HIV-1 Trafficking to the Dendritic Cell–T-Cell InfectiousSynapse Uses a Pathway of Tetraspanin Sorting to theImmunological Synapse

Eduardo Garcia1, Marjorie Pion1, AnnegretPelchen-Matthews2, Lucy Collinson2, Jean-Francois Arrighi1, Guillaume Blot1, FlorenceLeuba1, Jean-Michel Escola1, NicolasDemaurex3, Mark Marsh2 and Vincent Piguet1,*

1Department of Dermatology and Venereology, UniversityHospital of Geneva, Geneva, Switzerland2MRC Laboratory for Molecular Cell Biology and CellBiology Unit, and Department of Biochemistry andMolecular Biology, University College London, GowerStreet, London WC1E 6BT, UK3Department of Cell Physiology and Metabolism,University of Geneva Medical Center, Geneva,Switzerland*Corresponding author: Vincent Piguet,vincent.piguet@medecine.unige.ch

Dendritic cells (DCs) are essential components of theearly events of HIV infection. Here, we characterizedthe trafficking pathways that HIV-1 follows during itscapture by DCs and its subsequent presentation to CD4+

T cells via an infectious synapse. Immunofluorescencemicroscopy indicates that the virus-containing com-partment in mature DCs (mDCs) co-labels for thetetraspanins CD81, CD82, and CD9 but contains littleCD63 or LAMP-1. Using ratio imaging of pH-reportingfluorescent virions in live DCs, we show that HIV-1 isinternalized in an intracellular endocytic compartmentwith a pH of 6.2. Significantly, we demonstrate thatthe infectivity of cell-free virus is more stable at mildlyacidic pH than at neutral pH. Using electron micro-scopy, we confirm that HIV-1 accumulates in intra-cellular vacuoles that contain CD81 positive internalmembranes but overlaps only partially with CD63.When allowed to contact T cells, HIV-1-loaded DCsredistribute CD81, and CD9, as well as internalizedHIV-1, but not the immunological synapse markersMHC-II and T-cell receptor to the infectious synapse.Together, our results indicate that HIV-1 is internalizedinto a non-conventional, non-lysosomal, endocyticcompartment in mDCs and further suggest that HIV-1 isable to selectively subvert components of theintracellular trafficking machinery required for formationof the DC–T-cell immunological synapse to facilitate itsown cell-to-cell transfer and propagation.

Key words: dendritic cells, endosomes, HIV, infectioussynapse, trans infection

Received 14 March 2005, revised and accepted for pub-lication 23 March 2005, published on-line 29 April 2005

Dendritic cells (DCs) are believed to be crucial mediators

of the early events in HIV-1 infection following sexual

transmission [reviewed in (1,2)]. Dendritic cells reside in

the skin and mucosal tissues in a resting ‘immature’ state

until they encounter pathogens. Upon exposure to a

variety of stimuli, DCs are activated to a mature antigen

presenting state (3). Changes during DC maturation

considerably modify the DC intracellular trafficking

machinery allowing, for example, the rapid translocation

of MHC-II from lysosomes to the cell surface [reviewed in

(3)]. Maturation is closely linked with the migration of DCs

from peripheral tissues to secondary lymphoid organs.

Within these tissues, activated mature DCs (mDCs)

interact with antigen-specific T cells to initiate immune

responses (4,5).

HIV-1 infects Langerhans cells (LCs) and other types of

myeloid DCs both in vivo and in vitro [reviewed in (1,6)],

although this infection is inefficient compared with CD4þ

T cells. In addition, DCs can capture HIV-1 in an infectious

form and transfer this virus to CD4þ T cells in trans,

leading to massive levels of HIV-1 replication in DC–T-cell

clusters (7). Indeed, this DC-mediated trans infection is

believed to be the most efficient route for HIV-1 infection

of T cells [reviewed in (1,8)]. In immature DC (iDCs) sub-

types, the C-type lectin DC-SIGN (CD209) is the principal

molecule mediating HIV-1 trans infection to CD4þ T cells

(9,10). In DC-SIGN negative DC subsets, such as LCs, HIV

capture can occur through other C-type lectins, such as

the mannose receptor and Langerin (11).

Importantly, HIV-1 captured by DCs remains infectious for

several days in vitro, whereas free virus rapidly loses

infectivity (9,12). Although significant viral degradation

occurs after HIV-1 capture by DCs (13), the mechanisms

that mediate the prolonged retention of DC-associated

viral infectivity are currently unclear. Nevertheless, virus

internalization by DCs appears to be a prerequisite for

efficient transfer of HIV-1 infection to T cells in trans and

may explain why trypsin treatment of HIV-exposed DCs

does not decrease the efficiency of DC-mediated virus

transmission to T cells (12).

Upon contact with uninfected CD4þ T cells, internalized

HIV-1 recycles rapidly to sites of contact between DCs

and T cells (10,13,14). By analogy to the immunological

synapse involved in antigen presentation (15), these

sites of virus transfer have been termed ‘infectious’ or

‘virological’ synapses [reviewed in (16)]. The focusing of

Traffic 2005; 6: 488–501Copyright # Blackwell Munksgaard 2005

Blackwell Munksgaard doi: 10.1111/j.1600-0854.2005.00293.x

488

virions at the synapse may contribute to the observed

efficient infection of T cells by HIV-loaded DCs (7,17).

In order to understand the mechanisms involved in DC-

mediated trans infection in more detail, we here describe

a morphological study of a compartment into which HIV-1

is sequestered following capture and internalization in

monocyte-derived DCs. Using a combination of ratio

imaging of pH-reporting fluorescent virions, confocal and

electron microscopy, we show that HIV-1 is efficiently

captured and internalized by both immature and mature

DCs, at least in part via clathrin-mediated endocytosis.

Surprisingly, after internalization, HIV-1 does not accumu-

late in lysosomes but localizes in a mildly acidic compart-

ment (pH 6.2). Furthermore, confocal and electron

microscopic studies demonstrate that this compartment

is distinct from the classical late endosome/multivesicular

body (MVB) compartment but contains tetraspanins such

as CD81 and CD9. Finally, we show that upon contact

with T cells, internalized HIV-1 redistributes rapidly to

form infectious synapses in which the tetraspanins CD81

and CD9 are also observed. Given the apparent role of

CD81 as a component of the immunological synapse

(18,19), we suggest that HIV-1 is able to exploit a pathway

responsible for the delivery of key components involved in

immunological synapse formation and T-cell activation to

facilitate its transfer to CD4þ T cells.

Results

Mature DCs capture and transfer HIV-1 through an

infectious synapse

Peripheral blood monocytes were induced to differentiate

into iDCs in the presence of GM-CSF and IL-4 and subse-

quently activated with lipopolysaccharide (LPS) to obtain

mDCs (LPS-mDCs). As expected, mDCs expressed high

cell-surface levels of classical markers associated with DC

maturation such as CD83 (Figure 1A).

To examine whether mDCs could capture HIV-1, we used

a well-characterized FACS-based assay to measure viral

capture by detecting intracellular accumulation of the viral

p24gag protein. After incubating LPS-mDCs with HIV-1 for

2 h at 37 �C, p24gag could be detected in more than 50%

of the cells (Figure 1B). Similar results were obtained for

iDCs (data not shown). The observed FACS signals were

likely to indicate internalized HIV-1 because similar results

were seen when virus-pulsed DCs were treated with

trypsin to remove surface-bound HIV (data not shown).

We also tested whether LPS-mDCs pulsed with virus

could enhance transfer of HIV-1 infection to target cells

in trans as reported (7,9). For this purpose, we incubated

LPS-mDCs with HIV-1 for 2 h at 37 �C and then measured

viral transfer to Jurkat CD4þ T cells in a single round

infection assay. As expected, HIV-1 infection could be

transferred from mDCs to target cells in trans

(Figure 1C). To determine whether infection of T cells

occurred via formation of an infectious synapse between

DCs and CD4þ T cells, we analysed cell conjugates by

immunofluorescence. Monocyte-derived LPS-mDCs were

loaded with HIV-1, washed, and co-cultured with Jurkat

cells for 30 min before fixation, permeabilization, and

staining with appropriate antibodies. In mDCs, HIV was

taken up into a compartment that appeared clustered on

one side of the cell (Figure 1D, left). When the mDCs

encountered T cells, the virus re-distributed to the zone

of contact between the DC and the T cell. Infectious

synapses were considered to have formed when >75%

of the virus was focused in this contact zone. After 30 min

of incubation of HIV-1-pulsed LPS-mDCs and uninfected T

cells, approximately 40% of mDCs were observed trans-

ferring HIV-1 through infectious synapses to CD4þ T cells

as previously reported (10). Significantly, bona fide DC–T-

cell immunological synapse markers [MHC-II (HLA-DR)

and T-cell receptor (CD3)] were not enriched in the

DC–T-cell infectious synapse (Figure 1D, center and right).

HIV-1 accumulates in a mildly acidic endocytic

compartment

Measurement of the pH in endocytic organelles has

revealed that the acidity of the lumen increases from

pH 6.2–6.8 in early and recycling endosomes to 5.5–6.1

in late endosomes/MVBs and 5.0–5.5 in lysosomes

(20,21). To study the fate of HIV-1 internalized by DCs,

we used ratio imaging of pH-reporting fluorescent virions

in live DCs. For this purpose, we treated HIV-1 virions with

aldrithiol (AT)-2 to inactivate infectivity (for live experi-

ments). Then, we labeled AT-2-treated HIV-1 virions with

a pH-sensitive fluorescent probe (FITC) using a similar

method to that described for adenovirus (22). A significant

fraction of the AT-2-treated HIV-1 particles labeled with FITC

retained their capacity to interact with HIV-1 receptors and

undergo fusion (data not shown). Furthermore, AT-2-treated

HIV or SIV captured by DCs can recycle to DC–T-cell

infectious synapses (13), indicating that AT-2 treatment

does not alter the trafficking of HIV captured by DCs.

Subsequently, we incubated HIV-1-AT-2-FITC with iDCs or

LPS-mDCs for 2 h at 37 �C. The cells were then washed

with PBS and allowed to adhere to coverslips for 1 h. The

pH of the intracellular compartment containing HIV-1-AT-2-

FITC was assessed by ratio fluorescence imaging of the

internalized pH-sensitive FITC in living cells. HIV-1-AT-2-

FITC accumulated in structures that were more scattered

in iDCs (data not shown) and more clustered in mDCs

(Figure 2A). Strikingly, when we quantified the pH of inter-

nalized FITC-AT-2-HIV in endocytic organelles in mDCs,

we observed that the virus accumulated in an intracellular

compartment with a mean pH of 6.12. A Gaussian fit

analysis of the results confirmed that a majority of HIV-1

containing endocytic vesicles had a pH of 6.24 (Figure 2B).

Similarly, in iDCs, HIV-1-AT-2-FITC accumulated in a com-

partment with a pH of 6.22 (data not shown). This result

was significant because our previous studies, using a

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 489

similar method, demonstrated that the DC-specific recep-

tor DC-SIGN accumulated in vesicles with a pH of 5.47 in

iDCs (compatible with late endosomes/lysosomes) and

6.45 in mDCs (early endosomes) (23). Thus, HIV-1-AT-2-

FITC captured by DCs was targeted to an intracellular

endocytic compartment with an internal pH similar to

that of early endosomes but distinct from late endosomes

and lysosomes.

Because HIV-1 accumulates in organelles with a pH of

approximately 6.2, we tested the effect of different pH

media on HIV-1 infectivity. Infectious virus was incubated

for up to 5 days at 37 �C in media adjusted to pH values

ranging from 5.0 to 7.5. Surprisingly, though the infectivity

of virus incubated at all pHs declined, virus treated at

pH 5.5, 6.0, or 6.5 was preserved significantly longer

than virus incubated at neutral or above (pH 7–7.5) or

more acidic (pH 5.0) (Figure 2C).

Next, we compared the degradation of HIV-1 in DCs with

that of a well-characterized endocytic tracer, horseradish

peroxidase (HRP), using a FACS-based assay. In iDCs,

HRP was poorly degraded, but upon LPS-induced DC

maturation, HRP was degraded at a faster rate, in agree-

ment with the findings of (21) (Figure 2D). We also

observed that the internalized HRP co-localized at least in

part with LAMP-1 in iDCs and mDCs, consistent with

lysosomal degradation for this protein (data not shown).

Interestingly, no loss of signal was observed for HIV-1

over the first 4 h after internalization either in iDCs or

mDCs. However, after 24 h the cell-associated HIV-1

signal was decreased by approximately 90% in iDCs and

approximately 50% in mDCs (Figure 2D). Although we

cannot rule out in this assay that loss of signal is due to

HIV-1 recycling to the DC surface, it may be also due to

DC-mediated viral degradation. This result indicates that

HIV-1 virions are retained in iDCs and mDCs over the first

mDC mDC

mDC

T

T

HIV-1 HIV-1 HIV-1H

IV-1

HIV-1

HLA-DRHLA-DR CD3

DC-SIGN p24gag Moc

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83

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A B C

D

Figure 1: Mature dendritic cells

(mDCs) captureand transferHIV-

1 infection to CD4+ T cells via an

infectious synapse. A) FACS ana-

lysis of lipopolysaccharide-matured

mDCs. LPS-mDCs were positive

for DC-SIGN and CD83. B) LPS-

mDCs were pulsed with HIV-1 for

2 h at 37 �C, then fixed, stained

intracellular HIV p24gag, and ana-

lysed by FACS. About 50% of the

cells were positive for p24gag. C)

LPS-mDCs transfer HIV-1 infectiv-

ity to Jurkat CD4þ T cells in trans.

LPS-mDCs were incubated with

HIV-1 and co-cultured with non-

infected Jurkat cells treated with

Indinavir (1 mM). Forty-eight hours

post – co-culture, viral transfer was

determined by flowcytometric ana-

lysis of p24gag on CD3þ cells. D)

LPS-mDCs were incubated with

HIV-1 for 2 h at 37 �C. HIV-1 accu-

mulates in an intracellular ‘viral

endosome’ (D, left). Upon encoun-

tering Jurkat CD4þ T cells, HIV-1 is

redistributed from this intracellular

compartment to the zoneofcontact

(infectious synapse) between the

DC and the CD4þ T cell (D, center

and right). Immunological synapse

markers [MHC-II (HLA-DR, center)

and T-cell receptor (CD3, right)] do

not appear enriched in the infec-

tious synapse. This result is repre-

sentative of approximately 20

infectious synapses in each condi-

tion. (green, immunostaining of

HIV-1 p24gag; red (left and center),

HLA-DR; and red (right), CD3]

Bar ¼ 5 mm.

Garcia et al.

490 Traffic 2005; 6: 488–501

hours and are also detectable after 24 h in the absence of

viral replication. Furthermore, the loss of the HIV p24gag

signal differed from that of a classical endocytic tracer

targeted to lysosomes.

Intracellular localization of HIV-1 captured by mDCs

To further analyse the compartment in which HIV-1 accu-

mulates after internalization by DCs, we labeled cells with

several established markers of endocytic compartments,

including EEA1 (early endosomes), TGN46 (trans-Golgi

network), lysobisphosphatidic acid (LBPA), CD63 (late

endosome/MVB), CD81, CD9, HLA-DM, MHC-II (MHC-II

compartment) and LAMP-1 (lysosomes). HIV-1-GFP was

incubated with LPS-mDC for 2 h at 37 �C, to allow viral

capture and internalization. Cells were then washed with

PBS, allowed to adhere to coverslips for 1 h at 37 �C,fixed, and stained with appropriate antibodies and ana-

lysed by immunofluorescence microscopy. Most of the

cellular markers analysed did not show significant co-

localization with HIV-1 (Figure 3A). However, some

tetraspanins (CD81, CD82, CD9, and CD53) did show

significant overlap with internalized HIV-1 in the LPS-

mDC (Figure 3A and S1 available online at http://

www.traffic.dk/suppmat/6_6c.asp). By contrast, the inter-

nalized virus showed only limited overlap with the late

endosome/MVB marker CD63 and no co-staining with

LBPA or LAMP-1.

In iDCs, HIV-1 was transiently distributed to scattered

peripheral vesicles (Figure S2A available online at http://

www.traffic.dk/suppmat/6_6c.asp) that did not co-localize

with any of the marker antibodies tested (data not shown).

However, after 4–5 h incubation with iDCs, the virus

started to cluster in a perinuclear compartment (data not

shown). This clustering became very obvious after 24 h

(Figure 3B and S2 available online at http://www.traffic.dk/

suppmat/6_6c.asp), suggesting that the virus induced DC

maturation. At this point, the clustered intracellular HIV in

these HIV-1 treated DCs (HIV-mDCs) also co-localized

with CD81, but not with late endosome or lysosome

markers (CD63 and LAMP-1), with HLA-DM or with

MHC-II HLA-DR (Figure 3B). Some other morphological

7.0

6.0

5.0

pH

pH

50

40

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05.0 5.5 6.0 6.5 7.0

mDC

mDC

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102

101

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0 1 2 3 4 5

0 h

4 h

24 h

180

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100

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60

40

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HRP HIV

medium

Detection threshold

Days post-incubation iDC iDCmDC mDC

Num

ber

of F

ITC

+ v

esic

les

Tot

al s

igna

l (%

)

Infe

ctio

us u

nits

/ml

A B

C D

Figure 2: HIV-1 accumulates in a mildly acidic dendritic cell (DC) intracellular compartment. LPS-mDCs were incubated for 2 h at 37 ˚Cwith HIV-1-AT-2-FITC to allow internalization. The pH of HIV-1-AT-2-FITC positive vesicles was measured. A) A composite image of one

representative LPS-mDC integrating fluorescence and pH scaled in pseudocolors (side bar) is shown. The HIV-1-AT-2-FITC-positive vesicular

structures exhibit the blue coloration indicative of a pH of 6.1–6.2. Bar ¼ 5 mm. B) Distribution of HIV-1-AT-2-FITC-positive vesicles along the

endocytic pH gradient inmDCs,with Gaussian fit (red line). C)Mildly acidic pH stabilizes HIV-1 infectivity. Equal aliquots of HIV-1weremixedwith

DC culture medium adjusted to specific pH values ranging from 5.0 to 7.0 or with non-adjusted culture medium (pH 7.5–7.8) (medium) at 37 �Cfor various periods of time. Infectious units permillilitre contained in supernatantswere then determined. Each value represents themean of two

independent experiments. D) Quantitative decay of intracellular HRP and HIV-1 p24gag in DC-SIGN þ iDCs and LPS-mDCs. Horseradish

peroxidase and p24gag amounts measured at each time point are expressed as a percentage relative to the 100% starting points. Histograms

represent three independent experiments � SEM.

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 491

changes similar to those observed during LPS-induced DC

maturation (in the absence of HIV-1) were observed in HIV-

mDCs when compared to iDC, e.g. MHC-II was redistribu-

ted from intracellular compartments to the DC surface,

while CD81 and CD9 were removed from the cell surface

to an intracellular location (Figure 3A,B and S3 available

online at http://www.traffic.dk/suppmat/6_6c.asp). Thus,

after 24 h, HIV-1 induced at least partial maturation in the

DC (compared to full maturationwith LPS) (data not shown),

consistent with other studies (24,25).

To analyse the co-localization of HIV-1 with late endo-

somal/MVBmarkers more quantitatively, we used immuno

fluorescence and confocal microscopy. HIV-mDCs or LPS-

mDCs were processed for immunofluorescent labeling as

described above. In HIV-mDCs, pixel analysis indicated

that approximately 90% of HIV-1 co-localized with CD81,

approximately 20% of HIV-1 co-localized with CD63, and

less than 10% with LAMP-1, HLA-DM, or HLA-DR

(Figure 3C, center). As expected, approximately 70–80%

of HLA-DM and CD63 co-localized with LAMP-1 in these

cells, showing a typical late endosome/lysosome distribu-

tion (Figure 3C, right). Observations with LPS-mDCs were

similar to HIV-mDCs. Pixel analysis indicated that approxi-

mately 60–80% of HIV-1-GFP co-localized with CD81,

CD82, and CD9 (Figure 3C, left) and that up to 30–50%

of the total staining for CD81, CD82, CD53, and CD9

overlapped with HIV-1-GFP in LPS-mDCs (data not

shown). Only approximately 5% of HIV-1 co-localized

with LBPA (Figure 3C, left). HIV-1 co-localized partly with

CD63 (approximately 35% of virus overlapped with CD63).

However, the bulk of CD63 staining did not overlap with

HIV-1 (only 5% of the total CD63 staining co-localized with

HIV-1, data not shown) consistent with observations in

HIV-mDCs. These data indicate (i) that internalized HIV-1

reorganizes endocytic compartments in iDCs in a manner

that is similar to LPS and (ii) that HIV-1 is located in a ‘viral

endosome’ that is distinct from early endosomes (defined

by EEA1) or ‘classical’ late endosomes/lysosomes

(defined by CD63, LBPA, and LAMP-1). This subcompart-

ment is characterized by the presence of the tetraspanins

CD81, CD82, and CD9 and the absence of LAMP-1. Of

note, this CD81þ/LAMP-1– compartment is also present in

LPS-mDCs (in the absence of HIV-1, see below and

Figure 6). Therefore, we use in the present article the

term ‘viral endosome’ operationally, pending further func-

tional studies of this novel endocytic compartment.

Analysis of the HIV-1-containing endosome

compartment by electron microscopy

To examine the structure of the virus-containing endo-

somal compartments in more detail, we analysed iDCs or

EEA1 MHC-II TGN46

CD63 LBPA LAMP-1

CD53CD9CD81

A

Figure 3: Continued on next page.

Garcia et al.

492 Traffic 2005; 6: 488–501

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Percentage of HIV-1 co-localizedwith cellular markers

(LPS-mDC)

Percentage of HIV-1 co-localizedwith cellular markers

(HIV-mDC)

Percentage of cellular markers co-localized with LAMP-1

(HIV-mDC)

HIV-1

HIV-1

HIV-1

HIV-1

CD81

CD63

HLA-DM

HLA-DR

LAMP-1

LAMP-1

LAMP-1

LAMP-1

B

C

Figure 3: Analysis of HIV-1 intracellular compartment. Immature DCs were incubated for 24 h at 37 �C with HIV-1 (HIV-mDC), or

LPS-mDCs were incubated for 2 h at 37 �C with HIV-1, to allow internalization. A) LPS-mDCs loaded with HIV-1 were analysed by

immunofluorescence microscopy. One representative LPS-mDC is depicted here with the corresponding cellular markers [green, HIV-1-

GFP; red, cellular markers; and blue, DAPI (nucleus)] Bar ¼ 5 mm. B) HIV-mDCs loaded with HIV-1 were analysed by confocal microscopy.

One representative HIV-mDC is depicted here with the corresponding cellular markers (green, immunostaining for HIV-1 p24gag; red,

cellular markers; and blue, LAMP-1) Bar ¼ 5 mm. C) Quantification of the percentage of HIV-1 co-localized with the cellular markers in

LPS-mDCs (left) and in HIV-mDCs (center) (confocal images for LPS-mDCs and HIV-mDCs). Quantification of the percentage of LAMP-1

co-localized with the cellular markers in HIV-mDCs (right).

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 493

LPS-mDCs by immunolabeling and electron microscopy.

Cells were pulsed with HIV-1 for 2 h at 37 �C, fixed, andprocessed for cryosectioning and immunolabeling.

Labeling with antibodies against the viral matrix protein

(p17/MA) identified numerous virions as electron-dense,

slightly irregular particles of diameter 100–130 nm, some

of which contained a darker center representing the viral

core. Virus particles were found at the cell surface, often

tangled deeply among the numerous membrane protru-

sions and microvilli, or in pockets, folds or deeper invagi-

nations of the plasma membrane. In addition, viruses

were seen in coated vesicles, indicating that HIV-1 capture

and internalization by DCs occurred at least in part via

clathrin-mediated endocytosis (Figure 4).

In iDCs, some labeled virus particles were seen in small

vesicles (approximately 200-nm diameter) throughout the

cytoplasm but frequently found close to the plasma mem-

brane. By contrast, in the mDCs, large numbers of viruses

were observed in more complex vacuoles ranging in size

from 0.4 to 1.8 mm that are likely to represent the viral

endosome observed by immunofluorescence (Figure 5A).

These virus-containing structures often had a rounded or

elongated appearance and some seemed to consist of

clusters of several vacuoles, although these could be

interconnected in adjacent planes of section. Although

some of these virus-containing vacuoles were close to

the cell surface, they did not have obvious connections

to the plasma membrane; analysis of cells pulsed on ice

with HRP indicated that at least 20% of the virus vacuoles

were not accessible from the cell surface. The virus

vacuoles on mDCs often contained other intraluminal

membrane structures including small vesicles of 50–80-nm

diameter resembling the intraluminal vesicles of MVBs

(black arrow in Figure 5A). On some vacuoles, we observed

coated structures resembling clathrin-coated pits appar-

ently fusing into or budding away from the compartment

(see Figure 5B).

When LPS-mDCs cryosections were double stained for var-

ious cellular markers and HIV p17/MA, we found that the

virus-containing vacuoles consistently labeled for the tetra-

spanin CD81, which was usually seen on the small internal

vesicles (Figure 5B, black arrows). Similarly, we could

detect some CD63 on the small vesicles (Figure 5C, black

arrows). Although the CD63 gold particle densities on the

vesicles were comparable with the labeling seen for CD81,

the bulk of the cellular CD63 was seen over more juxta-

nuclear MVBs and lysosome structures, which were inten-

sely labeled but did not contain virus. Thus, as suggested by

the immunofluorescence labeling (Figure S3 available online

at http://www.traffic.dk/suppmat/6_6c.asp), virus-containing

vacuoles represent a subpopulation of the CD63 contain-

ing structures present in these cells. The virus-containing

endosomeswere also weakly labeled by an antibody against

MHC class II (Figure 5D). In contrast, prominent labeling

for this antigen was seen at the cell surface (Figure 5D,

white arrows), as expected for mDCs. The MHC class-II

staining in the virus-containing vacuoles appeared to be

associated mainly with the internal membranes (black

arrow) and not the limiting membrane, suggesting that

the virus-containing vacuoles are not continuous with

the plasma membrane but are discrete cytoplasmic

structures. The results described here are compared

with results from confocal immunofluorescence and

immunofluorescence on semithin crysection experiments

summarized in Table 1.

Thus, the compartment to which HIV-1 is sequestered

after internalization into mDCs has the appearance of a

MVB with internal membranes and small intraluminal vesi-

cles that contain various tetraspanin molecules and some

MHC class-II antigens (see Table 1). Although these vesi-

cles have characteristics similar to the vesicles in MVBs,

the virus-containing endosome appears to be distinct from

the main MVB and lysosome compartment in these cells.

We refer to this compartment as the ‘viral endosome’.

Figure 4: HIV is internalized by mDCs via clathrin-mediated endocytosis. Ultrathin cryosections of HIV-1-pulsed LPS-mDCs were

labeled with antibodies against HIV p17 (PAG 10 nm, left-hand panel) or p17 (PAG 15 nm) plus CD81 (PAG 5 nm, centre and right panels).

Virus particles could frequently be seen in coated vesicles, suggesting that HIV-1 is internalized, at least in part, through clathrin-

dependent endocytosis. Bars ¼ 100 nm.

Garcia et al.

494 Traffic 2005; 6: 488–501

HIV-1 and CD81 recycle to the infectious synapse

HIV-1 is rapidly routed to the DC surface when cells

pulsed with the virus encounter CD4þ T cells. To analyse

the pathway of HIV-1 trafficking from the viral endosome

to the DC–T-cell infectious synapse, we used our infec-

tious synapse assay (see above and Figure 1D). Because

HIV-1 did not co-localize in the infectious synapse either

with the T-cell receptor or MHC-II but shared trafficking

pathways with some tetraspanins such as CD81, we ana-

lysed the distribution of the tetraspanins CD81, CD9, and

CD63 as well as LAMP-1 at the infectious synapse. In

LPS-mDCs that had not been exposed to HIV, approxi-

mately 90% of the CD81 staining (quantified by confocal

microscopy) was in an intracellular compartment that did

not co-localize with LAMP-1, while 10% was at or close to

the cell surface. CD63 co-localized extensively with

LAMP-1 in the same conditions (Figure 6A, lines 1 and

2). Adding CD4þ T cells induced some re-location of

CD81 to the cell surface in a small proportion of the cells

but did not significantly alter the distribution of CD63 or

LAMP-1 (Figure 6A, lines 3 and 4). As staining for CD81

Table 1: Summary of co-localizations between cellular markers

and HIV-1 in LPS-mDCs

Cellular markers IF/confocal IF on cryosection EM

CD81 þþþ þþþ Yesa

CD9 þþþ þþþ Yesa

CD63 þ þ Yesb

CD53 þ ND ND

CD82 þþ ND ND

MHC-II – � Yesc

LAMP-1 – – ND

LBPA – – ND

EEA1 – ND ND

TGN46 – ND ND

þþþ, strong; þþ, medium; þ, weak, þ/–; or –, very weak or none;

ND: not defined, IF: immunofluorescence, EM: electronmicroscopy.aA majority of the intracellular maker co-localizes with HIV-1.bThe majority of CD63 is in MVB/lysosomes. Only a minority of

intracellular CD63 co-localizes with HIV-1.cThe majority of MHC-II is at the plasma membrane. Weak staining of

intracellularMHC-II is observedonlyon the internalmembranesof theviral

endosome and never on the limiting membrane of the viral endosome.

p17-10

p17 - 15

p17 - 15

p17 - 15

CD81 - 5

MHC-II - 5CD63 - 5

A B

C D

Figure 5: Ultrastructure of the viral endosome compartment. A) Ultrathin cryosections of HIV-1-pulsed LPS-mDCs were labeled with

antibodies against the HIV-1 matrix protein MA/p17 and PAG 10 nm. The large vacuole contains numerous labeled virus particles, while the

black arrow identifies one of the small internal vesicles. (B, C, D) Sections were double labeled for HIV-1 p17 (PAG 15 nm) and the cellular

markers B) CD81; C) D63; or D) MHC class II with PAG 5 nm. Black arrows show internal vesicles or membranes labeled with the markers.

Note the coated buds on the limiting membrane of the vacuoles shown in B (white arrowheads). In D), strong labeling for MHC-II is observed

at invaginations of the plasma membrane nearby (white arrows). Note the coated bud on this membrane (black arrowhead). Bars ¼ 200 nm.

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 495

CD81

CD63

CD81

CD63

CD63

CD81

CD81

CD63

LAMP-1

LAMP-1

LAMP-1

LAMP-1

LAMP-1

LAMP-1

LAMP-1

LAMP-1

HIV-1

HIV-1

HIV-1

HIV-1

+H

IV-1

–HIV

-1

+T

+T

A

B

Figure 6: HIV-1 subverts the trafficking pathways of components of the DC–T-cell immunological synapse. A) Distribution of CD81

and LAMP-1 (Lines 1 and 3) or CD63 and LAMP-1 (Lines 2 and 4) in LPS-mDCs alone (in absence of HIV-1, Lines 1 and 2) or after incubation

with Jurkat CD4þ T cells (Lines 3 and 4). B) LPS-mDCs were incubated with HIV-1 to allow internalization and incubated with Jurkat CD4þ T

cells for 30 min to allow infectious synapse formation. The pattern of HIV-1, CD81, and LAMP-1 (Lines 1 and 3) or HIV-1, CD63, and LAMP-1

(Lines 2 and 4) is shown in LPS-mDCs alone (Lines 1 and 2) or after incubation with the Jurkat CD4þ T cells (Lines 3 and 4). CD81

redistributes from its intracellular pool to the infectious synapse. This result is representative of three independent experiments. (green,

immunostaining of HIV-1 p24gag; red, cellular markers; and blue, LAMP-1). Bar ¼ 5 mm.

Garcia et al.

496 Traffic 2005; 6: 488–501

and CD9 was very weak in CD4þ T cells [Figure 6 (data not

shown)], our assay mainly follows CD81 and CD9 on the

DC side of the synapse.

In LPS-mDCs pulsed with HIV-1 (in the absence of T cells),

HIV co-localized with CD81, but not with CD63 or LAMP-1

(Figure 6B, lines 1 and 2). Strikingly, when the virus-pulsed

LPS-mDCs were incubated with CD4þ T cells, the intra-

cellular CD81 and CD9 disappeared and were completely

redistributed to the infectious synapses [Figures 6B (line

3) and 7]. In contrast, there was no apparent redistribution

of CD63 or LAMP-1 (Figure 6B, line 4). We quantified the

percentage of infectious synapses that showed redistribu-

tion and focusing of CD81 and CD9. Strikingly, 90–100%

of DC–T-cell conjugates presenting virus at their zone of

contact also relocated CD81 and CD9 in the synapse zone

(Figure 7). Interestingly, even in the absence of virus,

some DC–T-cell conjugates (approximately 30–40%)

showed a partial redistribution of CD9 or CD81 from intra-

cellular compartments to the DC–T-cell contact zone. This

indicates that HIV-1 stimulates the redistribution of CD9

and CD81 to the DC–T-cell contact zone in a similar way to

that which occurs during formation of antigen-dependent

immunological synapses (19).

Discussion

Our results demonstrate that, in immature and mature

DCs, intact HIV-1 particles are captured and internalized

into an intracellular endocytic compartment with novel

properties that may facilitate cell-to-cell transmission of

infectious virus. A major role for DCs in facilitating HIV-1

spread within infected individuals has been proposed

(7,17). Moreover, studies from several laboratories have

indicated that endocytosis of infectious virus is important

for this activity (12). However, except for the fact that HIV-

1 can be recycled to the specialized areas of DC–T-cell

zone of contact, termed infectious or virological synapses,

the fate of the internalized virus within DCs has remained

unclear (10,13,14).

Here, we show that the internalized virus accumulated in a

clustered intracellular compartment characterized by the

presence of the tetraspanins CD81, CD82, and CD9.

Although this compartment contained some (though not

the majority) of the cellular CD63, it was distinct from

HLA-DM and LAMP-1-containing lysosomes. Immuno-

electron microscopy confirmed that HIV-1 particles accu-

mulated in intracellular vacuoles that contained some

intraluminal vesicles reminiscent of exosomes. pH meas-

urements indicated that this compartment has a mildly

acidic pH, and studies with cell-free virus suggested that

this is the optimum pH to maintain HIV-1 infectivity. When

HIV-1-loaded DCs were allowed to contact T cells, the

virus, together with the markers CD81 and CD9, was

relocated to the infectious synapse.

In addition, we noticed that HIV-1 treatment could induce

reorganization of the endocytic compartments in iDCs

similar to that observed for LPS-induced activation. HIV-1

treatment induced the translocation of MHC-II to the cell

surface and the intracellular accumulation of the tetraspa-

nins CD81 and CD9. This result is consistent with the fact

that HIV might induce at least some degree of DC matura-

tion, possibly via Toll-like receptor 8 (26), in a similar

manner to LPS-induced maturation via TLR-4. Although

HIV-1-induced DC maturation is not as extensive as that

following LPS treatment (data not shown), several

changes associated with DC maturation have been

observed after HIV-1 binding, including cytokine secretion

and cell migration (24,25,27).

Maturation alters the endocytic trafficking in DCs, e.g.

shutting down some pathways such as macropinocytosis

(28). We, therefore, compared the degradation of HIV-1 in

DCs to that of HRP, a well-characterized endocytic tracer.

In iDCs, HRP was poorly degraded, but upon LPS-induced

DC maturation, HRP was degraded at a faster rate, con-

sistent with the finding that DC maturation activates lyso-

somal function (21). Interestingly, no loss of the HIV-1 p24

signal was observed over the first 4 h after internalization

either in iDCs or mDCs. However, after 24 h, DC-associated

HIV-1 degradation occurred faster in iDCs when compared

with LPS-mDCs, in agreement with (13). Although we

cannot rule out in this assay that loss of signal is due to

some HIV-1 recycling to the cell surface, DC-mediated

viral degradation is the most likely explanation for our

results. Together, our data indicate that the properties of

the viral endosome during DC developmental stages are

distinct from the ‘classical’ lysosomes to which HRP is

targeted. Interestingly, we could observe HIV-1 in coated

100

90

80

70

60

50

40

30

20

10

0

CD

63

CD

81

CD

81

CD

9

CD

9

CD

63

Mock +HIV-1

Red

istr

ibut

ion

(%)

Figure 7: HIV-1 stimulates the redistribution of tetraspanins

to the infectious synapse. Quantification of the redistribution of

tetraspanins from intracellular pools to DC–T-cell zone of contact

in the presence (right) or absence of virus (left). Results are

representative of three or four independent experiments including

SD.

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 497

vesicles (Figure 4), suggesting that, at least in part, HIV-1

could reach the viral endosome by clathrin-mediated endo-

cytosis, a pathway that is reported not to be affected by

DC maturation (28). However, the precise events that

allow HIV-1 to reach the viral endosome and avoid lyso-

somal degradation remain to be identified. One possibility

is that HIV-1 uses clathrin-mediated endocytosis to reach

the viral endosome directly. Alternatively, the virus may be

delivered to early endosomes, or even late endosomes,

and be actively sorted from these compartments to the

viral endosome.

After demonstrating that HIV-1 accumulates in a compart-

ment of pH of 6.1–6.2, we tested the direct effect of

media with a pH ranging from 5.0 to 7.5 on the infectivity

of cell-free HIV-1. Although most infectivity was lost over

time, we showed that virus incubated in mildly acidic pH

medium (approximately 6.0) retained infectivity signifi-

cantly longer than virus incubated in neutral/more alkaline

(7.5) or more acidic conditions (5.0) (Figure 2C). These

results may provide an alternative explanation for the

results of Kwon et al. (12), who showed that agents that

neutralize endosomal pH and affect the proper endosomal

trafficking in DCs also prevent HIV-1 transmission to T

cells. Correct trafficking of HIV-1 through the endocytic

pathway after internalization is obviously essential for

virus transmission, and perturbation of endosomal pH

might influence this trafficking. However, the finding that

HIV-1 infectivity is retained better at a mildly acidic pH,

similar to that found in viral endosomes, raises the possi-

bility that increasing endosomal pH could reduce the infec-

tivity of virus sequestered in the DC viral endosome. This

result is important, because even if a minimal fraction of

viral infectivity is retained at pH 6.0 after 3–4 days, very

small amounts of virus can be transferred from DCs to

T cells in trans, a process known as ‘trans enhancement of

HIV-1 infection to T cells’ (9,12,29). Nevertheless, given

that the contents of endosomal compartments and extra-

cellular culture supernatants are very different, the infec-

tivity of HIV-1 retained within the viral endosome will

require further analysis.

Characterization of the compartment where HIV-1 accu-

mulates by immunofluorescence showed that it shares

some features with late endosomes/MVBs in both LPS-

mDCs and HIV-mDCs, but that it is clearly distinct from

‘classical’ late endosomes or lysosomes. InternalizedHIV-1 did

not significantly co-localize with well-characterized cellular

marker proteins including EEA-1 (early endosomes),

TGN46 (trans-Golgi network), and LBPA or LAMP-1(lyso-

somes), extending the results from others showing that

HIV-1 did not co-localize with early endosomes (transfer-

rin) or lysosomes (LAMP-1) (12,13,30). However, HIV-1

did co-localize with a number of tetraspanins (CD81,

CD82, and CD9) and with a subpopulation of the cellular

CD63. Interestingly, recent observations demonstrated

that in HIV-infected human primary macrophages, a cell

type related to DCs, assembling HIV-1 can bud directly

into a late endosome/MVB compartment that also con-

tains CD63 and CD81 (31). Furthermore, the cellular

machinery involved in MVB formation (the ESCRT machin-

ery) has been found to be required to complete HIV-1

assembly (32). These observations have lead to the pro-

posal that HIV-1 might subvert similar trafficking pathways

for viral budding in macrophages and for transfer of viral

infection from DCs to T cells (31,33). However, in macro-

phages HIV-1 buds into the endosome compartment,

while in the DCs, the virus reaches its intracellular com-

partment by endocytosis in the absence of viral

replication.

We have also demonstrated that on encountering T cells,

DCs can translocate HIV-1 from this intracellular compart-

ment to the DC–T-cell infectious synapse. The presence of

a synapse between a virus-carrying cell and an uninfected

target cell is not restricted to HIV-1 in the DC–T-cell situa-

tion (7,10,13,14) and may well be a general mechanism of

viral propagation (34,35). Cell-to-cell transmission is likely

to favor HIV-1 replication because it avoids the rate-

limiting step of virus diffusion prior to attachment.

Furthermore, cell-to-cell transmission may reduce viral

neutralization by antibodies and complement (36) and

potentially allows for T-cell activation concurrently with

viral infection. As such, the presence of cellular antigens

implicated in T-cell activation in the infectious synapse is

potentially important.

Remarkably, upon contact with CD4þ T cells, HIV-1-pulsed

LPS-mDCss transported their intracellular pools of virus,

as well as the tetraspanins CD81 and CD9, to the infec-

tious synapse. The tetraspanin CD81 has been linked to

several functions including intracellular signaling (37) and

modulation of T-cell activation (18). Interestingly, in anti-

gen-presenting cells, CD81 facilitates MHC class-II-

mediated antigen presentation (38), and CD81 redistri-

butes to the central zone of the antigen-dependent immu-

nological synapse both on the APC side and on the T-cell

side (19). Interestingly, in the HIV-1-loaded DC, bona fide

immunological synapse markers such as HLA-DR and CD3

did not cluster in the infectious synapse. Further analysis

is required to determine the impact of the selective

recruitment of CD81 and CD9 to the DC–T-cell zone of

contact (in the absence of MHC-II and of the T-cell recep-

tor) on CD4þ T-cell activation and HIV-1 replication. The

fact that HIV-1 in DCs appears to follow, at least in part,

the trafficking pathway that CD81 uses to redistribute

from its intracellular pools to the immunological synapse

identifies a clear relationship between the DC–T-cell infec-

tious synapse and a DC–T-cell immunological synapse and

suggests that HIV-1 ‘highjacks’ a pathway involved in

trafficking components of the immunological synapse, in

order to mediate infection of T cells in trans.

In LPS-mDCs (in the absence of HIV-1), CD81 and CD9

were also observed clustered intracellularly in a similar

pattern and did not co-localize with CD63 or LAMP-1,

Garcia et al.

498 Traffic 2005; 6: 488–501

suggesting that HIV-1 might target a pre-existing tetra-

spanin-rich endosomal compartment. The function of this

CD81þ/CD9þ but CD63low/LAMP-1– vesicle-containing

endosome is unclear, but it might be implicated in antigen

processing, e.g. antigen degradation rates in this compart-

ment might be lower than in classical lysosomes, and this

may be a way to store antigens for prolonged periods of

time. Interestingly, much of the CD81 in the viral endo-

some was seen associated with the intraluminal exo-

some-like vesicles and not the limiting membrane.

Similarly, the MHC class II and CD63 labeling was also

mainly associated with the internal membranes. Whether

these antigens are released into the synapse as exosomes

remains unclear but warrants further investigation. The

tetraspanin-rich compartment may allow antigen sharing

between DCs by transferring some antigens from DCs to

other DCs or other antigen-presenting cells and might

then be exploited as an escape route for viruses such as

HIV-1 to avoid lysosomal degradation.

In conclusion, our studies identify a trafficking pathway

that is shared by molecules that function in the DC–T-cell

immunological synapse (CD81 and CD9) and by HIV-1

captured by DCs, allowing it to be transported in a retro-

grade manner from its viral endosome to the DC–T-cell

infectious synapse. The elucidation of HIV-1 trafficking in

DCs and of DC–T-cell infectious synapse formation begins

to provide us with insights into the interactions between

retroviruses and the highly organized endocytic machinery

of DCs, cells that are central for immune responses and

HIV-1 transmission.

Materials and Methods

Preparation of human primary DCsMonocytes from buffy coats were obtained according to institutional guide-

lines of the ethical committee of the University of Geneva. Monocytes

were induced to differentiate into iDC for 6 days with 50 ng/mL GM-CSF

and IL-4 or into mDC by further addition of LPS (20 ng/mL) for the last 2

days (LPS-mDC). Alternatively, iDCs were ‘matured’ by pulsing them with

HIV-1 for 24 h (MOI ¼ 5) and called HIV-mDC. Dendritic cells were har-

vested at day 6, analysed by flow cytometry, and used in subsequent

assays. Additional technical details are available in (10,29).

Viral stocksViral stocks production and viral titers were described previously (29). To

track HIV-1 particles, we prepared GFP-labeled HIV-1 X4 (HIV-1-GFP) by

incorporation of WxxF-GFP into virions through interaction with HIV-1-VPR

in a similar manner as in (39). HIV-1-AT-2-FITC was generated using a

modified version of the protocol used by Greber et al. to study adenoviral

entry (22) and described in supplemental online Material and Methods

available at http://www.traffic.dk/suppmat/6_6c.asp.

Antibodies and reagentsMost antibodies used in this study have been previously described (29).

The rabbit polyclonal anti-LAMP-1 was a gift from M. Fukuda (Cancer

Research Center, La Jolla, CA, USA) (40). Additional antibodies are

described in supplemental online Material and Methods available at

http://www.traffic.dk/suppmat/6_6c.asp.

Flow cytometric analysisFlow cytometric analysis was performed as described (10,29).

Viral capture and transfer assaysViral capture and transfer assays were performed as described previously

(10) with minor modifications available in supplemental online Material and

Methods available at http://www.traffic.dk/suppmat/6_6c.asp.

PH measurement studiesThe pH of the organelles to which internalized HIV-1-AT-2-FITC virions

were targeted was measured by ratio fluorescence imaging of a pH-

sensitive probe as previously described (23,41).

Variation of cell-free medium pH and effect on HIV

InfectivityAliquots of HIV-1 were added to DC culture medium adjusted to pH 5.0,

5.5, 6.0, 6.5, and 7.0 at 37 �C. Viral infectivity was monitored at 1-day

intervals using a single round infectivity assay on CD4þ HeLa P4-2 cells.

Proteolysis assaysDegradation assays in living cells were performed as in (21) with minor

modifications available in supplemental Material and Methods available at

http://www.traffic.dk/suppmat/6_6c.asp.

Immunofluorescence microscopy and confocal

microscopyTo localize HIV-1, LPS-mDCs (105 cells/condition) were loaded with HIV-1

GFP (MOI ¼ 10) for 2 h at 37 �C. HIV-mDCs were pulsed with HIV-1

(MOI ¼ 5) for 24 h, washed twice in PBS, and left to adhere on poly

L-lysine-treated (Sigma-Aldrich, St. Louis, MO, USA) glass coverslips for

1 h at 37 �C. Cells were then fixed 20 min at room temperature in 3%

paraformaldehyde, permeabilized with 0.05% saponin (Sigma-Aldrich), and

washed with PBS containing 0.2% bovine serum albumin (BSA; Sigma-

Aldrich) and human IgG (20 mg/condition). Cells were stained with primary

antibodies and secondary donkey anti-mouse coupled to rhodamine

(Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei

were stained with DAPI (Molecular Probes, Eugene, OR, USA).

Alternatively, triple labeling of HIV-mDCs was done as follows: iDCs pulsed

with HIV-1 (MOI ¼ 5) were stained with primary antibodies against CD81,

HLA-DM [both monoclonal and from BD PharMingen (San Diego, CA,

USA)], CD63 [monoclonal (1B5)] and LAMP-1 [polyclonal; a gift from

M. Fukuda (Cancer Research Center)]. After extensive washes in BSA/

saponin-containing PBS, cells were then stained with secondary donkey

anti-mouse antibodies coupled to rhodamine or secondary donkey anti-

rabbit antibodies coupled to Cy-5 (Jackson ImmunoResearch

Laboratories). In order to avoid unspecific labeling, cells were incubated

20 min at room temperature in PBS containing BSA, saponin, and mouse

serum (0.5 mg/mL). Finally, HIV-1-p24gag was detected using a monoclonal

anti-HIV-1-p24gag (KC57) coupled to FITC (Coulter, Miami, FL, USA).

Infectious synapse assays were performed as previously described (10)

with minor modifications available in supplemental Material and Methods

available at http://www.traffic.dk/suppmat/6_6c.asp.

Immunolabeling of cryosections for electron

microscopyImmunolabeling of cryosections for electron microscopy was performed

with minor modifications from (31). Details are available in supplemental

online Material and Methods available at http://www.traffic.dk/suppmat/

6_6c.asp.

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 499

Acknowledgments

We thank Q. Sattentau, D. Trono, and U. Greber for helpful discussions. We

thank J. Gruenberg for providing us with the anti-LBPA antibody (6C4) and

M. Fukuda for the polyclonal anti-LAMP-1 antibody. We thank S. Arnaudeau

for assistance during analysis of confocal images. This work was supported

by the Swiss National Science Foundation grant no. 3345–67200.01,

Leenaards Foundation, NCCR oncology and the Geneva Cancer League to

VP. VP is the recipient of a ‘Professor SNF’ position (PP00A�68785). MM,

AP-M, and LC are supported by the UK Medical Research Council.

References

1. Steinman RM, Granelli-Piperno A, Pope M, Trumpfheller C, Ignatius R,

Arrode G, Racz P, Tenner-Racz K. The interaction of immunodeficiency

viruses with dendritic cells. Curr Top Microbiol Immunol 2003;276:

1–30.

2. Pope M, Haase AT. Transmission, acute HIV-1 infection and the quest

for strategies to prevent infection. Nat Med 2003;9:847–852.

3. Mellman I, Steinman RM. Dendritic cells: specialized and regulated

antigen processing machines. Cell 2001;106:255–258.

4. Banchereau J, Steinman RM. Dendritic cells and the control of immu-

nity. Nature 1998;392:245–252.

5. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic

cells. Cell 2001;106:263–266.

6. Piguet V, Blauvelt A. Essential roles for dendritic cells in the pathogen-

esis and potential treatment of HIV disease. J Invest Dermatol

2002;119:365–369.

7. Pope M, Betjes MG, Romani N, Hirmand H, Cameron PU, Hoffman L,

Gezelter S, Schuler G, Steinman RM. Conjugates of dendritic cells and

memory T lymphocytes from skin facilitate productive infection with

HIV-1. Cell 1994;78:389–398.

8. Geijtenbeek TB, van Kooyk Y. DC-SIGN: a novel HIV receptor on DCs

that mediates HIV-1 transmission. Curr Top Microbiol Immunol

2003;276:31–54.

9. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ,

van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS,

KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y. DC-SIGN,

a dendritic cell-specific HIV-1-binding protein that enhances trans-

infection of T cells. Cell 2000;100:587–597.

10. Arrighi JF, Pion M, Garcia E, Escola JM, van Kooyk Y, Geijtenbeek TB,

Piguet V. DC-SIGN-mediated infectious synapse formation enhances

X4 HIV-1 transmission from dendritic cells to T cells. J Exp Med

2004;200:1279–1288.

11. Turville SG, Cameron PU, Handley A, Lin G, Pohlmann S, Doms RW,

Cunningham AL. Diversity of receptors binding HIV on dendritic cell

subsets. Nat Immunol 2002;3:975–983.

12. Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR.

DC-SIGN-mediated internalization of HIV is required for trans-

enhancement of T cell infection. Immunity 2002;16:135–144.

13. Turville SG, Santos JJ, Frank I, Cameron PU, Wilkinson J, Miranda-

Saksena M, Dable J, Stossel H, Romani N, Piatak M Jr, Lifson JD,

Pope M, Cunningham AL. Immunodeficiency virus uptake, turnover,

and 2-phase transfer in human dendritic cells. Blood 2004;103:

2170–2179.

14. McDonald D, Wu L, Bohks SM, KewalRamani VN, Unutmaz D,

Hope TJ. Recruitment of HIV and its receptors to dendritic cell-T cell

junctions. Science 2003;300:1295–1297.

15. Dustin ML, Colman DR. Neural and immunological synaptic relations.

Science 2002;298:785–789.

16. Piguet V, Sattentau Q. Dangerous liaisons at the virological synapse.

J Clin Invest 2004;114:605–610.

17. Cameron PU, Freudenthal PS, Barker JM, Gezelter S, Inaba K,

Steinman RM. Dendritic cells exposed to human immunodeficiency

virus type-1 transmit a vigorous cytopathic infection to CD4þ T cells.

Science 1992;257:383–387.

18. Miyazaki T, Muller U, Campbell KS. Normal development but differen-

tially altered proliferative responses of lymphocytes in mice lacking

CD81. Embo J 1997;16:4217–4225.

19. Mittelbrunn M, Yanez-Mo M, Sancho D, Ursa A, Sanchez-Madrid F.

Cutting edge: dynamic redistribution of tetraspanin CD81 at the central

zone of the immune synapse in both T lymphocytes and APC.

J Immunol 2002;169:6691–6695.

20. Gruenberg J. The endocytic pathway: a mosaic of domains. Nat Rev

Mol Cell Biol 2001;2:721–730.

21. Trombetta ES, Ebersold M, Garrett W, Pypaert M, Mellman I.

Activation of lysosomal function during dendritic cell maturation.

Science 2003;299:1400–1403.

22. Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP,

Greber UF. Microtubule-dependent plus- and minus end-directed

motilities are competing processes for nuclear targeting of adenovirus.

J Cell Biol 1999;144:657–672.

23. Engering A, Geijtenbeek TB, van Vliet SJ, Wijers M, van Liempt E,

Demaurex N, Lanzavecchia A, Fransen J, Figdor CG, Piguet V, van

Kooyk Y. The dendritic cell-specific adhesion receptor DC-SIGN interna-

lizes antigen for presentation to T cells. J Immunol 2002;168:2118–2126.

24. Granelli-Piperno A, Golebiowska A, Trumpfheller C, Siegal FP,

Steinman RM. HIV-1-infected monocyte-derived dendritic cells do not

undergo maturation but can elicit IL-10 production and T cell regulation.

Proc Natl Acad Sci USA 2004;101:7669–7674.

25. Fonteneau JF, Larsson M, Beignon AS, McKenna K, Dasilva I,

Amara A, Liu YJ, Lifson JD, Littman DR, Bhardwaj N. Human immuno-

deficiency virus type 1 activates plasmacytoid dendritic cells and

concomitantly induces the bystander maturation of myeloid dendritic

cells. J Virol 2004;78:5223–5232.

26. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C,

Akira S, Lipford G, Wagner H, Bauer S. Species-specific recognition

of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:

1526–1529.

27. Wilflingseder D, Mullauer B, Schramek H, Banki Z, Pruenster M,

Dierich MP, Stoiber H. HIV-1-induced migration of monocyte-derived

dendritic cells is associated with differential activation of MAPK path-

ways. J Immunol 2004;173:7497–7505.

28. Garrett WS, Chen LM, Kroschewski R, Ebersold M, Turley S,

Trombetta S, Galan JE, Mellman I. Developmental control of endo-

cytosis in dendritic cells by Cdc42. Cell 2000;102:325–334.

29. Arrighi JF, Pion M, Wiznerowicz M, Geijtenbeek TB, Garcia E,

Abraham S, Leuba F, Dutoit V, Ducrey-Rundquist O, van Kooyk Y,

Trono D, Piguet V. Lentivirus-mediated RNA interference of DC-SIGN

expression inhibits human immunodeficiency virus transmission from

dendritic cells to T cells. J Virol 2004;78:10848–10855.

30. Trumpfheller C, Park CG, Finke J, Steinman RM, Granelli-Piperno A.

Cell type-dependent retention and transmission of HIV-1 by DC-SIGN.

Int Immunol 2003;15:289–298.

31. Pelchen-Matthews A, Kramer B, Marsh M. Infectious HIV-1 assembles

in late endosomes in primarymacrophages. J Cell Biol 2003;162: 443–455.

32. Morita E, Sundquist WI. Retrovirus budding. Annu Rev Cell Dev Biol

2004;20:395–425.

33. Amara A, Littman DR. After Hrs with HIV. J Cell Biol 2003;162: 371–375.

34. Igakura T, Stinchcombe JC, Goon PK, Taylor GP, Weber JN,

Griffiths GM, Tanaka Y, Osame M, Bangham CR. Spread of HTLV-I

between lymphocytes by virus-induced polarization of the cytoskele-

ton. Science 2003;299:1713–1716.

35. Jolly C, Kashefi K, Hollinshead M, Sattentau QJ. HIV-1 cell to cell

transfer across an Env-induced, Actin-dependent synapse. J Exp

Med 2004;199:283–293.

Garcia et al.

500 Traffic 2005; 6: 488–501

36. Ganesh L, Leung K, Lore K, Levin R, Panet A, Schwartz O, Koup R,

Nabel G. Infection of specific dendritic cells by CCR5-tropic HIV-1

promotes cell-mediated transmission of virus resistant to broadly neu-

tralizing antibodies. J Virol (in press).

37. Tarrant JM, Robb L, van Spriel AB, Wright MD. Tetraspanins: molecular

organisers of the leukocyte surface. Trends Immunol 2003;24: 610–617.

38. Engering A, Pieters J. Association of distinct tetraspanins with MHC

class II molecules at different subcellular locations in human immature

dendritic cells. Int Immunol 2001;13:127–134.

39. Turelli P, Doucas V, Craig E, Mangeat B, Klages N, Evans R,

Kalpana G, Trono D. Cytoplasmic recruitment of INI1 and

PML on incoming HIV preintegration complexes: interference

with early steps of viral replication. Mol Cell 2001;7:

1245–1254.

40. Carlsson SR, Roth J, Piller F, Fukuda M. Isolation and characterization

of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2.

Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem

1988;263:18911–18919.

41. Piguet V, Gu F, Foti M, Demaurex N, Gruenberg J, Carpentier JL,

Trono D. Nef-induced CD4 degradation: a diacidic-based motif in Nef

functions as a lysosomal targeting signal through the binding of beta-

COP in endosomes. Cell 1999;97:63–73.

HIV-1 Localization in Human Dendritic Cells

Traffic 2005; 6: 488–501 501