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Real-Time Visualization of HIV-1 GAG Trafficking in Infected Macrophages

PLOS Pathogens

April 2008
4(3):e1000015

DOI:10.1371/journal.ppat.1000015

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PubMed

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CC0

Authors:

Karine Gousset

California State University, Fresno

Lori V Coren

U.S. Department of Health and Human Services

Akira Ono

University of Michigan

Show all 8 authorsHide

HIV-1 particle production is driven by the Gag precursor protein Pr55(Gag). Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse.

The MA-TC tag does not affect Gag function. (A) Schematic diagram of HIV-1 Gag indicating the position of TC tag insertion. The amino acid sequence of the TC tag is shaded. (B) Replication kinetics of WT HIV-1 vs. the MA-TC derivative in the Jurkat T-cell line and primary MDM. Jurkat and MDM experiments were performed with pNL4-3 and pNL(AD8) molecular clones, respectively. Media were obtained every two days for RT analysis. (C) Virus release efficiency of WT vs. MA-TC. HeLa cells were transfected with WT pNL4-3 or pNL4-3/MA-TC plasmids. Transfected cells were labeled with FlAsH or DMSO (control) for 5 min 24–48 hrs posttransfection and were washed for 20 min with 300 µM EDT/PBS. The cells were then metabolically labeled with [35S]Met/Cys for 2 hrs. Released virions were pelleted by ultracentrifugation, and both cell and virus lysates were immunoprecipitated with HIV-Ig and subjected to SDS-PAGE. Bands were quantified using a phosphorimager. +/− SD, n = 3.

…

The MA-TC tag does not affect Gag localization. HeLa cells (top panel) were transfected with pNL4-3/MA-TC or 1GA or 29KE/31KE derivatives. Cells were labeled with FlAsH for 5 min at 37°C, washed, fixed in 3.7% formaldehyde, and examined microscopically. MDMs (bottom panel) were infected with VSV-G- pseudotyped virus stocks that transduced NL4-3/MA-TC or 1GA or 29KE/31KE derivatives (see Materials and Methods). Infected cells were labeled and fixed as described above. Scale bars = 30 µm. For a 3D z-series of the 29KE/31KE-TC mutant in MDM, see Video S1.

…

Gag in MDMs colocalizes with tetraspanins at the cell surface and in apparently internal compartments. MDMs infected with VSV-G-pseudotyped NL4-3/MA-TC (A and C) or NL4-3/29KE/31KE-TC (B) were labeled with FlAsH (green) 20 hours post-infection, fixed, and further labeled with antibody against CD63 (red) (A and B) or CD81 (red) (C). The merged images of Gag-TC and CD63/CD81 are shown on the right, with yellow indicating colocalization between Gag and CD63/CD81. R = Pearson coefficient of correlation. Scale bars: panel A, 40 µm; panel B, 30 µm; panel C; 30 µm top, 20 µm bottom.

…

Gag localization in MDMs remains stable over time. (A) MDMs were labeled with FlAsH 20, 24, 48, 72, and 96 hrs after infection with VSV-G-pseudotyped NL4-3/MA-TC. At each time point, between 25 and 65 cells were categorized as having PM, intracellular (IC), or both PM+intracellular (PM+IC) Gag localization. (B) Live-cell analysis. MDMs infected with VSV-G-pseudotyped NL4-3/MA-TC virus were labeled with FlAsH 48 hrs postinfection, and immediately examined microscopically on the stage in a closed chamber (37°C/5% CO2). Time (t) represents time in min after FlAsH labeling. Scale bar = 30 µm.

…

Gag accumulates at the MDM/MDM synapse. Cells were infected with VSV-G-pseudotyped NL4-3/MA-TC virus, labeled with FlAsH 24–48 hrs post-infection, and fixed in 3.7% formaldehyde. (A) Gag accumulates at the synapse between MDMs. Individual macrophages are labeled “M”. Scale bar = 15 µm. (B) After FlAsH labeling, cells were fixed and stained with anti-CD81 antibody. The extensive overlap between Gag and CD81 at the MDM/MDM synapse is visualized as yellow in the merged panels. Scale bar = 30 µm. Far-right panels in (A and B) provide quantification of the Gag signals. The Surface Plot analyzing tool from the ImageJ software was used to obtain a three-dimensional graph of the pixel intensities in grayscale. The x- and y- axes represent the length of the region analyzed in pixels, and the z-axis represents the pixel intensity of the Gag signal. (C) EM analysis of VLPs at the synapse. Infected MDMs were fixed with 2% glutaraldehyde and processed for EM. Fully assembled VLPs can be visualized at the MDM/MDM junctions. Red boxes represent regions that are enlarged in the adjacent panels.

…

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Real-Time Visualization of HIV-1 GAG Trafficking in

Infected Macrophages

Karine Gousset

, Sherimay D. Ablan

, Lori V. Coren

, Akira Ono

, Ferri Soheilian

, Kunio Nagashima

David E. Ott

, Eric O. Freed

1 Virus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland, United States of America, 2 AIDS Vaccine Program, SAIC-

Frederick, Inc., National Cancer Institute, Frederick, Maryland, United States of America, 3 Department of Microbiology and Immunology, University of Michigan Medical

School, Ann Arbor, Michigan, United States of America, 4 Image Analysis Laboratory, Advanced Technology Program, SAIC-Frederick, National Cancer Institute at

Frederick, Frederick, Maryland, United States of America

Abstract

HIV-1 particle production is driven by the Gag precursor protein Pr55

Gag

. Despite significant progress in defining both the

viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of

assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived

macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in

this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag

was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere

with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo

detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma

membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular

bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and

uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected

macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse.

Citation: Gousset K, Ablan SD, Coren LV, Ono A, Soheilian F, et al. (2008) Real-Time Visualization of HIV-1 GAG Trafficking in Infected Macrophages. PLoS

Pathog 4(3): e1000015. doi:10.1371/journal.ppat.1000015

Editor: Thomas J. Hope, Northwestern University, United States of America

Received October 2, 2007; Accepted Jan uary 30, 2008; Published March 7, 2008

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public

domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Funding: This research was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH, by the Intramural

AIDS Targeted Antiviral Program, and with federal funds from the National Cancer Institute, NIH, under contract N01-CO-12400.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: efreed@nih.gov

Introduction

The human immunodeficiency virus type 1 (HIV-1) Gag

polyprotein precursor, Pr55

Gag

, plays an essential role in virus

assembly and release. Its expression alone is able to generate virus-

like particles (VLPs) [1,2]. All four domains of Pr55

Gag

–matrix

(MA), capsid (CA), nucleocapsid (NC) and p6–play important roles

in particle assembly and release [1,3]. The MA domain regulates

the association of Gag with the host cell plasma membrane (PM);

this membrane-binding activity is provided primarily by a myristic

acid moiety covalently attached to the N-terminus of MA and a

highly basic patch of amino acid residues that interacts with acidic

phospholipids, including phosphatidylinositol-(4,5)-bisphosphate

[PI4,5)P

] on the inner leaflet of the PM [4,5,6,7]. CA and NC

promote Gag-Gag interactions during assembly [8], in part

through the ability of NC to interact with nucleic acid [2,9].

Finally, the p6 domain of Gag stimulates virus release by

interacting with components of the cellular endosomal sorting

machinery [10,11,12].

Although significant progress has been made in elucidating the

viral and cellular factors necessary for Gag membrane binding,

Gag multimerization, and virus release, the subcellular location of

HIV-1 assembly has been the subject of controversy and the

itinerary of Gag trafficking to the site of assembly remains to be

defined. Mutational studies have shown that the viral determinants

for Gag targeting to the PM reside in the MA domain of Gag. A

large deletion in MA redirects HIV-1 assembly to the endoplasmic

reticulum [13,14], whereas point mutations, particularly in the

highly basic domain of MA, shift the site of assembly from the PM

to internal compartments [15,16,17] defined as late endosomes or

multivesicular bodies (MVBs) [18].

HIV-1 was long assumed to follow the classically defined ‘‘C-

type’’ pathway in which Gag assembly and release take place at

the PM [2]. This dogma was challenged by a number of studies

suggesting that HIV-1 assembly takes place in an endosomal

compartment and that particle release from the infected cell

follows the ‘‘exosomal’’ pathway in which virus-containing

endosomes fuse with the PM to release their contents

[19,20,21,22,23,24]. This endosomal model was then subsequently

contested by several studies showing PM-based HIV-1 assembly

and release [25,26,27,28,29]. The nature of the HIV-1 assembly

site in primary monocyte-derived macrophages (MDMs) has been

a matter of particular interest [30]. Early electron microscopy

(EM) observations in HIV-1-infected MDMs revealed an abun-

dance of virions assembling and budding into intracellular

vacuoles [31,32]. In later studies, it was observed that the virus-

containing internal compartments in MDMs bore markers

characteristic of late endosomes or MVBs; e.g., major histocom-

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patibility complex II (MHC II) and tetraspanins CD63, CD81, and

CD82 [18,33,34]. Furthermore, virions derived from MDMs

packaged late endosome/MVB markers, suggesting that these

virions originated from a late endosomal compartment [33,35,36].

In an intriguing refinement of the model that HIV-1 assembles in

MVBs in primary macrophages, it was demonstrated that at least

some of the virus-positive, ‘‘intracellular’’ structures in MDMs were

actually connected to the PM. These apparently internal structures

may therefore represent PM invaginations that are positive for

tetraspanin markers [37,38]. Elucidating the virus assembly

pathway in primary MDMs is highly significant since this cell type

represents one of the major targets for HIV-1 infection in vivo [39].

One of the difficulties in evaluating previous studies focused on

defining the Gag assembly/release pathway in MDMs is the

absence of live-cell imaging data in this cell type that allow the

trafficking of Gag to be visualized in real time. To this end, we

developed a system for visualizing in living cells the localization

and trafficking of Gag expressed in the context of a fully infectious

and replication-competent HIV-1 molecular clone. We used the

biarsenical-tetracysteine labeling method first described by Tsien

and colleagues [40,41,42]. This system is based on the insertion of

a small tetracysteine (TC) motif into a protein of interest. Cells

expressing the TC-tagged protein are treated with a membrane-

permeable biarsenical dye [e.g., green (FlAsH) or red (ReAsH)]

that fluoresces upon binding to the TC tag. The advantages of this

method are that the TC tag is very small and that labeling occurs

immediately upon binding of the dye to the TC tag. Recently, this

system was used to label Gag expressed from non-infectious clones

in HeLa, Mel Juso and Jurkat T cells [23,29]. We introduced the

TC tag near the C-terminus of the MA domain of Gag in the

context of the full-length infectious HIV-1 molecular clone pNL4-

3. Insertion of the TC tag had no significant effect on HIV-1 Gag

function. By using VSV-G-pseudotyped viruses, we were able to

infect and follow Gag trafficking in primary MDMs. Our data

indicate that in MDMs Gag accumulates both at the PM and in an

apparently internal MVB-like compartment. Although we ob-

tained no evidence for constitutive movement of the internal Gag

to the PM, or for internalization of the PM-localized Gag to

apparently internal structures, we observed rapid relocation of the

internal population of Gag to the site of cell-cell contact following

addition of susceptible T cells to the infected macrophage cultures.

These findings support a model whereby newly assembled virus

particles are sequestered in infected macrophages and then

efficiently presented to susceptible target cells following synapse

formation.

Results

Introduction of a TC tag near the C-terminus of the MA

domain of HIV-1 Gag does not disrupt virus replication,

assembly and release, or Ga g trafficking

The MA domain of HIV-1 Gag performs several important

functions in virus assembly and release [1]; however, deletion of a

number of C-terminal residues (amino acids 116-128) [43], or the

insertion of a Myc or green fluorescent protein (GFP) tag near the

C-terminus of MA [44] does not block virus assembly and release,

suggesting that the C-terminus of MA is relatively insensitive to

mutation. Thus, to facilitate the study of HIV-1 Gag trafficking,

we deleted codons 121–128 of MA and inserted a TC tag in the

full-length molecular clone pNL4-3 to generate pNL4-3/MA-TC

(Fig. 1A).

To determine the effects of the TC tag on virus replication, we

transfected the Jurkat T-cell line with WT pNL4-3 or with pNL4-

3/MA-TC and monitored virus replication over time by

measuring the levels of reverse transcriptase (RT) activity in the

medium (Fig. 1B). We observed that replication of NL4-3/MA-

TC was comparable to that of WT in Jurkat T-cells. To test the

replication of MA-TC in primary MDMs, the MA-TC tag was

introduced into the macrophage-tropic pNL4-3 derivative

pNL(AD8) [45,46]. Virus stocks were prepared and used to infect

MDMs. As indicated in Fig. 1B, the NL(AD8)/MA-TC virus

replicated with kinetics indistinguishable from those of WT

NL(AD8) in this physiologically relevant primary cell type.

The ability of MA-TC virus to replicate efficiently in both T-cell

lines and primary MDMs suggested that the insertion of the TC

tag near the C-terminus of MA does not affect HIV-1 assembly or

release. To test this directly, we transfected HeLa cells with WT

pNL4-3 or pNL4-3/MA-TC. One day posttransfection, the cells

were labeled for 5 minutes with or without FlAsH, washed with

ethanedithiol (EDT), and metabolically labeled for 2–3 hrs with

[

S]Met/Cys. Cell and virion lysates were prepared, immuno-

precipitated with anti-HIV immunoglobulin (HIV-Ig), subjected to

SDS-PAGE, and bands were quantitated by phosphorimager

analysis (Figure 1C) (see Materials and Methods). The results

indicated that insertion of the MA-TC tag had no significant effect

on virus particle production and that the FlAsH dye caused no

measurable disruption of HIV-1 particle production. We note that

insertion of the TC tag in MA resulted in increased labeling of the

MA protein with [

S]Met/Cys due to the additional Cys residues

(Fig. 1C). We also compared the single-cycle infectivity of WT and

MA-TC Gag in the TZM-bl indicator cell line [47] and observed

no effect of the MA-TC tag on virus infectivity (data not shown).

Together, these data demonstrate that the MA-TC tag does not

disrupt normal HIV-1 Gag function.

We previously reported a number of mutations within the MA

domain of Gag that alter normal HIV-1 Gag trafficking and

localization [15,16,18]. For example, mutation of the site of Gag

myristylation (1GA; [15]) results in a diffuse cytosolic Gag

localization. Mutations in the MA highly basic domain (e.g.,

29KE/31KE) retarget Gag to MVBs [16,18]. To validate further

the TC labeling approach, we sought to confirm that the effect of

these mutations on Gag localization in the context of otherwise

WT Gag would be recapitulated in the context of MA-TC Gag.

We introduced the 1GA and 29KE/31KE MA mutations into

Author Summary

The viral Gag protein is both necessary and sufficient for

the assembly of a new generation of virus particles. There

has been a significant amount of debate in recent years

regarding the site in the cell at which HIV-1 assembly takes

place. Of particular interest has been the site of assembly

in macrophages, a cell type that serves as an important

target for HIV-1 infection in vivo. In this study, we examine

the site of Gag localization and virus assembly in primary

human macrophages in living cells by using biarsenical

dyes that become fluorescent when they bind a small

target sequence introduced into HIV-1 Gag. We observe

Gag localization both at the plasma membrane and in an

apparently internal compartment that bears markers

characteristic of multivesicular bodies (MVBs). Significantly,

when infected macrophages are cocultured with uninfect-

ed T cells, the apparently internal Gag moves rapidly to the

contact site, or synapse, between the macrophage and the

T cell. These findings support the hypothesis that infected

macrophages sequester assembled HIV-1 particles in an

internal compartment and that these particles move to

synapses where cell–cell transmission can occur.

HIV-1 Gag in Macrophages

PLoS Pathogens | www.plospathogens.org 2 2008 | Volume 4 | Issue 3 | e1000015

MA-TC and analyzed Gag localization within cells using a rapid

FlAsH labeling method. Transfected HeLa cells were labeled for

5 min with FlAsH and washed for 20 min in EDT. Cells were then

fixed and either mounted or processed further for antibody

labeling. Similar to our previous results obtained with antibody

labeling [16,48], MA-TC Gag was found primarily in a punctate

pattern at the cell surface. In contrast, MA-TC/1GA was diffusely

localized throughout the cytosol and MA-TC/29KE/31KE was

found in internal compartments (Fig. 2, top). We previously

observed that the internal compartment to which 29KE/31KE

localizes in HeLa cells is positive for the MVB marker CD63 [18].

To verify that this was also the case in the context of MA-TC Gag,

we examined the colocalization of the 29KE/31KE-TC mutant,

labeled with ReAsH, with CD63 in transfected HeLa cells. We

observed nearly complete colocalization between 29KE/31KE

Gag and CD63 (Fig. S1). These results confirm the biochemical

experiments indicating that the addition of the TC tag had no

effect on Gag trafficking, assembly, or release in HeLa cells.

Since our goal was to visualize Gag trafficking in physiologically

relevant primary cells, we analyzed the localization pattern of MA-

TC Gag in infected MDMs. Cells were infected with VSV-G-

pseudotyped virus stocks obtained from transfected 293T cells.

Infected MDMs were then labeled with FlAsH and fixed 24 to

72 hours post-infection. Infection efficiencies, as determined by

Gag staining, typically ranged between 2 and 10%. MA-TC Gag

localized both to the PM and to an apparently internal

compartment (Fig. 2, bottom). In agreement with our previous

results obtained by antibody labeling [16,18,49], the localization of

MA-TC-derived 1GA and 29KE/31KE Gag in MDMs was

similar to that observed in HeLa cells: 1GA-TC was diffusely

distributed throughout the cytoplasm, and 29KE/31KE-TC was

almost exclusively found in apparently internal compartments

(Fig. 2, bottom). To provide a clearer visualization of the internal

localization of 29KE/31KE-TC Gag in MDM, we obtained a z-

series reconstruction by using the Maximum Intensity Projection

mode from the image processing software OsiriX (Video S1). The

results presented in Fig. 2 demonstrate that introduction of the TC

tag near the C-terminus of MA (MA-TC) allows HIV-1 Gag to be

readily visualized in infected primary MDMs at early time points

postinfection.

In MDM, Gag localizes to both the PM and to an internal,

tetraspanin-positive compar tment

We and others have previously reported that the apparently

internal vesicles to which HIV-1 Gag localizes in MDMs bear

Figure 1. The MA-TC tag does not affect Gag function. (A) Schematic diagram of HIV-1 Gag indicating the position of TC tag insertion. The

amino acid sequence of the TC tag is shaded. (B) Replication kinetics of WT HIV-1 vs. the MA-TC derivative in the Jurkat T-cell line and primary MDM.

Jurkat and MDM experiments were performed with pNL4-3 and pNL(AD8) molecular clones, respectively. Media were obtained every two days for RT

analysis. (C) Virus release efficiency of WT vs. MA-TC. HeLa cells were transfected with WT pNL4-3 or pNL4-3/MA-TC plasmids. Transfected cells were

labeled with FlAsH or DMSO (control) for 5 min 24–48 hrs posttransfection and were washed for 20 min with 300 mM EDT/PBS. The cells were then

metabolically labeled with [

S]Met/Cys for 2 hrs. Released virions were pelleted by ultracentrifugation, and both cell and virus lysates were

immunoprecipitated with HIV-Ig and subjected to SDS-PAGE. Bands were quantified using a phosphorimager. + /2 SD, n = 3.

doi:10.1371/journal.ppat.1000015.g001

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tetraspanin markers, suggesting that they are MVBs or MVB-like

structures [18,33,34]. To define the site of Gag localization in

MDMs at early time points postinfection using TC-tagged Gag,

we infected MDMs and examined the localization of Gag and

tetraspanins (CD63 and CD81) at 20 hrs postinfection. As

previously observed with fully WT Gag [18], MA-TC Gag

displayed a localization pattern that partially overlapped with that

of CD63 (Fig. 3A). The colocalization pattern in these cells was

very heterogeneous, with some cells displaying a high degree of

Gag/CD63 colocalization (Fig. 3A, top panels) and other cells

showing a lower level of colocalization (Fig. 3A, lower panels).

29KE/31KE-TC Gag also overlapped with a subset of CD63 in

infected MDMs (Fig. 3B). Both MA-TC (Fig. 3C) and 29KE/

31KE-TC Gag (data not shown) showed much more extensive

colocalization with CD81 than with CD63. We note that some

cells displayed a high level of Gag and CD81 costaining at the PM

(Fig. 3C, lower panel), consistent with HIV-1 assembly occurring

in tetraspanin-enriched microdomains at the cell surface [19,50].

To quantitatively compare the degree of Gag/CD63 vs. Gag/

CD81 colocalization in MDMs, we measured the Pearson

correlation coefficient (R) values (see Materials and Methods) for

these two sets of colocalizing proteins in a total of 75 cells. The

results confirmed the higher degree of Gag/CD81 compared to

Gag/CD63 colocalization (Fig. S2). We observed that 71% of cells

displayed a Gag/CD63 R-value of ,0.6, whereas 91% of cells

showed a Gag/CD81 R-value of .0.6 (Fig. S2).

As indicated in Fig. 3, WT Gag was localized both to an internal

tetraspanin-positive compartment and to the PM in infected

MDMs. Very few cells showed exclusively PM staining; instead,

the vast majority of cells showed either an internal localization or

both PM and internal staining. To determine whether the

distribution changed over time, we classified cells as displaying

uniquely PM, intracellular, or both PM and intracellular Gag

localization at 20, 24, 48, 72, and 96 hrs postinfection. The

percentage of cells within these three categories remained

essentially unchanged over time (Fig. 4A).

To visualize Gag movement in living MDMs, cells were infected

with MA-TC virions pseudotyped with VSV-G and were labeled

for 5 min with FlAsH 24 to 72 hrs post-infection. After washing,

labeled MDMs were immediately placed in a microscope chamber

(37uC/5% CO

) and imaged over time. Interestingly, no clear

movement of Gag between PM and apparently intracellular

compartments was observed during the time course (Fig. 4B); i.e.,

no obvious internalization of Gag from the PM was visualized, nor

was there clear movement of internal Gag puncta to the PM.

These results suggest that Gag can assemble both at the PM and in

internal compartments in infected MDMs. As stated in the

Materials and Methods, prior to 20 hrs post-infection we were not

able to definitively distinguish between specific Gag staining and

the diffuse, low-level background.

Gag and CD81 accumulate at sites of cell-to-cell contact

During the course of our analyses, we frequently observed

concentrated Gag staining at the contact sites formed between

infected and uninfected MDMs (Fig. 5A). 3D z-stack reconstruc-

tions illustrating this phenomenon are presented in Figs. S3A and

Video S2). These Gag-enriched cell-cell junctions also displayed a

high degree of staining for the tetraspanin markers CD81 and

CD82 (Fig. 5B and data not shown). Analogous junctions have

been reported to form between HIV-1-treated dendritic cells and

T-cells; because these junctions bear markers (e.g., tetraspanins

and adhesion molecules) found at immunological synapses

[51,52,53] they have been named ‘‘infectious’’ or ‘‘virological’’

synapses [54,55,56,57,58]. A concentration of budding and

released virions was also observed in the vicinity of cell-cell

contact sites by transmission electron microscopy (EM) (Fig. 5C).

To quantify the localization of Gag at the synapse observed in our

EM analysis, we counted the number of virus particles and

budding structures at synapse vs. non-synapse regions of the

plasma membrane. More than 60 cells were scored for this

analysis. The results indicated a markedly (5-6-fold) higher density

Figure 2. The MA-TC tag does not affect Gag localization. HeLa cells (top panel) were transfected with pNL4-3/MA-TC or 1GA or 29KE/31KE

derivatives. Cells were labeled with FlAsH for 5 min at 37uC, washed, fixed in 3.7% formaldehyde, and examined microscopically. MDMs (bottom

panel) were infected with VSV-G- pseudotyped virus stocks that transduced NL4-3/MA-TC or 1GA or 29KE/31KE derivatives (see Materials and

Methods). Infected cells were labeled and fixed as described above. Scale bars = 30 mm. For a 3D z-series of the 29KE/31KE-TC mutant in MDM, see

Video S1.

doi:10.1371/journal.ppat.1000015.g002

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of particles and budding events at synapse vs. non-synapse regions

of the cell surface, consistent with the immunofluorescence data

presented above.

To extend the analysis of Gag concentration at the cell-cell

synapse to include junctions formed between infected MDMs and

uninfected T-cells, we performed the following analysis: infected

MDMs were labeled with FlAsH and then incubated at 37uC for

2 hours with Jurkat T-cells. The cells were then fixed and, when

necessary, labeled with anti-CD81 antibodies. Gag was frequently

detected at the synapses between infected MDMs and uninfected

Jurkat T-cells (Fig. 6A). 3D z-stack reconstructions are provided in

Videos S3 and S4. Furthermore, as we observed for MDM/MDM

junctions, MDM/T-cell synapses also displayed a high degree of

colocalization between Gag and tetraspanin markers (Fig. 6B). We

also observed that Gag concentrated at synapses formed between

infected MDMs and primary T cells (data not shown). Overall,

these data show that HIV-1 Gag, along with CD81, are recruited

to the synapses formed between infected macrophages and

uninfected macrophages or T-cells. To quantify the concentration

of Gag at the synapse, we used the ImageJ software to determine

the pixel intensity for Gag staining at the MDM/MDM and

MDM/T-cell synapses compared to the overall pixel intensity in

each infected cell. The results confirmed a high degree of Gag

concentration at cell-cell junctions, with approximately 80% of the

total Gag signal localized to the synapse (Figs. 6C, S3A).

Movement of Gag to the synapse in MDM is not Env-

dependent but is disrupted by mutations in MA

To analyze further the process of Gag recruitment to the

synapse in infected MDMs, we determined whether Gag was

recruited to cell-cell junctions in the context of proviral clones

carrying additional mutations. We first examined the localization

of Gag in the absence of Env expression by using the Env(-) MA-

TC mutant KFS/MA-TC. Examining a possible role for Env in

Gag recruitment to the MDM synapse was of interest as it has

been reported that Env is required for synapse formation between

infected and uninfected T cells [59] and also plays a role in the

formation of filopodial bridges that can facilitate transfer of

retroviruses between cells [60]. In contrast to these prior findings

in non-monocytic cell types, we observed that Gag was efficiently

localized to both MDM/MDM and MDM/T-cell synapses in the

absence of Env expression (Fig. 7A and B). This concentration of

Gag to the synapse was quantified as described above, confirming

the high degree of localization of Gag to the cell-cell junction

independent of Env expression (Fig. 7C, S3B). 3D z-stack

reconstructions are provided in Video S5. The data indicated no

statistically significant difference between Gag concentration at the

MDM/T-cell vs, MDM/MDM synapse, or in the presence or

absence of Env expression (compare Figs. 6C and 7C).

We previously reported that mutations in the highly basic

domain of MA (e.g., 29KE/31KE) redirect Gag to MVBs [18].

Here, we observed that in MDMs the 29KE/31KE-TC mutant

displayed nearly complete localization to an apparently internal

compartment that stained positive for CD63 and CD81 (Figs. 2

and 3 and data not shown). In contrast, MA-TC Gag displayed a

mix of PM and internal staining (e.g., Fig. 2–4). It was therefore of

interest to examine whether 29KE/31KE-TC Gag could

redistribute from its normally internal site of localization to the

cell surface upon synapse formation. Interestingly, we observed

that in contrast to MA-TC Gag, 29KE/31KE-TC Gag did not

relocalize to either MDM/MDM (Fig. 8; Video S6) or MDM-T-

cell (data not shown) synapse. Instead, in cells expressing 29KE/

31KE-TC Gag, both Gag and CD81 remained deep within the

infected cell (Fig. 8). In four independent experiments with 29KE/

31KE-TC, Gag accumulation was never observed at the synapse.

These data suggest the possibility that the apparently internal

compartments to which WT and 29KE/31KE Gag localize are

distinct.

Real-time visualization of Gag movement to the

infectious synapse in living MDM

The data presented above using fixed infected cells and EM

techniques demonstrate the accumulation of Gag and virus

particles at the junction between infected MDMs and uninfected

MDMs or T-cells. To visualize the movement of Gag to the cell-

cell contact site, we used FlAsH labeling and live-cell imaging in

infected MDMs. For these experiments, infected MDMs were

labeled with FlAsH for 5 minutes 24 to 72 hours post-infection,

washed, and imaged over time. When visualizing MDM/T-cell

junctions, Jurkat T-cells were added to the infected cells post-

Figure 3. Gag in MDMs colocalizes with tetraspanins at the cell

surface and in apparently interna l compartment s. MDMs

infected with VSV-G-pseudotyped NL4-3/MA-TC (A and C) or NL4-3/

29KE/31KE-TC (B) were labeled with FlAsH (green) 20 hours post-

infection, fixed, and further labeled with antibody against CD63 (red) (A

and B) or CD81 (red) (C). The merged images of Gag-TC and CD63/CD81

are shown on the right, with yellow indicating colocalization between

Gag and CD63/CD81. R = Pearson coefficient of correlation. Scale bars:

panel A, 40 mm; panel B, 30 mm; panel C; 30 mm top, 20 mm bottom.

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FlAsH labeling and imaged under the same conditions. After

addition of the Jurkat cells, incubation periods of approximately

30–60 min were required for stable MDM/T-cell synapses to

form. As mentioned above, we observed no clear evidence of

movement of apparently internal Gag to the PM, or vice versa, in

MDMs not actively engaged in cell-cell contact. Interestingly,

however, upon addition of Jurkat T-cells to the infected MDM

cultures, we observed rapid movement of apparently internal Gag

to the MDM/T-cell synapse. The infected macrophage (‘‘M1’’) in

Fig. 9A is surrounded by uninfected macrophages (e.g., ‘‘M2’’) and

Jurkat T-cells (‘‘T1’’ and ‘‘T2’’). In this particular time course,

40 min after adding Jurkat T cells to the MDMs (t = 0 min), Gag

has already accumulated at the contact site between M1 and T1.

Gag-containing compartments are also rapidly recruited to the site

of M1/T2 contact. Movement of other Gag-containing compart-

ments toward the site of MDM-MDM (M1/M2) contact can be

observed starting at time t = 25 min and is complete at t = 40 min.

These data demonstrate that Gag present in internal compart-

ments can be rapidly redistributed to the site of contact with

uninfected cells. After its movement to the MDM/MDM synapse,

Gag can be seen moving along the surface of macrophage M2

(e.g., at 45 and 50 min). A movie of Gag movement to the synapse

can be viewed at Video S7. Intriguingly, we frequently observed

an apparent preference for MDM/T-cell synapse formation at

sites close to high levels of Gag concentration (Fig. 9B). In this

gallery, time t = 0 represents cells 90 min post-FlAsH labeling and

70 min after addition of T-cells. One of the surrounding T-cells

(‘‘T ‘‘) at time t = 0 min extends on top of the infected MDM

toward the site of Gag accumulation. This resulted in the

movement and attachment of the T-cell with the infected MDM

near the site of Gag accumulation.

Discussion

Most studies that have examined HIV-1 Gag trafficking have

used non-infectious constructs in which codon-optimized Gag is

fused to fluorescent proteins such as green or red fluorescent

protein (GFP or RFP). Although these studies provided important

insights, disadvantages of using GFP and its derivatives in protein

trafficking analyses include the large size of the fluorescent protein

and the fact that achieving their fluorescent state requires time-

dependent chromophore maturation [61,62]. We have also

observed that Gag expressed from some codon-optimized

constructs assembles relatively inefficiently and forms perinuclear

cytosolic aggregates not typically observed with WT Gag

(unpublished results). In this study, we describe the application

of the biarsenical labeling system to visualize HIV-1 Gag

trafficking in primary MDMs. We show here that the TC tag

Figure 4. Gag localization in MDMs remains stable over time. (A) MDMs were labeled with FlAsH 20, 24, 48, 72, and 96 hrs after infection with

VSV-G-pseudotyped NL4-3/MA-TC. At each time point, between 25 and 65 cells were categorized as having PM, intracellular (IC), or both

PM+intracellular (PM+IC) Gag localization. (B) Live-cell analysis. MDMs infected with VSV-G-pseudotyped NL4-3/MA-TC virus were labeled with FlAsH

48 hrs postinfection, and immediately examined microscopically on the stage in a closed chamber (37uC/5% CO

). Time (t) represents time in min

after FlAsH labeling. Scale bar = 30 mm.

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Figure 5. Gag accumulates at the MDM/MDM synapse. Cells were infected with VSV-G-pseudotyped NL4-3/MA-TC virus, labeled with FlAsH

24–48 hrs post-infection, and fixed in 3.7% formaldehyde. (A) Gag accumulates at the synapse between MDMs. Individual macrophages are labeled

‘‘M’’. Scale bar = 15 mm. (B) After FlAsH labeling, cells were fixed and stained with anti-CD81 antibody. The extensive overlap between Gag and CD81

at the MDM/MDM synapse is visualized as yellow in the merged panels. Scale bar = 30 mm. Far-right panels in (A and B) provide quantification of the

Gag signals. The Surface Plot analyzing tool from the ImageJ software was used to obtain a three-dimensional graph of the pixel intensities in

grayscale. The x- and y- axes represent the length of the region analyzed in pixels, and the z-axis represents the pixel intensity of the Gag signal. (C)

EM analysis of VLPs at the synapse. Infected MDMs were fixed with 2% glutaraldehyde and processed for EM. Fully assembled VLPs can be visualized

at the MDM/MDM junctions. Red boxes represent regions that are enlarged in the adjacent panels.

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that serves as the binding site for the biarsenical dye FlAsH is

remarkably well tolerated with respect to preserving Gag function

when introduced near the C-terminus of the MA domain. The

MA-TC tagged Gag produces virus particles with WT efficiency,

and these particles are fully infectious in both single-cycle assays

and in spreading infections. The MA-TC Gag can be readily

delivered to primary cells as a VSV-G pseudotype. Overall, this

system provides a rapid and efficient method for observing WT

Gag trafficking in living cells.

The biarsenical labeling system employed in this study allowed

us to examine Gag localization after ,20 hrs postinfection, the

earliest time point at which Gag expression could be readily and

consistently visualized. At this early time point, we observed a mix

of PM and apparently internal Gag staining. Between 20 and

Figure 6. Gag accumulates at the MDM/T-cell synapse. (A) Infected MDMs were labeled with FlAsH and Jurkat cells were added to the cultures.

One to two hours after addition of the T-cells, the co-cultures were fixed with 3.7% formaldehyde and imaged or (B) stained with anti-CD81 antibody.

In panel B, Gag/CD81 colocalization is indicated as yellow in merge. M = MDMs; T = Jurkat T-cells. Scale bars in panels A and B = 15 mm. (C)

Quantification of Gag concentration at the synapse. The microscopy images were opened in ImageJ and a plot profile was obtained. A column

average plot was generated, in which the x-axis represents the horizontal distance through the image and the z-axis the vertically averaged pixel

intensity. The % Gag at the synapse = (pixel intensity of Gag signal at synapse)/(total intensity of Gag in the cell+synapse)6100. N values indicate the

number of cells analyzed in each data set. For more information on how the Gag quantification was performed, see Fig. S3A.

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96 hrs postinfection, we did not observe a shift in the percentage of

cells displaying PM, intracellular, or PM+intracellular staining

(Fig. 4) nor did we observe a time-dependent accumulation of

internal Gag. These results differ from those of a recent study in

which intracellular Gag-GFP staining increased over time [27].

We note that the TC-tagged Gag in the current study is fully

functional for particle assembly and release and produces

infectious virions. Furthermore, our TC-tagged Gag is expressed

in the context of a full-length molecular clone that encodes all the

HIV-1 accessory proteins including Vpu. Indeed, elimination of

Vpu expression led to a time-dependent accumulation of Gag in

internal compartments (unpublished results), consistent with recent

reports [26,28]. In agreement with the study of Jouvenet et al.

[27], we did not observe an effect on virus release of treating

infected cells with U18666A, a drug that arrests endosome motility

(unpublished results). This observation supports the hypothesis

that release of HIV-1 in MDMs occurs from PM-assembled VLPs.

Although the FlAsH method can be accompanied by high

background staining, we observed that this problem is largely

mitigated by using very brief labeling periods. We also observed

Figure 7. Gag recruitment to the synapse in MDMs is Env-independent. (A) MDMs were infected with VSV-G-pseudotyped NL4-3/KFS/MA-TC

virus (which is defective for HIV-1 Env expression) and were labeled with FlAsH 24–48 hrs postinfection. Boxes indicate regions enlarged on the right.

Scale bars = 15 mm. (B) MDMs were infected as in (A), and Jurkat T cells were added after FlAsH labeling. Far-right panels in (A) and (B) provide

quantification of the Gag signals, determined as indicated in the Fig. 5 legend. Scale bars = 15 mm. (C) Quantification of Gag concentration at the

synapse, determined as described in the Fig. 6C legend. For more information on how the Gag quantification was performed, see Fig. S3B.

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that background staining in MDM is less evident in MDM than in

HeLa cells.

The most significant finding in this study is the visualization of

apparently internal Gag moving to the site of cell-cell contact after

synapse formation with uninfected T cells. It therefore appears

that the tetraspanin-rich, apparently internal compartment in

which HIV-1 assembles in MDMs can serve as a storage

compartment for rapid presentation of virus particles at cell-cell

junctions. These findings have clear implications for HIV-1

transmission from MDMs. In this regard, it is interesting to note

that the virions in these internal vesicles reportedly remain

infectious for weeks postinfection [63] and that virus transmission

between infected MDMs and T cells is extremely rapid [64]. In

several respects, our observations with infected MDMs are similar

to those made with dendritic cells treated with HIV-1. Binding of

HIV-1 virions to dendritic cells can lead to transfer of virus to

uninfected T-cells through the formation of a virological or

infectious dendritic cell/T-cell synapse without the dendritic cell

itself being productively infected [55,56,57,58,65,66]. Virions

bound to the dendritic cell are reportedly internalized into an

internal compartment that is strongly positive for CD81 but only

weakly positive for CD63. Synapse formation induces the

redistribution of virus particles and CD81 to the site of cell-cell

contact, presumably facilitating transfer of virus to the T cell. The

internal virus-containing compartment in dendritic cells is weakly

acidic, as also reported for the virus-positive compartment in

MDMs [67]. Thus, it appears that HIV-1 has evolved to subvert a

pathway in both MDMs and dendritic cells that allows infectious

virus particles to be retained in an apparently internal compart-

ment and then redistributed to the cell surface following infectious

synapse formation. Transfer of HIV-1 between T cells also

involves the formation of a synapse that bears tetraspanin markers

[68]; however, there is currently no evidence for long-term

retention of infectious virus particles within an internal compart-

ment in T cells. Interestingly, whereas the generation of T-cell/T-

cell infectious synapses [59] and formation of cell-cell filopodial

bridges that allow intercellular transfer of virus particles [60] have

been reported to require Env expression, we found no such Env

dependence for Gag translocation to the synapse formed between

MDMs and T cells (Fig. 7A).

We previously reported that mutations in the highly basic

domain of MA (e.g. 29KE/31KE) induce a shift in Gag

localization in HeLa cells and T cells from PM to MVBs [18].

In MDMs, both WT and 29KE/31KE Gag localize to an

apparently internal, tetraspanin-positive compartment [18]. This

finding is confirmed here (e.g., Fig. 2 and 8). Interestingly, while

WT MA-TC Gag rapidly translocates to the MDM/T-cell

junction after synapse formation, we did not observe significant

movement of 29KE/31KE-TC Gag to the MDM/T-cell synapse

(Fig. 8). These results imply that WT and 29KE/31KE Gag

localize to distinct tetraspanin-positive compartments in MDMs. A

possible interpretation of these observations is that 29KE/31KE

Gag localizes to ‘‘true’’ MVBs which do not move to the synapse,

whereas WT Gag localizes to a compartment that is apparently

internal but is connected to the PM [37,38]. It is this PM-

connected tetraspanin-positive compartment that undergoes a shift

in localization following synapse formation, thereby allowing virus

particle movement to the site of cell-cell contact. Surprisingly, we

frequently observed that T-cells made contact with regions of the

MDM PM under which Gag was concentrated, and in many cases

the T-cells formed pseudopodia to contact this site (e.g., Fig. 9B).

These results imply that the T cell can ‘‘sense’’ regions of the PM

that overlie the putative invaginations in which assembled virus

particles are concentrated. These regions of the PM may be

enriched in lipid rafts and/or tetraspanin-enriched microdomains.

These observations are somewhat reminiscent of previous studies

on the recruitment of uninfected T cells into infected cell syncytia

[69]. A future challenge will be to characterize in greater detail the

membrane composition at the site of MDM/T-cell contact and

elucidate the signals that induce the movement of the newly

assembled, internally sequestered virus particles to the infectious

synapse.

Figure 8. Gag recruitment to the synapse in MDMs is blocked by the 29KE/31KE MA mutations. MDMs were infected with VSV-G-

pseudotyped NL4-3/29KE/31KE-TC virus, labeled with FlAsH 24–48 hrs postinfection, fixed, and stained with anti-CD81 antibody. Gag/CD81

colocalization is indicated as yellow in merge. Scale bars = 30 mm. Far-right panels show distribution of Gag signal; plots were obtained as described

in the Fig. 5 legend. Note the centrally located (non-synapse) concentration of Gag in these cells expressing the 29KE/31KE MA mutant.

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Materials and Methods

Plasmids and preparation of virus stocks

Plasmids pNL4-3/MA-TC and pNL(AD8)/MA-TC were

constructed as follows: for pNL4-3/MA-TC, nucleotides 1250–

1273 (encoding DTGNNSQV Gag codons 121–128) were deleted

in the MA-coding region of the full-length HIV-1 molecular clone

pNL4-3 [70] and the TC tag GSMPCCPGCCGSM was inserted

in its place using overlap-extension PCR [71]. The MDM-tropic

pNL(AD8)/MA-TC clone was constructed by exchanging the

EcoRI-XhoI fragment of pNL4-3/MA-TC with that from the

CCR5-tropic clone pNL(AD8) [45]. Construction of molecular

clones expressing pNL4-3 MA mutants 1GA and 29KE/31KE

was described previously [15,16]. The molecular clones pNL4-3/

1GA-TC and pNL4-3/29KE/31/KE-TC were constructed by

exchanging the BssHII-SphI fragments of pNL4-3/1GA or pNL4-

Figure 9. Real-time trafficking of Gag to the synapse in infected MDMs. (A) MDMs infected with VSV-G-pseudotyped NL4-3/MA-TC virus

were labeled with FlAsH 48 hrs postinfection. Jurkat T-cells were added to the FlAsH-labeled cultures and incubated on the microscope stage in a

closed chamber (37uC /5% CO

). Time t = 0 min is 40 min (gallery A) or 70 min (gallery B) after addition of Jurkat cells. M1 = infected MDM; M2 = non-

infected MDM; T1 and T2 are Jurkat T-cells. Scale bars = 20 mm in A and 40 mm in B. For a movie of Gag movement to the synapse, derived from the

experiment presented in Fig. 9A, see Video S7.

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3/29KE/31KE with the corresponding fragments from MA-TC.

To construct the Env(-) pNL4-3 construct, pNL4-3/KFS-TC, we

exchanged the EcoRI-XhoI fragment from the Env(-) molecular

clone pNL4-3/KFS [72] with the corresponding fragment from

pNL4-3/MA-TC. Finally, we constructed pNL4-3/Vpu(-)/MA-

TC by replacing the BssHII-EcoRI fragment from Vpu-DEL-1

[73] (kindly provided by K. Strebel), with the corresponding

fragment from pNL4-3/MA-TC. VSV-G-pseudotyped virus

stocks were prepared by transfecting 293T cells with the Gag/

Pol expression vector pCMVNLGagPolRRE [74], the VSV-G

expression vector pHCMV-G [75], and the indicated HIV-1

molecular clones by using Lipofectamine 2000 (Invitrogen),

according to the manufacturer’s protocol.

Cells, transfections, and infections

HeLa and Jurkat T cells were cultured as previously described

[15]. MDMs were prepared by culturing elutriated monocytes

[45] in RPMI-1640 medium, supplemented with 10% fetal bovine

serum, for 5 to 7 days on ultra-low attachment plates (Costar).

HeLa cells were transfected by using the calcium phosphate

method, as previously described [15]. Jurkat T-cells were

transfected by using the DEAE-dextran procedure as previously

reported [15]. Infection of MDMs was performed as follows:

MDMs were detached from the ultra-low attachment plates

(Fisher Scientific, Pittsburgh, PA) and plated onto tissue culture

dishes or microscope culture chambers Fisher Scientific, Pitts-

burgh, PA). Virus stocks, pseudotyped with VSV-G, were

incubated with MDMs for 5-6 hours. 2610

counts/minute

(cpm) of reverse transcriptase (RT) activity was used per well of

4-well Nunc chambers, 10

RT cpm/well for 8-well Nunc-

chambers, and 4610

RT cpm/well for 6-well plates.

Virus replication and infectivity assays

Virus replication assays in the Jurkat T-cell line were performed

as previously described [15]. Briefly, Jurkat cells were transfected

in parallel with WT pNL4-3 or MA-TC using the DEAE-dextran

method. Cells were split 1:3 every two days and an aliquot of

medium was reserved at each time point for RT assay [76].

MDMs in 6-well plates were infected with 2610

RT cpm/well

with WT pNL(AD8) or MA-TC(AD8) virus stocks. Medium in the

infected MDM cultures was changed every two days and an

aliquot was reserved for RT activity. For single-cycle infectivity

assays, 4610

HeLa-derived TZM-bl cells [47] (obtained from J.

Kappes through the NIH AIDS Research and References Reagent

Program) per well were infected with 2610

RT cpm virus stocks.

Infection efficiency was determined by measuring luciferase

activity 2 days post-infection, as described previously [77].

Biarsenical labeling

Adherent cells cultured in Lab-Tek chamber slides (Nunc) or 6-

well plates were labeled 24–72 hours posttransfection/infection.

All labeling steps were performed at 37uC in the dark. The cells

were washed twice with Opti-MEM I (Invitrogen, Carlsbad). For

each experiment, biarsenical labeling solutions were freshly

prepared immediately prior to use. Wash solutions of 300 mM

and 100 mM 1,2-ethaneditiol (EDT) (Aldrich Chemical Company,

Inc., Milwaukee) were prepared in phosphate-buffered saline

(PBS) and 0.2 mM Lumio Green (FlAsH) was prepared in

dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Inc., St Louis). Before

labeling, 2 ml of 1 mM EDT was mixed quickly with 4.7 mlof

0.2 mM FlAsH or Lumio Red (ReAsH) and immediately added to

400 ml Opti-MEM I. This solution was added to cells, which were

incubated for 5 min at 37uC. After the 5 min biarsenical labeling,

cells were washed with 300 mM EDT/PBS for 8 min and 100 mM

EDT/PBS for 10 min at 37uC. Cells were then washed further 3X

with PBS and either fixed with 3.7% formaldehyde prior to

antibody labeling or incubated with Opti-MEM I for live cell

imaging or addition of Jurkat T cells. The levels of background in

MDMs and in Hela cells were greatly reduced with the addition of

EDT in our labeling solutions. We also observed that keeping the

biarsenical labeling time short (5 min) was enough to obtain a

strong Gag signal, while limiting non-specific background staining.

As early as 20 hrs post-infection, specific Gag staining could be

readily detected. However, at earlier time points, the cytosolic Gag

signals were too low to be clearly distinguishable from background

fluorescence, and therefore no data were acquired before 20 h

postinfection.

Fluorescence microscopy and EM

For fluorescence microscopy, 24–48 hours post transfection/

infection, cells were labeled using the biarsenical method and either

fixed using 3.7% formaldehyde/PBS for 20 min or Jurkat T-cells

were added to the MDMs (in Opti-MEM I) for 2 hours, then fixed

with formaldehyde. The cells were then permeabilized with 0.1%

Triton-X100/PBS and incubated with 0.1 M glycine for 10 min at

room temperature to quench free aldehyde groups. The cells were

then blocked with 3% bovine serum albumin (BSA)/PBS, incubated

with either mouse monoclonal anti-CD63 (Santa Cruz Biotechnol-

ogy) or mouse monoclonal anti-CD81 (BD Pharmingen) for 1 hr at

room temperature, washed and incubated with Alexa-594 or 488-

conjugated anti-mouse IgG (Invitrogen) for 30 min at room

temperature. The cells were then washed and mounted with Aqua

Poly Mount (Polysciences Inc., Warrington, PA). For live cell

imaging, the labeled cells were imaged in a temperature-controlled

chamber (37uC/ 5% CO

) in Opti-MEM I. For both fixed and live-

cell microscopy, the cells were imaged using an Olympus 1X-71

inverted deconvolution microscope and analyzed with Delta Vision

software (Applied Precision Inc., Seattle, WA). To quantify the

degree of relative colocalization, we obtained the Pearson

correlation coefficient (R) values, which are standard measures of

colocalization [78]. The R values were calculated using the

softWORx colocalization module which generates a ‘‘colocalized’’

image from two channels. A scatter plot of the two intensities on a

pixel-by-pixel basis is then plotted and the R value is calculated by

dividing the covariances of each channel by the product of their

standard deviations. For EM, infected cells were fixed and processed

as previously described [15].

Metabolic labeling and radioimmunoprecipitation

analysis

Metabolic radiolabeling, preparation of cell and viral lysates,

and immunoprecipitation assays were performed as previously

described [15]. Briefly, transfected HeLa cells, or infected MDMs,

labeled with the biarsenical dyes or DMSO (control) were

metabolically labeled with [

S] Met/Cys for 2 hours, 24-48 hours

posttransfection/infection, and released virions were pelleted by

ultracentrifugation. Cell and virus lysates were immunoprecipitat-

ed with HIV immunoglobulin (HIV-Ig), obtained from NABI and

the National Heart Blood and Lung Institute through the NIH

AIDS Research and Reference Reagent Program. Immunopre-

cipitates were subjected to SDS-PAGE followed by fluorography.

Quantitative analysis of the bands visualized by radioimmunopre-

cipitation was performed using a Bio-rad phosphorimager.

Supporting Information

Figure S1 Colocalization of 29KE/31KE-TC Gag with CD63

in HeLa Cells. Cells transfected with pNL4-3/29KE/31KE-TC

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were labeled for 5 min with ReAsH (Gag) for 5 min at 37 uC,

washed, fixed in 3.7% formaldehyde and then stained with anti-

CD63 Ab (CD63). Lower panels show the overlay between the

Gag and CD63 staining. Scale bars = 15 mm.

Found at: doi:10.1371/journal.ppat.1000015.s001 (4.20 MB TIF)

Figure S2 Quantification of Gag/CD63 and Gag/CD81

Colocalization. MDMs infected with VSV-G-pseudotyped were

analyzed for Gag, CD63 and CD81 localization as described in

the Fig. 3 legend. Pearson coefficient (R) values were obtained for

a total of 75 cells and were plotted on a scale of 0 to 1 (x-axis). The

y-axis indicates the number of cells scored within each R-value

range.

Found at: doi:10.1371/journal.ppat.1000015.s002 (2.57 MB TIF)

Figure S3 Sample Images Used to Quantify the Localization of

Gag at the Synapse. Illustrates method used for data presented in

Fig. 6C (panel A) and Fig. 7C (panel B). The microscopy images

were opened in ImageJ and a plot profile was obtained. A column

average plot was generated, in which the x-axis represents the

horizontal distance through the image and the y-axis the vertically

averaged pixel intensity. The % Gag at the synapse was calculated

as: (pixel intensity of Gag signal at synapse)/(total intensity of Gag

in the cell+synapse)6100. N values indicate the number of cells

analyzed in each data set. Scale bar = 10 mm.

Found at: doi:10.1371/journal.ppat.1000015.s003 (3.67 MB TIF)

Video S1 Analysis of 29KE/31KE-TC Gag localization in

MDM in 3D. The z-series reconstructions were obtained by using

the Maximum Intensity Projection mode from the image

processing software OsiriX. The data correspond to the MDM

29KE/31KE-TC Gag panel in Fig. 2.

Found at: doi:10.1371/journal.ppat.1000015.s004 (0.18 MB

MOV)

Video S2 Analysis of Gag localization in 3D. The z-series

reconstructions were obtained by using the Maximum Intensity