbreviations are as follows: AHV1: Alcelaphine herpesvirus 1 (wildebeest

herpesvirus); AtHV3: Ateline herpesvirus 3; BHV1/4/5: Bovine herpesvirus 1/4/5; CercoHV1:

Cercopothecine herpesvirus 1 (Simian herpes B virus); CercoHV9: Cercopothecine herpesvirus 9 (Simian

varicella virus); CHV3: Callitrichine herpesvirus 3 (marmoset lymphocryptovirus); EBV: Epstein-Barr

virus; EHV1/2/4: Equine herpesvirus 1/2/4; GHV1/3: Gallid herpesvirus 1/3; HCMV: Human

cytomegalovirus; HHV6/7/8: Human herpesvirus 6/7/8; HSV1/2: Herpes simplex virus 1/2; MCMV:

Murine cytomegalovirus; MDHV1: Meleagrid herpesvirus 1; MD: Marek’s disease; MMR: Macca

Mulatta rhadinovirus; PongHV4: Pongine herpesvirus 4; PorcHV1/2: Porince herpesvirus 1/2; RCMV:

Rat cytomegalovirus; RhCMV: Rhesus cytomegalovirus; SuidHV1: Pseudorabies virus; THV1: Tupaiid

herpesvirus 1; VZV: Varicella-Zoster virus.

80


Sequence alignments revealed a high degree of variation amongst the isolates (Figure

12), especially in a region that has been associated with oligomerization of the UL84

protein (Colletti, 2004). In particular, deletions of glutamine residues in three clinical

isolates (16B, 21B and 30E) were observed in a region that was previously associated

with nuclear import (Xu, 2002) and is part of a previously described hydrophobic region

(He, 1992). Two overlapping CKII phosphorylation sites (consensus site = S/TxxD/E)

(Pinna, 1990) TLQE and SQEE, were detected flanking this region (Figure 13) and

interestingly, three strains contained only a single CKII site; two strains (AD169 and

70A) had substitutions which abolished the second motif while another isolate (77A)

abolished the first motif. ScanProsite also detected a previously described leucine zipper

domain (He, 1992) at the amino-terminus of UL84.

81

η η η η δ α δ η γ γ γ η γ δ UL84 1 11 21 31 41 51 61 71 81 91

consensus MPRVDPNLRNRARRPRARRGGGGGVGSNSSRHSGKCRRQRRALSAPPLTFLATTTTTTMMGVASTDDDSLLLKTPDELDKHSGSPQTILTLTDKHDIRQP

AD169 ................................................................................Y...................

Merlin ............................................T.......................................................

Towne ...A................................................................................................

44A ....................................................................................................

70A ................................................................................Y...................

77A ............................................T.......................................................

90A ............................................T.......................................................

91A ............................................T.......................................................

16B ..........................T.........................................................................

21B ..........................T.........................................................................

4E ........................................................R...........................................

30E ..........................T.........................................................................

ε γ γ γ 101 111 121 131 141 151 161 171 181 191

consensus RVHRGTYHLIQLHLDLRPEELRDPFQILLSTPLQLGEANGESQTAPATSQEEETAASHEPEKKKEKEE-KKEEDEDDRNDDRERGILCVVSNEDSDVRPA

AD169 .......................................D........L.................Q.................................

Merlin ....................................................................K.E.............................

Towne ...........................................................L........K.E.............................

44A ............................................DL.....................K................................

70A ................................................L.................Q.................................

77A ..............................................TM....................K.E.............................

90A ..............................................T.....................K.E.............................

91A ....................................................................K.E.............................

16B ...................................................................-................................

21B ...................................................................-................................

4E ....................................................................................................

30E ...................................................................-................................

82

γ α γ γ 201 211 221 231 241 251 261 271 281 291

consensus FSLFPARPGCHILRSVIDQQLTRMAIVRLSLNLFALRIITPLLKRLPLRRKAAHHTALHDCLALHLPELTFEPTLDINNVTENAASVAD AESTDADLTP

AD169 .........................................................................................T..........

Merlin .........................................................................................T..........

Towne .........................................P...V...............M..........S................A..........

44A .................................................................................................... 70A .........................................................................................T..........

77A ....................................................................................V.----.....T....

90A .........................................................................................T..........

91A .........................................................................................T..........

16B ......................................................................................A..A..........

21B ......................................................................................A..A..........

4E .....................................................................................TA..A..........

30E ......................................................................................A..A..........

δ δ δ δ γ α β 301 311 321 331 341 351 361 371 381 391

consensus TLTVRVRHALCWHRVEGGISGPRGLTSRISARLSETTAKTLGPSVFGRLELDPNESPPDLTLSSLTLYQDGILRFNVTCDRTEAPADPVAFRLRLRRETV

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne .........V.............................................................M............................

44A .......................................................................M............................

70A ....................................................................................................

77A .......................................................................M............................

90A ....................................................................................................

91A ....................................................................................................

16B .........................................................................................T..........

21B .........................................................................................T..........

4E ....................................................................................................

30E .........................................................................................T..........

83

δ δ δ γ 401 411 421 431 441 451 461 471 481 491

consensus RRPFFSDAPLPYFVPPRSGAADEGLEVRVPYELTLKNSHTLRIYRRFYGPYLGVFVPHNRQGLKMPVTVWLPRSWLELTVLVSDENGATFPRDALLGRLY

AD169 ....................................................................................................

Merlin ....................................................................................................

Towne ....................................................................................................

44A ....................................................................................................

70A ....................................................................................................

77A ...........................H........................................................................

90A ....................................................................................................

91A ....................................................................................................

16B ....................................................................................................

21B ....................................................................................................

4E ...........................................................C........................................

30E ....................................................................................................

δ η η α δ 501 511 521 531 541 551 561 571 581

consensus FISSKHTLNRGCLSAMTHQVKSTLHSRSTSHSPSQQQLSVLGASIALEDLLPMRLASPETEPQDCKLTENTTEKTSPVTLAMVCGDL

AD169 .......................................................................................

Merlin .......................................................................................

Towne .......................................................................................

44A .......................................................................................

70A ..............................Y........................................................

77A .......................................................................................

90A .......................................................................................

91A .......................................................................................

16B .......................................................................................

21B .......................................................................................

4E ...................................................................M....G..............

30E .......................................................................................


Residues highlighted in yellow indicate functionally significant motifs (Colletti, 2004; He, 1992; Lischka, 2006) while underlined sequences represent putative motifs

identified with ScanProsite. α = N-linked glycosylation site (N-x-T/S-x), β = cAMP-dependent protein kinase phosphorylation site (RR/KK/RK-x-S/T), γ = Casein kinase II

phosphorylation site (S/T-xx-D/E), δ = Protein kinase C phosphorylation site (S/T-x-R/K), ε = Leucine zipper (L-xxxxxx-L-xxxxxx-L-xxxxxx-L), η = N-myristoylation site

(G-A/N/C/Q/G/I/L/M/S/T/V-xx-all except P).

84

Consensus PATSQEEETAASHEPEKKKEKEE-KKEEDEDDRNDDRERGILCVVSNEDSDVRPA

AD169 PATLQEEETAASHEPEKKKEKQEKKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

Merlin PATSQEEETAASHEPEKKKEKEEKKEEEDEDDRNDDRERGILCVVSNEDSDVRPA

Towne PATSQEEETAASHELEKKKEKEEKKEEEDEDDRNDDRERGILCVVSNEDSDVRPA

44A LATSQEEETAASHEPEKKKEKEKKKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

70A PATLQEEETAASHEPEKKKEKQEKKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

77A PTMSQEEETAASHEPEKKKEKEEKKEEEDEDDRNDDRERGILCVVSNEDSDVRPA

90A PTTSQEEETAASHEPEKKKEKEEKKEEEDEDDRNDDRERGILCVVSNEDSDVRPA

91A PATSQEEETAASHEPEKKKEKEEKKEEEDEDDRNDDRERGILCVVSNEDSDVRPA

16B PATSQEEETAASHEPEKKKEKE-KKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

21B PATSQEEETAASHEPEKKKEKE-KKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

4E PATSQEEETAASHEPEKKKEKEEKKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

30E PATSQEEETAASHEPEKKKEKE-KKEE-DEDDRNDDRERGILCVVSNEDSDVRPA

**** ************* *** **** **********************

Figure 13 Overlapping putative casein kinase-2 (CKII) phosphorylation sites of UL84

CKII

85

3.4 Discussion

Several putative motifs were identified using protein scanning software and although

the core HCMV replication genes have already been characterized (Anders, 1988; Ertl,

1992; Pari, 1993; Smith, 1995a; Smith, 1996; Weiland, 1994), their significance in

replication was mainly predicted from similarities with the HSV counterparts (Anders,

1988; Pari, 1993). By combining the novel approach of motif scanning and phylogenetic

analysis between isolates and across species homologues, we have identified several

putative functional motifs in HCMV that may contribute to a complete understanding of

the CMV replication cycle.

One particular class of motifs has gained much attention in recent times. CKII is a

cellular serine-threonine kinase that has multiple target substrates involved in nucleic

acid replication, transcription, and protein synthesis among other biological processes

(Meggio, 2003). Active CKII is incorporated into the HCMV virion resulting in

immediate post-infection phosphorylation of cellular proteins that enhance HCMV

replication (Nogalski, 2007). There is also increasing evidence that CKII enhances

HCMV replication through phosphorylation of several HCMV proteins involved in

DNA replication and other processes (Alvisi, 2005; Barrasa, 2005; Jarvis, 2004). This

includes the UL44 DNA polymerase accessory protein that contains a (S/T)xx(D/E)

consensus CKII phosphorylation motif (also confirmed here) and is phosphorylated at

serine and/or threonine residues (Alvisi, 2005). In this study, we have identified

interesting and potential CKII phosphorylation motifs in the HCMV helicase (UL105)

and putative initiator protein (UL84), the latter recently shown to be a substrate of CKII

in pull-down assays (Gao, 2007). Although the previously described putative NLS

spanning residues 160-171 (Xu, 2002) has been disproved of its role in nuclear import

(Lischka, 2003), the position of the CKII phosphorylation site relative to this putative

NLS in UL84 (Xu, 2002) resemble a pattern shared by the CKII enhancing the NLS of

UL44 (Alvisi, 2005), which also binds with high affinity to the importin α/β

heterodimer (Alvisi, 2005). In addition, both these motifs fit the ‘CcN’ profile described

for phosphorylation regulated nuclear localization (Jans, 1996).

UL84 is an essential regulatory protein that is required for oriLyt-dependent DNA

replication (Lischka, 2003; Xu, 2004; Yu, 2003). Of the six replication genes studied, it

86

also had the lowest amino acid identity when compared against clinical isolates with a

score ranging from 97.46% to 100% identity. Initial characterization of UL84 identified

two potential leucine zippers between amino acids 114 to 135 and 325 to 373 (He,

1992), as well as a highly charged region starting at amino acid 161 (He, 1992).

Coincidentally, this region was initially associated with nuclear import (Xu, 2002),

although subsequent studies later mapped nuclear localization to a complex domain

downstream of this region (Lischka, 2003). In addition, two nuclear export signals (NES)

starting at amino acids 229 and 360 were also found to act as a non-conventional NLS

which allowed pUL84 to shuttle to the cytoplasm (Lischka, 2006). Recent studies have

shown that several regions of pUL84 share similar homology to the DExD/H box family

helicases (Colletti, 2005) while another bioinformatic study has shown homology to

dUTPases (Davison, 2005), both of which support the hypothesis that pUL84 acts as an

initiator protein for viral-DNA synthesis of HCMV (Xu, 2004a). PROSITE scanning for

potential motifs yielded three potential N-linked glycosylation sites while in earlier

studies, five were detected (He, 1992). However, on closer inspection one of the

original five N-linked glycosylation sites, beginning at amino acid 354, would have

been discounted as having functional significance on the basis of proline being present

within the consensus sequence (Bause, 1983; Gavel, 1990). BLAST searching revealed

only four sequence homologues, though when aligned showed a high degree of

conservation for both of the NES as well as a putative CKII phosphorylation site at

UL84 amino acid residue 474 identified in our studies. In addition, the putative CKII

phosphorylation site lies upstream of a motif suggested to be associated with helicase

function (Colletti, 2005) and may have a regulatory role. The putative leucine zipper

starting at amino acid 114 in HCMV was also detected in our scanning programs, while

the putative zipper starting at residue 325 was not, albeit neither were conserved

structures when aligned against the BLAST result homologues.

Among the ten HCMV isolates studied for UL44, variations in peptide sequence

occurred outside of regions currently known to have a functional role with the exception

of a K431R substitution in the previously identified NLS (Alvisi, 2005). Both the

HCMV UL44 and the HSV-1 homolog (UL42) share a “connector loop” structure that

plays a crucial role in binding to the DNA polymerase (Appleton, 2004; Bridges, 2001;

Loregian, 2004a). However, the sequence structure for the HCMV connector loop is

better conserved across the species, with more than 50% of the sequence alignments

87

having a similar structure of at least 57% sequence homology in that region. Although

the crucial functions of UL44 has previously been mapped to the N-terminal two-thirds

of the gene (Weiland, 1994), more than half of the potential motifs identified for UL44

in this study were found in the C terminal third of the gene, which has been determined

to be functionally dispensable for replication (Ertl, 1992). However, a recent study

elucidating the presence of a nuclear localization signal located at residues 425-431 of

the C-terminus (Alvisi, 2005) suggests that further study into this region needs to be

done, considering that the putative casein kinase-2 (CK2) site enhancing the NLS

(Alvisi, 2005) was also identified in our motif scans. Further analysis revealed an

unusual integrin binding motif in UL44 that was later shown to be conserved in pongine

Herpesvirus 4 as well. Integrins are known to mediate cell-cell association as well as

interactions between cells and extra-cellular proteins (Haynes, 2002). In addition, a

previous study on the murine CMV M44 homologue protein identified a putative

integrin binding RGD motif that was capable of mediating cell adhesion (Loh, 2000),

suggesting that UL44 could play a novel role in CMV infection.

Structure-function prediction for the UL57 single stranded DNA binding protein in

HCMV were largely based on studies of the HSV-1 homolog ICP8, which has so far

mapped the cooperative DNA binding region as well as the nuclear localization signal

to the C-terminus (Dudas, 1998; Gao, 1992; Mapelli, 2000), while an intranuclear

localization site was found to be located between residues 1080-1135 (Taylor, 2003). A

putative zinc binding motif between amino acids 499-512 in ICP8 (Gao, 1988; Gupte,

1991) was also found to be conserved in 16 of the 35 (46%) homolog sequences for the

single stranded DNA binding protein, albeit it was found to be non-essential for DNA

binding in HSV-1 (Wang, 1990). Although formal elucidation of its function in other

species of herpesvirus is yet to be validated, the structural nature of the motif is missing

key elements including a conserved phenylalanine and leucine involved in stabilizing

the motif (Jasanoff, 1993; Michael, 1992). However, the N terminal fragment of the

putative zinc finger beginning at amino acid 472 of UL57 showed a potential disulfide

bond that was conserved in 34 of the 35 (97%) homolog alignments. The conservation

of this putative disulfide bond motif across the species indicates an evolutionarily

conserved functional domain. Currently, the exact DNA binding region has yet to be

elucidated with numerous studies detailing a range of potential active sites (Gao, 1989;

Wang, 1990; White, 1999). The presence of a micro-bodies targeting signal observed in

88

the C-terminus of UL57, while yet to be functionally elucidated, suggests this protein

may be involved with the non-viral, non-lysosomal microbodies which have been

observed in CMV infected cells for decades (Craighead, 1972; Ruebner, 1966).

The primase-helicase complex of HCMV consisting of UL70, UL102, and UL105 is

considered to be homologous to the HSV-1 primase-helicase complex consisting of

UL52, UL8, and UL5 respectively (McMahon, 2002; Pari, 1993; Smith, 1996). All

three genes were highly conserved amongst the clinical isolates ranging from 98% to

100% sequence identity and 99 to 100% amino acid identity. Although the HCMV

primase (UL70) has not been formally characterized, it shares two functionally distinct

motifs that have been documented in its HSV-1 counterpart, UL52. The DxD catalytic

motif is essential for initiating DNA synthesis via the primer synthesis (Dracheva, 1995;

Klinedinst, 1994) while substitutions of the third and fourth conserved cysteines in the

zinc-finger at the C terminal end resulted in the loss of primase, helicase, ATPase, and

DNA-binding activities of the HSV helicase-primase complex (Biswas, 1999). The zinc

binding residues CHCC were found to be conserved in 26 of the 30 (87%) herpesvirus

homologues, while a key phenylalanine involved in structural stability (Michael, 1992)

was conserved in 22 of the 30 (73%) sequences. Interestingly, putative zinc fingers

lacking the conserved phenylalanine or an ‘aromatic swap’ (Kochoyan, 1991)

equivalent have been shown to be non-essential for key biological functions such as

DNA binding and replication (Hui, 2003; Wang, 1990). Analysis of the UL70

homologues show a highly conserved cluster of aromatic residues preceding a

conserved cysteine which has been associated with conferring resistance to a compound

which currently inhibits the HCMV primase (Chen, 2007). Aromatic interactions have

been attributed to involve up to 60% of the aromatic residues in a protein of which 80%

of these contribute to tertiary structure (Burley, 1985) and may provide some insight

into the structure-function relationship of that domain. Further analysis also revealed a

pair of cysteines conserved across all 30 homologues while a previously

uncharacterized domain spanning amino acids 631-646 contained several highly

conserved residues amongst the isolates and homologues in an otherwise poorly

conserved region (Figure 14B). Protein disulfide isomerase (PDI), with the canonical

CxxC motif, is the most efficient known catalyst of oxidative protein folding

(Weissman, 1993). Our phylogenetic analyses uncovered two highly conserved cysteine

residues forming a CxC motif in a previously uncharacterized domain of UL70 primase

89

and 30 primase homologues. The conservation of this CxC motif suggest that the CMV

primase may utilize a rare thiol-disulfide exchange mechanism (Woycechowsky, 2003)

that is present in only a handful of organisms including the Mengo encephalomyelitis

virus coat protein (Krishnaswamy, 1990), Bacillus Ak.1 protease (Smith, 1999) and the

E.Coli chaperone, Hsp33 (Jakob, 1999). The CxC motif is known to mediate disulfide

bond interactions between other conserved cysteines to form the well defined tertiary

structure for the chemokine class of proteins (Fernandez, 2002), and hence may have a

similar role in stabilizing the UL70 tertiary structure of HCMV here. In addition, an

earlier study of the HSV homologue demonstrated that substitutions of the arginine

residue seven amino acids downstream of the putative domain decreased the replication

efficiency of HSV by more than 50% (Klinedinst, 1994), suggesting that functionally

significant residues lie in close proximity to the CxC motif.

UL102 is a positional homolog of the HSV-1 UL8 primase associated factor (PAF) and

is shown to be involved in modulating primase-helicase activity (Barnard, 1997). Apart

from it’s initial characterization (Smith, 1995a), there have been few studies on UL102,

which, along with UL44 is the least well conserved of the core replication proteins in

HCMV, with BLAST searches yielding only 12 and 14 homologues respectively.

However, the functional domains of HSV-1 UL8 has been mapped to the regions

spanning amino acids 6–23 at the N-terminus, 718–750 at the C-terminus, and 78–339

internally where deletion mutants inhibit the ability of UL8 to support origin-dependent

DNA synthesis (Barnard, 1997). Several variations involving the distribution of glycine

residues were observed between residues 625-635 (Figure 14E) amongst the isolates

while the short serine-rich domain spanning residues 795-836 (Figure 14F) may have

implications for phosphorylation (Barrasa, 2005). Although UL102 was highly

conserved amongst the clinical isolates, alignments with the BLAST results showed no

distinguishable motifs conserved across the homologues. However, similar to UL44, an

unusual integrin binding motif was identified in the N-terminal of UL102 (Figure 14D),

although whether it plays a role in infection has yet to be seen.

UL105 is predicted to encode the HCMV helicase protein, based on sequence homology

to its HSV-1 UL5 counterpart which also contains six motifs typical of superfamily-1 of

helicase proteins (Gorbalenya, 1989; Graves-Woodward, 1997). However, of the six

motifs found to be essential for helicase activity in HSV-1 (Zhu, 1992), only two have

90

been functionally characterized. Motif I (also called the Walker A motif) follows the

consensus sequence GxxGxGKT/S and is believed to be involved in binding ATP

(Walker, 1982) while motif II (also called the Walker B motif) contains a series of

hydrophobic residues terminated by an aspartic and glutamic acid residue and is

believed to be involved in stabilizing Mg2+ (Walker, 1982). The functional significance

of Motifs III, IV, V, and VI is yet to be elucidated although a G815A substitution in

motif V of HSV-1 UL5 altered its ability to bind ATP, but otherwise left primase and

helicase activities intact (Graves-Woodward, 1996). In this study, a putative

cAMP/cGMP-dependent protein kinase phosphorylation site was detected in Motif IV

suggesting that this domain may interact with the cAMP signaling pathway as part of its

function. These six motifs were found to be highly conserved amongst the HCMV

clinical isolates. Comparisons with homologues revealed a high degree of conservation

for the six motifs amongst all 33 sequence homologues, emphasizing the strict structure-

function relationship of this class of proteins. In addition to the six helicase motifs, two

potential N-linked glycosylation sites as well as a potential casein-kinase 2

phosphorylation site was found to be highly conserved in the helicase homologues. All

three motifs were previously identified from the results of PROSITE scanning (Figure

14M/N/O). N-linked glycosylation sites follow a distinct Nx(T/S)x motif and have been

associated with aiding correct protein folding as well as cell-cell adhesion while casein

kinase 2 phosphorylation sites follow a (S/T)xx(D/E) consensus motif (Pinna, 1990) and

has already been shown to be involved with several biological processes of HCMV

replication (Alvisi, 2005; Barrasa, 2005; Jarvis, 2004).

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Figure 14 Isolate variation in the HCMV primase-helicase complex.

Green lines represent amino acid substitutions amongst isolates while pink segments represent known

functional domains and tan segments represent interesting regions identified by phylogenetic analysis: A)

Catalytic domain (Dracheva, 1995), B) Novel conserved domain, C) Putative zinc finger motif (Biswas,

1999) D) Putative integrin binding motif, E) Glycine-rich domain, F) Serine-rich domain, G-L) Helicase

motifs I-VI (Zhu, 1992), M-N) Putative N-linked glycosylation sites, O) Putative CKII phosphorylation

sit

The functional domains of a protein are often found to be highly conserved amongst

homologues (Dracheva, 1995; Gorbalenya, 1988), with the implication that these

domains remained unchanged over time and during species differentiation due to the

crucial role they play in propagating the organism. In this study we have identified

nearly 300 potential motifs spanning five core and one non-core replication gene of

HCMV, many of which are ubiquitous commonly-occurring sequences. Motif numbers

can be overestimated by ScanProsite in glycine rich regions, where a string of glycines

can give rise to the same motif repeated several times. The 27 N-myristoylation sites

identified for UL44, for example, may be an overestimation, and caution should be

applied when using ScanProsite to determine a definitive number of particular motifs

within a given gene. However, as we have demonstrated, motif scanning programs

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combined with phylogenetic analysis are useful tools for the discovery of previously

unidentified motifs likely to be important for overall protein activity. This provides

focus areas for future studies of these important HCMV replication proteins including

investigations of potential antiviral inhibitors.

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4 HCMV primase expression

4.1 Introduction

Despite the efficacy of HCMV anti-viral therapy in treating HCMV-related disease

(Goldberg, 2003; Salzberger, 2005), long-term administration of ganciclovir (GCV) or

foscarnet (FOS) can lead to severe toxicity or the emergence of drug-resistant virus

strains (Baldanti, 1995). Therefore, there is a need to develop new therapeutic drugs

that combine efficacy and safety with a novel mechanism of action, thus excluding the

possibility of cross-resistance with existing therapeutics (Biron, 2006; Wathen, 2002).

Among the new anti-viral therapies currently being developed, Maribivir targets the

CMV protein kinase gene (Evers, 2004) and has been shown to be effective against

strains resistant to GCV, FOS, and Cidofovir (CDV) (Drew 2006). However, resistant

strains have already begun to emerge, with mutations conferring resistance identified in

the UL27 and UL97 genes (Chou, 2007; Komazin, 2003).

The HCMV primase, encoded by the UL70 ORF, shares 27% homology with the HSV-

1 primase, UL52 (Chee, 1990) and is an essential component of the viral DNA

replication apparatus (Pari, 1993). All known DNA polymerases are unable to initiate

synthesis of a new DNA strand de novo, while almost all require the activity of a DNA

primase to prime the template (Arezi, 2000). However, unlike eukaryotic primases,

which initiate primer synthesis at preferred but nonspecific template sequences,

prokaryotic and viral primases usually have specific template sequence requirements for

primer initiation (Frick, 2001). The HCMV primase is part of a trimeric complex which

also includes the helicase (UL105) and the primase-associated protein (UL102)

(McMahon, 2002). The complex is thought to unwind and prime the DNA template for

replication, which has been shown to be the case for homologous proteins of the HSV-1

(Graves-Woodward, 1997; Ramirez-Aguilar, 2004). Currently, high-throughput

screening has identified a series imidazolyl-pyrimidine compounds that can inhibit the

primase (Cushing, 2006) Although the mechanism of action of these compounds are

largely unknown, resistance conferred by a P570S point mutation in UL70 suggests that

the primase is inactivated via irreversible binding of the primase with the compound

(Chen, 2007). The importance of the primase-helicase complex in CMV DNA

replication presents as a logical target for future anti-viral therapeutics. Compounds

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which can target either the primase activity or its ability to associate with complement

members of the essential complex can provide a novel mechanism of action that is

unique from existing treatments. In addition, they may be used in conjunction with

existing anti-viral therapies to provide effective treatment and reduce the incidence of

specific resistance mutants that often emerge with long term single course treatments.

By elucidating the functional aspects of the primase, we can increase our understanding

of the complex CMV replication cycle and provide the groundwork for developing new

anti-viral strategies in the future.

Due to the large genome size and prolonged replication cycle of CMV, generating

recombinant HCMV has been difficult and time-consuming (Wang, 2004), while

previous efforts to study the HCMV helicase–primase proteins in particular have been

hampered by their low abundance in HCMV-infected cells (McMahon, 2002). However,

the advent of Bacterial Artificial Chromosome (BAC) technology solves these problems

as well as providing a robust genetic system for constructing viral mutants (Jarvis 2007).

BAC technology has already been used in several studies ranging from systematic

profiling of the CMV genome (Dunn, 2003; Yu, 2003) to elucidating mutations

conferring anti-viral resistance (Chou, 2007a; Martin, 2006). We proposed using the

BAC system for the purpose of creating recombinant mutants to study the importance of

the HCMV primase for viral replication. Furthermore, we pursued the expression of

wild-type and mutant UL70 from baculovirus constructs in insect cells, as well as in

vitro expression using a streamlined TNT Quick Coupled Transcription/Translation Kit

(Promega, USA), with a view towards functional and protein-protein interaction

analyses.

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4.2 Methods

4.2.1 Cloning of UL70 into high-copy pBluescript II primase vector

4.2.1.1 Viral stocks and DNA extraction

Viral DNA from high-passage HCMV isolates 29A and 5B were extracted from frozen

cell-associated viral stock using the QIAamp DNA Mini Kit (Qiagen, USA) as per

manufacturer’s instructions. Briefly, specimens were incubated in lysis buffer and

Proteinase K to release cellular DNA. Ethanol was used to precipitate the DNA which

was subsequently captured in microfuge spin columns (Qiagen, USA). The DNA was

then washed and eluted in 50µl of buffer AE after 5 min incubation instead of the

recommended 200µl to increase DNA concentration.

4.2.1.2 Polymerase chain reaction and purification of UL70 for pBluescript

The full length ORF encoding the HCMV primase (UL70) was amplified from the DNA

of high-passage isolates using primers containing the SpeI and HindIII restriction sites

in the 5’ and 3’ ends respectively (Table 10). Two sets of primers were created such that

UL70.SpeI and UL70.HindIII produced a ~3kb fragment containing only the UL70

ORF (UL70a) while UL70.3SpeI and UL70.4HindIII produced a ~5kb fragment that

contained the UL70 ORF as well as approximately 1kb genomic material flanking the 5’

and 3’ ends (UL70b) (Table 10). A single round PCR was carried out in a 50µl reaction

consisting of 1x buffer (supplemented with 1.75mM MgCl2), 400µM dNTPs, 300nM

primers, 100µg/ml bovine serum albumin (BSA) (Promega, US), 3.75U ExpandLong

template enzyme (Roche) and 5µl DNA template. The single round cycling conditions

consisted of denaturation at 94°C for 3 min; 10 cycles of 94°C for 10 sec, 65°C for 30

sec, 68°C for 4 min; 20 cycles of 94°C for 15 sec, 65°C for 30 sec, 68°C for 4 min

(increasing by 20 sec for each subsequent cycle); and a final elongation at 68°C for 7

min. PCR products were visualized following electrophoresis on 0.75% agarose gels

and purified using polyethylene glycol (PEG). Briefly, this involved mixing the PCR

product with 2x PEG (26.7% PEG 8000, 0.6 M NaOAc (pH 5.2), 6.5 mM MgCl2) in a

1:1 ratio. The mixture is incubated at room temperature for 10 min and centrifuged at

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8000xg, with the supernatant removed afterwards. The DNA pellet was then washed

with 96% ethanol, air-dried and re-suspended in 20µl distilled water (Baxter, Australia).

Table 10 Nucleotide primers used for UL70/pBluescript cloning

Primer Name Primer sequence*

UL70.SpeI 5' - GCACTAGTTGACGTCGGTCCGAAACCTCC

UL70.HindIII 5' - CGCAAGCTTATCGTGCGGGTTGACGGGTAG

UL70.3SpeI 5' - TAGAGGACTAGTCAGCACGTAATCGGCCACAGG

UL70.4HindIII 5' - CGATGAAAGCTTTATAGATGAGACCGCTGCCGG

UL70.4 5' – CCCAACTGGATCTGCGTAACCTGCT

T3 5' - AATTAACCCTCACTAAAGGG

T7 5' - GTAATACGACTCACTATAGGGC

*Underlined sequences denote restriction sites

4.2.1.3 Molecular cloning of UL70 into pBluescript II

Both the 3kb and 5kb UL70 PCR products as well as the pBluescript II vector were

double digested with SpeI and HindIII restriction enzymes in separate 20µl reaction

consisting of 1x buffer B (Promega, US), 2 µg/µl acetylated BSA (Promega, US), 5U

SpeI (Promega, US), 5U HindIII (Promega, US), and ~1µg DNA, or a 5U/µg over-

digest. Restriction digest reactions were incubated at 37°C for 1.5hrs followed by heat

inactivation at 74°C for 15 min. Ligation was performed immediately after in a 10µl

reaction consisting of 1x ligase buffer (Stratagene, USA), 1mM rATP (Stratagene,

USA), 2U T4 DNA ligase (Stratagene, USA), and DNA in an insert to vector ratio of

3:1 as calculated by the following formula:

Figure 15 Insert to vector ratio formula

A reaction consisting of pUC18 plasmid in place of the sample was used as a ligation

control. The ligation reactions were incubated at room temperature for 1 hr followed by

transformation to XL10-Gold Ultracompetent Cells (Stratagene, USA) as per

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manufacturer’s protocol. Briefly, this involved thawing a 100µl aliquot of cells on ice

before adding 4µl β-Mercaptoethanol and 2µl ligation mixture. The reaction was heat

pulsed at 42°C for 30 sec followed by the addition of 900µl NZY broth. The cells were

grown in a shaking incubator at 37°C and 225rpm for 1 hr prior to plating on LB-

Ampicillin agar plates supplemented with 100µl 1M IPTG and 20µl X-Gal and

incubated at 37°C for 16hrs. Plates were then placed in a 4°C fridge for 1 hr to enhance

the blue/white screening process.

Single white colonies were re-streaked and screened via colony PCR using an internal

UL70 primer and a pBluescript T7 primer (Table 10). A single round colony PCR was

carried out in a 50µl reaction consisting of 1x GoTaq Flexi buffer (Promega), 4mM

MgCl2, 200µM dNTPs, 0.2µM primers, 1.25U GoTaq DNA polymerase (Promega), and

5µl DNA template. The cycling conditions consisted of an initial denaturation at 95°C

for 2 min followed by 30 cycles at 95°C for 1 min, 65°C for 1 min, 72°C for 2 min and

a final elongation at 72°C for 5 min. PCR products were visualized following

electrophoresis on 1.5% agarose gels. PCR positive colonies were grown overnight in

2ml NZY broth supplemented with 100µg/ml Ampicillin at 37°C with 225rpm shaking.

Plasmid DNA was extracted using a Wizard Plus Minipreps DNA Purification System

(Promega, USA) as per manufacturer’s instruction and sequenced to check for genomic

integrity of the vector/insert construct. Sequencing reactions consisted of Big Dye

Terminator (Applied Biosystems, USA), premix buffer, 0.25µM primer (Table 7 and

Table 10), and approximately 75ng/300bp of DNA. Cycling conditions consisted of 25

cycles of 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min. Sequencing products

were purified and analyzed at the University of New South Wales DNA analysis facility

(Figure 16).

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Figure 16 Overview of cloning UL70 into pBluescript

99

4.2.2 Construction of baculovirus entry clone for HCMV primase expression


Plasmids containing the UL70 ORF were extracted from the UL70a pBluescript II stock

using the Wizard Plus Midipreps DNA Purification System (Promega, USA) as per

manufacturer’s instruction. Briefly, plasmids were inoculated and grown overnight in

5ml NZY broth supplemented with 10mg/ml Ampicillin at 37°C with 225rpm shaking.

The cells are centrifuged at 10,000xg for 10 min and the supernatant is discarded. Cell

re-suspension, lysis, and neutralization solutions are added to lyse the cells and

precipitate the protein from solution. The lysate was centrifuged at 14,000xg for 15 min

and the supernatant is decanted to a new microfuge tube for purification. The DNA is

transferred to a Wizard midi-column (Promega, USA) and purified under vacuum with

diatomaceous resin and washed with ethanol before a final elution with 100µl hot (65°C

-70°C) distilled water (Baxter, Australia).

4.2.2.2 Polymerase chain reaction and purification of UL70 for pDONR

The full length UL70 ORF was amplified from midipreps of the UL70a pBluescript

stock with primers which incorporated attachment sites into the product (Table 11). A

single round PCR to amplify the ~3kb product was carried out in a 50µl reaction

consisting of 1x High Fidelity buffer (Invitrogen, USA), 0.2mM dNTPs, 2mM MgSO4,

400nM primers, 1U Platinum Taq High Fidelity enzyme (Invitrogen, USA) and 5µl

DNA template. The single round cycling conditions consisted of denaturation at 94°C

for 3 min followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C for 3 min.

PCR products were visualized following electrophoresis on 1.0% agarose gels and

purified using a 30% PEG 8000/30mM MgCl2 Solution (Invitrogen, USA). Briefly, this

involved diluting the PCR product 4-fold with TE buffer, followed by adding 0.5 times

volume of 30% PEG 8000/30mM MgCl2 solution. The mixture is centrifuged for 15

min at 8000xg, with the supernatant removed afterwards and the clear pellet suspended

in 20µl TE.

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Table 11 Nucleotides primers used for UL70/pDONR cloning

Primer Name Primer Sequence*

UL70attb1 5' - GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTGCATCGTGCCGGCGCGACG

UL70attb2 5' - CCCCTGGTGAAACATGTTCTTTCGACCCAGCGCCGTGGAAAGTGAGGCTAG

UL70.3 5' - GCGAGCCCAGTAGCAGACGCGCGAA

UL70.4 5' - CCCAACTGGATCTGCGTAACCTGCT

M13 Forward 5' - GTAAAACGACGGCCAG

M13 Reverse 5' - CAGGAAACAGCTATGAC

* Underlined sequences denote attachment sites

4.2.2.3 pDONR recombination and molecular cloning of UL70

The UL70 PCR product was recombined with the pDONR donor vector (Invitrogen,

USA) (Figure 17) using a Gateway BP Clonase II enzyme (Invitrogen, USA).

Figure 17 pDONR/Zeocin Vector

pDONR/Zeocin vector with key features including: pUC Ori: pUC Origin; Zeocin: Zeocin resistance gene;

T1/T2: Transcription termination sequence; attp1/2: Recombination sites; ccdB: ccdB gene (negative

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selection of plasmid); CmR: Chloramphenicol resistance gene; M13 Forward/Reverse: Sequencing

priming sites.

A 10µl BP reaction consisting of 20-50fmol PCR product (Figure 18), 150ng pDONR

vector, TE Buffer, and 2µl BP Clonase II enzyme mix (Invitrogen, USA) was incubated

overnight at 25°C, followed by the addition of 1µl Proteinase K (Invitrogen, USA) and a

further incubation of 37°C for 10 min. A pEXP7-tet positive control was used in

conjunction with each experiment. A 1-2µl sample of the BP reaction was then

immediately transformed to Library Efficient DH5α competent cells (Invitrogen, USA).

Briefly, this involved thawing a 50µl aliquot of cells on ice before adding the sample

and heat shocking the reaction at 42°C for 30 sec followed by the addition of 250µl

SOC media pre-warmed to 37°C. The cells were grown in a shaking incubator at 37°C

and 225rpm for 1 hr prior to plating on LB-Zeocin agar plates while positive controls

were plated on LB-tetracycline plates. All plates were incubated overnight at 37°C.

Figure 18 converting femtomoles to nanograms

Single colonies were re-streaked and screened via colony PCR using an internal UL70

primer and a pDONR M13 primer (Table 11). A single round colony PCR as outlined in

section 4.2.1.3 was carried out to check for the insert. PCR positive colonies were

grown overnight in 5ml SOC media supplemented with 50µg/ml Zeocin at 37°C with

225rpm shaking. Plasmid DNA was extracted using a Wizard Plus Midipreps DNA

Purification System (Promega, USA) as per manufacturer’s instruction and sequenced

to check for genomic integrity of the construct. Sequencing reactions and cycling

conditions were previously outlined in section 4.2.1.3. A brief overview of the process

is outlined in Figure 19.

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Figure 19 Overview of UL70/pDONR recombination

4.2.3 In vitro UL70 primase expression using TNT Quick Coupled

Transcription/Translation


Plasmids containing the UL70 ORF were extracted from the UL70a pBluescript II stock

using the Wizard Plus Midipreps DNA Purification System (Promega, USA) as per

manufacturer’s instruction and as outlined in previous sections.

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4.2.3.2 Polymerase chain reaction and purification of UL70

The full length UL70 ORF was amplified from midipreps of the UL70a pBluescript

stock using primers which incorporated the BamHI and SalI restriction sites at the 5’

and 3’ ends of UL70 respectively (Table 12). A single round PCR to amplify the ~2.9kb

product was carried out in a 50µl reaction consisting of 1x High Fidelity buffer

(Invitrogen, USA), 0.2mM dNTPs, 2mM MgSO4, 400nM primers, 1U Platinum Taq

High Fidelity enzyme (Invitrogen, USA) and 5µl DNA template. The single round

cycling conditions consisted of denaturation at 94°C for 3 min followed by 35 cycles of

94°C for 30 sec, 60°C for 30 sec, and 68°C for 3 min. PCR products were visualized

following electrophoresis on 1.0% agarose gels and purified using a Wizard SV Gel and

PCR Clean-Up Kit (Promega, USA) as per manufacturer’s protocols. Briefly, the PCR

products were mixed in a 1:1 ratio with Membrane Binding Solution (Promega, USA),

incubated at room temperature and transferred to a SV mini-column (Promega, USA)

where it is centrifuged at 16,000xg to collect the DNA in the membrane. The membrane

is washed several times with Membrane Wash Solution (Promega, USA) before a final

elution of 30µl with nuclease-free water (Promega, USA).

Table 12 Nucleotide primers used for UL70/pCITE cloning

Primer Name Primer Sequence*

UL70pCITE.3 5' - ATTGGATCCCTGCATCGTGCCGGCGCGACG

UL70pCITE.4 5' - ATTGTCGACTGACGGCGGTCGCCGGCGGCAT

UL70.3 5' – GCGAGCCCAGTAGCAGACGCGCGAA

T7 5' - GTAATACGACTCACTATAGGGC

* Underlined sequences denote restriction sites

4.2.3.3 Molecular cloning of UL70 into pCITE 4A

Both the UL70 PCR product and pCITE vector were sequentially digested, initially with

SalI followed by BamHI. Restriction digests with SalI were carried out in a 20µl

reaction consisting of 1x NEBuffer 3 (New England BioLabs (NEB), USA), 100µg/ml

BSA (NEB, USA), 30U SalI (NEB, USA), and ~3µg DNA, or a 10U/µg DNA over-

digest. Digest reactions were incubated at 37°C for 3 hrs followed by heat inactivation

at 65°C for 20 min. Digest products were then purified using the Wizard SV Gel and

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PCR Clean-Up Kit (Promega, USA) as per manufacturer’s protocols outlined in the

previous section prior to the second digest. Restriction digests with BamHI were also

carried out in 20µl reaction consisting of 1x NEBuffer 3 (NEB, USA), 100µg/ml BSA

(NEB, USA), 20U BamHI (NEB, USA), and ~2µg DNA, or a 10U/µg DNA over-digest.

Digest reactions were incubated at 37°C for 2 hrs and immediately followed by

processing with the Wizard SV Gel and PCR Clean-Up Kit (Promega, USA) as a means

to stop the digest reaction and purify the restriction products. Products were ligated in a

20µl reaction consisting of 1x ligase duffer (Stratagene, USA), 1mM rATP (Stratagene,

USA), 4U T4 DNA ligase (Stratagene, USA), and DNA in an insert to vector ratio of

3:1 as calculated by the formula in section 4.2.1.3. A reaction consisting of pUC18

plasmid in place of the sample was used as a ligation control. The ligation reactions

were incubated at room temperature for 16 hrs followed by transformation to Library

Efficient DH5α Competent Cells (Invitrogen, USA) as per manufacturer’s protocol,

albeit omitting the dilution steps. Briefly, this involved thawing a 100µl aliquot of cells

on wet ice before adding 1-10ng DNA and incubated on ice for 30 min. The mixture

was heat shocked at 42°C for 45 sec followed by the addition of 900µl SOC media. The

cells were grown in a shaking incubator at 37°C and 225rpm for 2 hrs prior to plating on

LB-Ampicillin agar plates.

Single colonies were re-streaked and screened via colony PCR using an internal UL70

primer and a pCITE T7 primer (Table 12). A single round colony PCR as outlined in

section 4.2.1.3 was carried out to check for the insert. PCR positive colonies were

grown overnight in 5ml SOC media supplemented with 100µg/ml Ampicillin at 37°C

with 225rpm shaking. Plasmid DNA was extracted using a Wizard Plus Midipreps

DNA Purification System (Promega, USA) as per manufacturer’s instruction and

sequenced to check for genomic integrity of the construct. The sequencing reaction and

cycling conditions used were previously outlined in section 4.2.1.3. An overview of the

process is outlined in Figure 20.

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Figure 20 Overview of cloning UL70 into pCITE expression vector

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4.2.3.4 Protein expression and detection

UL70 expression was carried out using a TNT Quick Coupled Transcription/Translation

Kit (Promega, USA) designed to express protein in an in vitro environment. Protein

expression was carried out in 50µl reactions consisting of 40µl TNT Quick Master-Mix

(Promega, USA), 20µM methionine (Promega, USA), and ~1ng DNA. The reaction was

incubated at 30°C for 90 min, followed by a 1:4 dilution with 4X loading dye and

finally denatured at 100°C for 5 min. A T7 luciferase which produced a 61kDa

monomeric protein was used as a translation control. Translation products were

visualized with a 10% SDS-PAGE resolving gel using a Mini-PROTEAN 3 Cell

electrophoresis system (Biorad, USA). The protein samples were loaded alongside 10µl

Kaleidoscope markers (Biorad, USA) and Magic Mark protein markers (Novagen,

Germany). All samples were run through the 5% stacking gel at a constant voltage of

80V for 30 min prior to running the samples through the resolving gel at a constant

voltage of 110V until the dye front left the gel. Protein bands were transferred from the

gel to nitrocellulose membrane using Western blotting techniques. The transfer was run

overnight at a constant current of 90mA (30V) using a magnetic stirrer and ice-pack to

keep the system from overheating. The gel was stained with 20ml Gelcode Blue Stain

(Pierce, USA) post-transfer to check for total protein. The S-Tag and His Tag antibodies

were used to detect for the UL70 protein product. Briefly, the nitrocellulose membrane

was incubated with gentle shaking in blocking solution for 1 hr, followed by thorough

washing with PBST solution. For membranes being detected with the S-Tag, the S-

Protein Alkaline Phosphatase Conjugate (Novagen, Germany) was diluted 1:5000 with

PBST and incubated with the membrane at room temperature for 30 min with gentle

shaking. Detection with the S-Tag did not require a secondary antibody. For membranes

being detected with the His-Tag, c-terminal Anti-His antibodies (Invitrogen, USA) were

diluted 1:5000 with PBST and incubated with the membrane at room temperature for 1-

2 hrs with gentle shaking. The membrane was washed thoroughly with PBST prior to

incubation with the secondary antibody. Immun-Star goat anti-mouse IgG-HRP

(Australian Laboratory Services, Australia) was diluted 1:4000 with blocking solution

and incubated with the membrane at room temperature with gentle shaking for 1 hr. The

membranes are thoroughly washed again with PBST after antibody incubation and

prepared for chemiluminescence visualization with SuperSignal West Pico

Chemiluminescent Substrate (Pierce, USA), as per manufacturer’s instructions. Briefly,

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a working solution is prepared by mixing the Luminol/Enhancer Solution and the Stable

Peroxide Solution in a ratio of 1:1. The working solution is then carefully applied over

the membrane using a pipette and incubated at room temperature for 5 min. The excess

reagent is drained and the membrane is exposed to a camera set to detect

chemiluminescence (Figure 21). The membrane was also stained with Ponceau S

(Sigma-Aldrich, USA) on several occasions to check for total protein.

Figure 21 Overview of UL70 in vitro protein expression

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4.3 Results

4.3.1 UL70 was cloned into pBluescript II high-copy vector

Both the UL70 ORF spanning 2,985bp from high-passage isolate 5B and the 5,250bp

fragment from high-passage isolate 29A were successfully cloned into pBluescript and

designated as UL70a and UL70b respectively. UL70a consisting of the 3kb pBluescript

vector and the 3kb UL70 insert (Figure 22A) produces a 6kb band when extracted from

a plasmid preparation. In addition, we also detected an extra band several kilo-bases

heavier than our anticipated product indicating that nicked DNA may also be present

(Figure 22B). However, sequencing of the plasmid confirmed the insert ligated correctly

into the vector without mutations.

A B

Figure 22 pBluescript UL70a

(A) UL70a plasmid map indicating position of UL70 (red) relative to key vector domains (orange). (B)

UL70a produces two products bands, the anticipated plasmid (II) and nicked DNA (I). Lambda DNA

HIndIII markers (Promega) on the left are indicated in base-pairs.

UL70b consists of the 3kb pBluescript vector and the 3kb UL70 ORF as well as

approximately 1kb of flanking genomic region either end of the UL70 ORF (Figure

23A). Plasmid preparations produces an anticipated band of approximately 8kb as well

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as a high molecular weight product predicted to be nicked DNA (Figure 23B).

Sequencing of the plasmid confirmed that the 5kb insert ligated into pBluescript and

without mutations.

A B

Figure 23 pBluescript UL70b

(A) UL70b plasmid map indicating position of UL70 (red) relative to key vector domains (orange). (B)

UL70b produces two products bands, the anticipated plasmid (II) and nicked DNA (I). Lambda DNA

HIndIII markers (Promega) Markers on the right are indicated in base-pairs.

110

4.3.2 Generating Baculovirus entry clones via BP recombination

Experiments to create a UL70/pDONR recombinant have been unsuccessful with

several instances of the PCR incorporated attachment site failing to bind to their correct

complement on the pDONR vector, resulting in recombinants which truncated the UL70

ORF. As a result, subsequent experiments to recombine the pDONR/UL70 entry clone

with the baculovirus expression DNA have not been pursued. The length of the excised

regions varied, with designate samples BPR4, BPR5, and BPR8 missing 1,919bp, 36bp,

and 505bp respectively, all from the 3’ end of UL70. However, the 5’ end of UL70

recombined with pDONR without problems. Other attempts to generate an entry clone

failed to produce colonies at the screening stage despite the transformation controls

producing visible colonies. Experiment BPR1 and BPR2 both used BP Clonase I

(Invitrogen, USA) in the BP recombination reaction (c.f. BP Clonase II in later

experiments), with the former transformed into One Shot TOP10 competent cells

(Invitrogen, USA) while latter experiments used the DH5α competent cells. BPR3 was

carried out as a time-course experiment to determine the optimal BP reaction time, and

though colonies were generated at the longest incubation, all samples were found

negative for UL70. Samples BPR4 and BPR5 were generated using increasing amounts

of total DNA during the BP reaction, with BPR5 grown for an additional hour during

the transformation step to increase the number of cells prior to plating. BPR4 was

selected from several colonies of which only one produced a band at the expected size

of 780bp when screened using M13 forward and UL70.3 reverse primers (Figure 24A).

However, plasmid preparations of the construct revealed a prominent band at 2.3kb, the

expected band of 4.9kb, as well as a band exceeding 9kbp predicted to be nicked DNA

(Figure 24B).

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A B

Figure 24 Screening of entry clone BPR4

(A) PCR screening of BPR4 generated the expected 780bp fragment. (B) Plasmid preparation of BPR4

revealed 3 products: a band >9kbp predicted to be nicked DNA (I), band at ~6kbp predicted to be the

expected entry clone, and an unknown prominent band at 2.3kbp (III). Lambda DNA HIndIII Markers

(Promega) (right) and BenchTop pGEM DNA markers (Promega) (left) sizes are indicated in base-pairs.

Sequence analysis of the BPR4 plasmid preparation revealed a truncated 3kb product

with UL70 missing the C-terminal 1,919bp, albeit retaining frame with the rest of

pDONR. The initial PCR screening was a false positive as the region amplified did not

involve the truncation. In addition, the truncation removes the catalytic motif and the

putative zinc finger binding domain (Figure 25).

Figure 25 The BPR4 truncation removes key motifs from UL70

Key UL70 motifs highlighted in purple while red box denotes section of gene truncated during

recombination. Orientation and length of te UL70 protein is denoted by numbers.

112

Despite the unfavorable recombination in BPR4, another attempt was carried out on the

assumption that increasing the overall number of cells prior to plating would increase all

populations universally, including the constructs which contained our desired product.

In addition, PCR screening of the colonies involved the use of both the M13

forward/UL70.3 reverse primers as well as a set of internal UL70 primers such that

colonies containing the truncation in BPR4 would be selected out. BPR5 was selected

on the basis of colony PCR (Figure 26).

Figure 26 Colony PCR screening for entry clone BPR5

Internal UL70 primers were used to amplify a 1.2kbp region as well as intrinsically select out

recombinants carrying the BPR4 truncation. BenchTop pGEM DNA marker (Promega) sizes on the right

are indicated in base-pairs.

However, after sequencing several BPR5 samples, a design flaw was identified at the 3’

end of the UL70 insert product, which caused a short truncation and amplification of

non-specific DNA (Figure 27). An inverted attachment sequence was identified for the

3’ primer, resulting in complications during the amplification as well as the subsequent

BP recombination.

113

UL70 junk Primer attb1 pDONR

-----------------------|----------|------------------------|------|---------------------

Figure 27 The BPR5 truncation of UL70

A primer design flaw caused the 36bp truncation of UL70 as well as the amplification of non-specific

sequences (green). Key UL70 motifs highlighted in purple while red box denotes section of gene

truncated during recombination. Orientation and length of the UL70 protein is denoted by numbers.

Subsequent experiments, including BPR6, BPR7 and BPR8 were carried out using a

modified primer with the correct attachment sequence. BPR6 and BPR7 failed to

generate colonies despite using the previously optimized protocol while BPR8,

transformed in a new batch of DH5α cells produced several colonies. PCR positive

colonies were sequenced, revealing another novel truncation at the 3’ end of UL70. A

505bp fragment at the c-terminal end of UL70 was shown to be excised, removing a

putative zinc finger domain in the process (Figure 28).

Figure 28 The BPR8 truncation removes the putative zinc finger from UL70

Key UL70 motifs highlighted in purple while red box denotes section of gene truncated during

recombination. Orientation and length of the UL70 protein is denoted by numbers.

114

4.3.3 UL70 was cloned into pCITE expression vector

The full length UL70 ORF was successfully cloned into expression vector pCITE 4A

with a functional S-Tag and c-terminal His-tag (Figure 29A). The original UL70 stop

codon, TGA, was replaced with TCA to ensure that subsequent translation incorporated

the down-stream His-tag (Figure 29A). Plasmid preparations of the DNA gave a distinct

band at the expected size of 6.7kbp (Figure 29B) while sequencing confirmed that the

3kbp insert ligated into pCITE without mutations.

A B

Figure 29 UL70/pCITE expression vector

(A) UL70/pCITE plasmid map indicating position of UL70 (red) relative to key vector domains (orange).

(B) UL70/pCITE produces one distinct band at 6.7kbp. Lambda DNA HIndIII markers (Promega) on the

right are indicated in base-pairs.

115

4.3.4 UL70 in vitro expression using the TNT system

The expression vector created in 4.3.3 was used with the TNT Quick Coupled

Transcription/Translation Kit (Promega, USA) to express the HCMV primase. However,

attempts at protein expression failed to produce a protein of the predicted 107kDa size.

In addition, an unknown product of ~55kDa was detected in all lanes (Figure 30). The

method of detection was by using the S Tag protein, which is present in the pCITE

expression vector, but not in the luciferase control DNA, hence the control reaction was

not carried out.

Figure 30 Expression of UL70 in TNT system

Typical protein expression produces an unknown product of ~55kDa (I) in both the UL70 sample (A) and

the negative control (B). Precision Plus Kaleidoscope markers (Biorad) on the left are given in Kilo-

Daltons (kDa).

Since previous attempts were based on a single incubation time of 90 min as

recommended per manufacturer’s instructions, we carried out a time course experiment

to determine the incubation time that would yield the optimal amount of protein.

116

However, samples taken at 30 min intervals up to duration of 3 hrs yielded similar

results to incubations at 90 min (Figure 31).

Figure 31 Time course expression of UL70 with TNT system

Samples were taken at 30 min intervals starting at 1hr (A) to 3 hrs (E). The negative control was taken at

3 hrs (F). Markers on the left are given in kDa.

117

4.4 Discussion

The ability to replicate the UL70 ORF at high yields using a high copy vector as

opposed to traditional cell culturing is an invaluable tool for elucidating the functions of

the primase in future expression studies. UL70b, which contains flanking regions either

side of the 5’ and 3’ ends of the UL70 ORF, was designed as a precursor to express the

UL70 gene in a Bacterial Artificial Chromosome (BAC) system. The BAC system has

been shown to be an effective tool for studying the importance of various genes in

HCMV (AuCoin, 2006; Hahn, 2002; Hahn, 2004; Jiang, 2008; Komazin, 2004; Lorz,

2006; Martin, 2006; Michel, 2005; Stropes, 2008; Xu, 2004) and the creation of a UL70

precursor may serve as a contribution to future studies on the HCMV helicase-primase

complex, which has yet to be studied in detail.

Protein expression involving baculovirus requires the creation of an expression virus

through recombination between an entry clone and the baculovirus linear DNA. The

entry clone is created through another recombination reaction involving an entry vector

and a PCR generated DNA fragment utilizing attachment sites. In this study, attempts to

generate entry clones for this process resulted in recombinants that truncated UL70 and

although initial results reflected the consequences of a faulty primer, later experiments

persisted with the truncation of the 3’ end. The original 3’ primer sequence was found to

have an inverted attachment sequence, which resulted in the PCR fragment failing to

recognize the complement sequence on pDONR. Initially this was thought to have

caused the truncations as demonstrated in BPR4 and BPR5. However, closer inspection

of the plasmid sequences suggests that the truncations may have been caused by the

recognition sequences binding to domains within UL70 that were similar to the

attachment sites (Figure 32). During sequence analysis we noticed that the

recombination site remained continuous between UL70 and pDONR, indicating that the

plasmid was circularized. In addition, BPR4 remained in frame with pDONR,

suggesting that expression of the partial UL70 protein should still be possible, and

indeed could be used to determine if the N-terminal third of the HCMV primase played

a role in primase function or in interacting with other proteins, in particular, other

members of the primase-helicase complex. Unfortunately, the design of the baculovirus

system meant that BPR4 could not be carried forward to express protein since the

recombination between the entry clone and baculovirus linear DNA requires another

118

recombination sequence that is created upon successful recombination between the PCR

fragment and pDONR.

A

GGACGTCATGCAAAAATACTTCTCGCTCGACAACTTTCTACACGATTACGTGGAGACGCATCTACTACGT

| | || |||||| | |

CCGTCCTAGCCTCACTTTCCACGGCGGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTG

B AAGCCAACTTTATCGAGACGCGCTCGCTTAACGTGACGCGTTATCGACGCCGCGGTCTCACCGAGGTGCT

| ||||| | | | |||| || | | |

AGCCTCACTTTCCACGGCGGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCA

Figure 32 Sequence analysis of BPR4 and BPR8

Comparisons of the recombination site for BPR4 (A) and BPR8 (B) reveals a degree of similarity

between the expected recombination sites (grey) and the random sequence within UL70 (as underlined)

that marked the recombination site for BPR4 and BPR8 with pDONR (purple).

Experiments to express the HCMV primase failed to produce detectable levels of the

107kDa protein. The absence of detectable protein could have been the result of low

translation efficiency caused by contaminants. In particular, the presence of calcium

may reactivate nucleases in the TNT lysate mix that were used to remove endogenous

mRNA and result in degradation of the DNA template (TNT Technical manual). The

unexpected 55kDa band which appeared during expression may indicate that another

peptide was translated from the template due to translation initiating at internal

methionines or that an artifact was created by the methods used for expression.

Increasing the fidelity for the correct initiation methionines can done by optimizing the

Mg2+

or K+ concentration (Hurst, 1996). Antibody detection was done using the S-Tag

system exclusively, which was a unique feature of the pCITE vector. The S-Tag system

is based on the interaction between a 15 amino acid S-Tag peptide and a ribonuclease S-

protein, which differs from current conventional His-Tag antibody detection in that it

does not require a secondary anti-body and hence thought to reduce the amount of

background on the Western Blot.

119

5 Concluding discussion and future directions

In this study we have shown that essential HCMV replication genes UL44, UL57, UL70,

UL102, UL105, and UL84 are highly conserved amongst isolates with variations

occurring outside of known or otherwise predicted functional domains. We have also

shown that the certain components of HCMV primase-helicase complex, UL70 and

UL105, as well as the single-stranded DNA binding protein (UL57) were highly

conserved amongst homologues, affirming the premise that functional domains remain

conserved as species diverged. Then putative functional motifs predicted from protein

motif scanning software were used as a basis for identifying potential functional

domains amongst the homologues under this premise.

We identified two N-linked glycosylation sites and a casein-kinase II phosphorylation

site that were highly conserved amongst 33 homologues of the HCMV helicase

(UL105). In addition, a cAMP-dependent protein kinase phosphorylation site was

identified in motif IV of the six putative helicase motifs previously predicted to exist in

UL105 (Graves-Woodward, 1996), providing some insight into the mechanistic

properties of that domain. In UL70, we identified a highly conserved domain consisting

of aromatic residues preceding a conserved C570 residue that has been implicated in the

structural inhibition of a compound currently being studied for its inhibitory effects on

the HCMV primase (Chen, 2007). In addition, a pair of highly conserved cysteines was

also identified although guesses as to their function, if any, are highly speculative at this

stage of the study. On one side, the domain is reminiscent of a rare PDI motif found in

only a handful of organisms in nature (Woycechowsky, 2003) while on the other, it may

be an important motif for protein structure, interacting with other conserved cysteines,

possibly with C570, or cysteines from the putative zinc finger domain (Chen, 2005), to

stabilize the tertiary structure in a manner that is similar to that of the chemokine class

of proteins (Fernandez, 2002). Similar conserved cysteine residues were previously

predicted to reside in UL57 (Gao, 1988) and were shown here to be highly conserved

across 35 homologues. In particular, a classic disulfide bond motif with the canonical

CxxC sequence was shown to be present in 34 of the 35 homologues studied and may

serve as the starting point for understanding the structural properties for this class of

proteins.

120

While conservation across homologous proteins may act as an indicator of

evolutionarily conserved functional domains, the lack of homology may also

demonstrate the ability of genes to adapt over time to specific roles unique to a

particular species. The DNA polymerase accessory proteins of HSV (UL42) and HCMV

(UL44) are both well characterized and document this distinction well. While

homologous in function and position (Ertl, 1992), UL42 operates as a monomer

(Gottlieb, 1990) and relies on basic interactions (Bridges, 2001; Zuccola, 2000), while

UL44 was shown to form a dimer and rely on hydrophobic interactions (Appleton,

2004). Similarly, UL102 shares no homology with its HSV equivalent, UL8, despite the

primase and helicase components sharing homologies of 27% and 34% respectively to

the HSV equivalents (Chee, 1990) while UL84 has no homolog equivalent in other

herpesviruses, despite being an essential component of HCMV DNA replication (Pari,

1993). Although we cannot use conservation as a means to identify putative domains for

these genes, the putative motifs predicted by the scanning software in this study can be

used as a reference for future studies elucidating protein function.

In this study, three different protein expression techniques were explored and though we

failed to express the HCMV primase, insights can be gained from the methods used in

constructing the vectors and attempts at expression in vitro. In addition, the creation of a

high copy vector for UL70 featuring recombinant arms can be the basis of future

experiments to express the HCMV primase in a BAC system.

121

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