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Traffic 2003; 4: 902–910 Blackwell Munksgaard

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Blackwell Munksgaard 2003

doi: 10.1046/j.1600-0854.2003.00145.x

HIV-1 Egress is Gated Through Late Endosomal Membranes Sascha Nydegger1, Michelangelo Foti2, Aaron Derdowski3, Paul Spearman3 and Markus Thali1,* 1

Department of Microbiology and Molecular Genetics, College of Medicine and CALS, 318 Stafford Hall, University of Vermont, Burlington, VT 05405, USA 2 Department of Morphology, University of Geneva, CH-1211 Geneva, Switzerland 3 Department of Pediatrics and Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232, USA * Corresponding author: Markus Thali, [email protected] HIV-1 buds from the surface of activated T lymphocytes. In macrophages, however, newly formed HIV-1 particles amass in the lumen of an intracellular compartment. Here, we demonstrate by live-cell imaging techniques, by immunocytochemistry and by immuno-electron microscopy that HIV-1 structural proteins, particularly the internal structural protein Gag, accumulate at membranes of the late endocytic compartment in a variety of cell types and not just in monocyte/macrophage-derived cells. Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in retroviral budding. Together with those observations, our study suggests that HIV-1 morphogenesis is thoroughly rooted in the endosomal system. Key words: human immunodeficiency virus type 1 (HIV-1), late endosome, multivesicular body (MVB), retroviral assembly and release Received 27 July 2003, revised and accepted for publication 22 September 2003

and must contain the cellular machinery that allows macrophage-tropic HIV-1 to bud into the lumen of these late endosomes. The same machinery may also be responsible for the biogenesis of cellular internal vesicles in MVBs, both in macrophages and in other cell types. MVBs are destined to fuse with lysosomes, thus allowing for degradation of cargo carried by the intraluminal vesicles (5). Alternatively, MVBs can migrate to the cell surface and secrete their content, the intraluminal vesicles, which are then called exosomes [reviewed in (6,7)]. Exosome-assisted release of proteins is best documented for the major histocompatibility complex class II (MHC class II) (6,8). Yet another potential fate of MHC class II-containing internal vesicles was revealed in dendritic cells, where exosomes, before being released, can be re-adsorbed by the limiting membrane, resulting in the display of MHC class II at the plasma membrane (9). Importantly, in either of the two latter cases and most likely in all cell types, the limiting membrane of the MVB can fuse with the plasma membrane and thus become an integral part of it. Whether patches of limiting membrane, upon integration into the plasma membrane in T lymphocytes or epithelial cells, still harbor the vesiculation machinery is not known. If so, HIV-1 and other viruses could potentially use this machinery for their egress at the plasma membrane. Here, we hypothesize that the limiting membrane of late endosomes/MVBs provides platforms for HIV-1 assembly and escape not only in macrophages but that this endosomal membrane contributes to the formation of zones of viral budding also in other cell types. We demonstrate that viral Gag, the internal structural protein of retroviruses, accumulates at or adjacent to constituents of late endosomal membranes in all cell types analyzed, independent of whether HIV-1 egress is observed at the plasma membrane or at intracellular membranes. Our results suggest that HIV-1 does not just hijack a few MVB constituents for its budding, but that viral egress at the cell periphery is gated through segments of plasma membrane which are derivatives of the limiting membrane of late endosomes/MVBs.

Retroviral release, unlike egress of some nonenveloped viruses, does not depend on cell lysis. Retroviruses bud either through internal cellular membranes or through the plasma membrane of the host cell, thus producing the virions. Such exit by budding allows infected cells to shed viral particles without disintegrating. In principle, infected cells can thus serve as reservoirs for virus progeny until senescence (1).

Results

HIV-1 budding is observed primarily at the plasma membrane of infected T lymphocytes. In macrophages, however, this virus accumulates in an intracellular, vacuole-like compartment [e.g. (2)]. Recent ultrastructural studies identified this compartment as late endosomes/multivesicular bodies (MVBs) (3,4). The outer membrane of MVBs, the so-called limiting membrane, thus serves as a platform for viral budding

Host cell-dependent subcellular accumulation of HIV-1 Gag To investigate if T-cell-tropic HIV-1 always exits via the plasma membrane, we transfected different human cell lines with plasmids harboring the provirus of T-cell-tropic HIV-1 (NL4-3), and we analyzed the subcellular localization of newly synthesized viral Gag and envelope glycoprotein

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(Env) by staining with anti-Gag and anti-Env antibodies or with serum from an HIV-1-infected individual. Figure 1A illustrates that this virus accumulated preferentially in intracellular compartments of human erythroleukemic K562 cells, whereas the virus localized predominantly at the surface of either Jurkat T cells or HeLa cells. Intermediate phenotypes were observed in human monocytic THP-1 cells and in the human melanoma cell line Mel JuSo. Like other melanocytes, these latter cells are known to have a high capacity to recycle material from late endosomes/ melanosomes to the surface [e.g. (10)]. Importantly, the amount of virus released from Mel JuSo cells, relative to the entire production of HIV-1 Gag, was higher than what was observed in HeLa cells. HIV-1 is thus not retained in the intracellular compartments, but exits from these cells very efficiently (see last panel in Figure 1A). We conclude that T-cell-tropic HIV-1 either predominantly associates with intracellular membranes or accumulates at the cell surface, depending on the cell type in which it is expressed. Retroviral Gag proteins are competent to form virus-like particles (VLPs) in the absence of other viral components, and Gag thus directs the process of particle assembly and release (11). To monitor the association of HIV-1 Gag with cellular membranes in live cells, we expressed green fluorescent protein (EGFP)-tagged HIV-1 Gag of another T-cell-tropic virus (SF2) in the same cell types described above. EGFP-tagging does not interfere with the overall subcellular distribution, nor with particle formation (12–14). Formation of EGFP-tagged VLPs thus faithfully mimics the viral assembly/release process. As observed for the whole virus (Figure 1A), whether EGFP-tagged Gag accumulated preferentially at intracellular membranes or at the surface depended on the cell type in which it was expressed (Figure 1B and supplemental data available at http:// www.traffic.dk/suppmat/4_12.asp). All together, these data reveal that the determination of the subcellular site where Gag of T-cell-tropic HIV-1 predominantly accumulates is a function of the host cell. HIV-1 Gag associates with membranes belonging to the late endosomal compartment and with segments of the plasma membrane harboring late endosomal proteins The host cell dependence of the subcellular targeting of HIV-1 Gag could be due to host cell elements interacting with Gag’s targeting signals in a cell type-specific manner. Alternatively, the different sites through which Gag is chaperoned during the late phase of the viral replication cycle may be related, i.e. they may have elements in common, and some of those common elements may be required for anchoring viral Gag for subsequent budding. To distinguish between these two possibilities, we characterized the membranes towards which Gag was targeted. Because Mel Juso cells displayed substantial Gag accumulation at intracellular membranes and at the surface, we focused our analysis on this cell type, though results were substantiated by continued examination of other cell types. Traffic 2003; 4: 902–910

Figure 1: Subcellular localization of HIV-1 and HIV-1 Pr55GagEGFP, and virus release. Cells were analyzed by fluorescence microscopy either directly or after staining with patient serum and a FITC-conjugated anti-human secondary antibody. Median sections of cells are shown. Scale bar ¼ 5 mm. (A) T-cell tropic HIV-1 (NL4-3) was expressed in different cell lines. The lower right panel shows the relative amount of particles released from HeLa cells and Mel JuSo cells as measured by HIV-1 p24 ELISA. (B) Expression of EGFP-tagged Pr55Gag (SF2) in the absence of other viral proteins.

The size (Ø 0.5–1.5 mm), perinuclear distribution, overall appearance and the number (100–300 entities per individual cell) of intracellular vesicles harboring HIV-1 Gag in Mel JuSo cells, suggested that these vesicles are late endosomes (Figure 1). To confirm or refute this hypothesis, cyan fluorescent protein (ECFP)-tagged HIV-1 Gag was coexpressed with GFP-tagged MHC class II (b chain-GFP), a marker for the late endocytic pathway. It has been demonstrated that newly synthesized MHC class II-GFP in transit to the cell surface of Mel JuSo cells resides in late endosomes/MVBs (15,16). Figure 2A reveals that a significant proportion of MHC class II-GFP-containing vesicles also harbored Gag-ECFP, suggesting that the two biosynthetic routes intersect in late endosomes. Late endosomes/MVBs display a distinct motility [e.g. (16)]. To characterize the motions of HIV-1 Gag-associated vesicles, we analyzed cells expressing Gag-EGFP by time-lapse fluorescence microscopy. Migration of vesicles associated with Gag-EGFP was analyzed by generating images, in a single optical section, every 3 s for a total of 400 s (Figure 2B and supplemental movie for Figure 2, movies available in 903

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is involved in controlling the trafficking of late endosomes/ MVBs (19,20). RILP stabilizes the interaction of late endosomes with the dynein-dynactin motor complex that carries these vesicles towards the minus end of microtubules. Therefore, when overexpressed in cells, RILP inhibits transport of late endosomes and lysosomes towards the plus end of microtubules, and these vesicles cluster perinuclearly, at the microtubule organizing center (MTOC). To test if MVBs that transport HIV-1 Gag associate with Rab7, we coexpressed either the Gag-EGFP fusion protein and/or the complete virus, together with RILP in Mel JuSo cells. While the subcellular distribution of Gag-EGFP was altered only slightly in RILP-expressing cells (data not shown), most of the MVBs transferring viral Gag expressed in the context of the entire virus were clustered in the perinuclear area (Figure 2C). Coexpression of the C-terminal half of RILP, a dominant negative mutant of this protein, led to the opposite phenotype, i.e. HIV-1 Gag-carrying MVBs were dispersed throughout the cell. We conclude that HIV-1 Gag, if expressed in the context of the complete virus, is directed through Rab7-regulated late endosomes/MVBs.

Figure 2: Targeting of HIV-1 and HIV-1 Pr55Gag-EGFP to MHC Class II containing, Rab-7 controlled intracellular vesicles. (A) Mel JuSo cells expressing a GFP-tagged version of MHC class II were transfected with Pr55Gag-ECFP expressor plasmids. (For consistency, colors were switched during image processing.) (B) Time-lapse analysis of Mel JuSo cells expressing Pr55Gag-EGFP. Pictures were taken every 3 s to track the movement of intracellular particles associated with viral Gag (arrows). (C) Mel JuSo cells expressing HIV-1 together with Pr55Gag-EGFP (for visualization) and either RILP (myc-tagged) or DN-RILP (vsv-tagged). Cells were fixed and stained with anti-myc or anti-VSV antibodies and TexasRed-conjugated secondary antibody. Scale bar ¼ 5 mm.

the video gallery at www.traffic.dk). Gag-containing vesicles were recorded to move with velocities of up to 1 mm/s. Vesicle movement was both erratic and bi-directional. These findings, together with the notions about size, subcellular distribution and the approximate number of Gag-containing vesicles, corroborate that HIV-1 Gag associates with late endosomes/MVBs in Mel JuSo cells. The small GTP-binding protein Rab7 is known to coordinate the transport of cargo to late endosomes (and lysosomes) as well as movements within the late endosomal compartment (17,18). Consequently, the Rab7 effector protein RILP 904

To further confirm that the intracellular structures at which Gag assembles are late endosomes/MVBs, we used antibodies against various endosomal marker proteins. Mel JuSo cells coexpressing HIV-1 and Gag-EGFP were fixed and stained with antibodies against EEA1, Lamp-2 or CD63, markers primarily hosted by early endosomes, lysosomes as well as the limiting membrane of MVBs, and the intraluminal vesicles in MVBs, respectively. Very little overlap between EEA1 and Gag was observed (Figure 3A). In contrast, a substantial fraction of Gag-containing structures exactly matched CD63-containing vesicles, lending support to the hypothesis that Gag is targeted to late endosomal membranes. An intermediate phenotype was observed with regard to Lamp-2-containing membranes. While some of those vesicles overlapped with structures associated with Gag, a significant fraction of the Lamp-2-containing vesicles, larger structures in particular, did not colocalize with HIV-1 Gag in these cells. No colocalization of viral Gag with markers for the endoplasmic reticulum or the Golgi apparatus was evident (data not shown). Most importantly, colocalization of Gag and CD63 or Gag and Lamp-2 took place not only at late endosomes/ MVBs but also at the plasma membrane and in close proximity to the plasma membrane. Figure 3B documents a partial overlap of Gag and CD63 signals also at the periphery of Jurkat T lymphocytes coexpressing Gag-EGFP (as marker) together with HIV-1 Gag in the context of the complete virus.

Viral budding is restricted to distinct cortical microdomains To corroborate, with higher spatial resolution, that HIV-1 on its way to be released from cells associates with late endosomal membranes, we performed an immunoelectron microscopic analysis. Ultrathin cryosections of K562 Traffic 2003; 4: 902–910

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Figure 3: Colocalization of HIV-1 and HIV-1 Pr55Gag-EGFP with markers of the endosomal compartment. (A) Mel JuSo cells or (B) Jurkat T cells expressing HIV-1 and HIV-1 Pr55Gag-EGFP were fixed and stained with antibodies against markers of the endosomal compartment and with TexasRed-conjugated secondary antibodies and examined by fluorescence microscopy. Scale bar ¼ 5 mm. Colocalization is revealed by yellow and its frequency was assessed utilizing colocalizations software (relative colocalization values were 4  3%, 17  10%, 35  14%, for EEA1, Lamp-2 and CD63, respectively; see Materials and Methods for details).

and HeLa cells producing Gag-EGFP VLPs were labeled with antibodies against HIV-1 Gag and with anti-CD63 antibodies or an antibody against lysobisphosphatidic acid (LBPA), a phospholipid implicated in the regulation of cholesterol transport and known to segregate into the internal vesicles of MVBs (21). Cryosections were examined following incubation with secondary, colloidal gold-coupled antibodies. Figure 4A documents in K562 cells that Gag-EGFP VLPs accumulated in either multilaminar or multivesicular compartments. Both their distinct morphology, as well as the presence of CD63 and LBPA, identify these compartments as late endosomes/MVBs. Only a few of the Gag-VLP-carrying MVBs also contained LBPA, but most showed the presence of CD63. The majority of Gag staining was found associated with late endosomes/MVBs in K562 cells, suggesting that newly assembled VLPs primarily accumulate in this compartment, consistent with our fluorescence microscopic analysis (Figure 1B). Occasionally Gag-EGFP was also found associated with curved tubular compartments, Traffic 2003; 4: 902–910

Figure 4: Ultrastructural analysis of Pr55Gag-EGFP localization and VLP formation. Ultrathin cryosections of K562 cells (A) or HeLa cells (B) expressing Pr55Gag-EGFP were double immunogold labeled for HIV-1 Gag (15 nm) and CD63 (10 nm) or LBPA (10 nm). Scale bar ¼ 200 nm. (C) Mel JuSo cells expressing Pr55Gag-EGFP were fixed and embedded in Epon resin and processed for conventional EM analysis. Dashed lines demarcate zones of viral budding in the upper panel. Scale bar ¼ 1 mm.

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probably early or recycling endosomes, or budding from the cell cortex (Figure 4A, right panel). It was not possible to get high-quality ultrathin cryosections of transiently transfected Mel JuSo cells. Therefore, we tested in HeLa cells, which were amenable to such analysis, if late endosomes/MVBs can provide attachment sites for GagEGFP in cells that release virus predominantly from the plasma membrane. In concordance with the fluorescence microscopy data documented in Figure 1, Gag accumulation as well as budding structures were frequently observed at the surface of these cells (Figure 4B, upper right panel). Additionally, however, and also in agreement with results shown in Figure 1, some Gag-EGFP also accumulated in late endosomes/MVBs. We detected fully formed Gag-VLPs within the lumen of this compartment (Figure 4B, left panel and lower right panel), albeit much less frequently than in K562 cells, and we also observed Gag anchoring at the cytosolic face of the limiting membrane (arrows, Figure 4B, lower right panel). Significantly, VLPs produced into MVBs as well as virus budding from the surface of HeLa cells colocalized with, and incorporated, CD63. If late MVBs (or lysosomes) fuse with the plasma membrane, their limiting membrane becomes part of the cell cortex, thus forming, at least transiently, small islands consisting of endocytic membrane [e.g. (22,23)]. Based on the fluorescence microscopic analysis as well as the examination of the cryosections, we hypothesized that newly synthesized HIV-1 Gag associates with sections of the limiting membrane of late endosomes/MVBs either before MVBs fuse with the plasma membrane or after MVBs have merged with the plasma membrane. Either scenario predicts that virus exit is confined to distinct zones at the surface of virus producing cells, interspersed within areas free of particle formation. When we examined thin sections of HIV-1 Gag-EGFP expressing Mel JuSo cells by conventional electron microscopy we found that particle formation at the surface of these cells was indeed confined to distinct domains, scattered within overall larger areas free of viral buds (Figure 4C). The islands where budding took place extended from 1 to 3 mm, comparable to the fluorescent zones visualized in Figure 1(B) and compatible with the hypothesis that they arise from 10 to 20 mm2 segments of MVB limiting membrane that have inserted into the plasma membrane.

Discussion It is amply documented that HIV-1 Env, during the viral assembly/release process, interacts with cellular components that target this protein to the endocytic compartment in T lymphocytes and other cell types [referenced in (24), also (25,26)]. More recently, HIV-1 Gag was also found to interact with cellular apparatuses that either guide 906

proteins to the endosomal compartment or reside in that compartment and select cargo for the internal vesicles of MVBs (27–33). Here, we report that viral Gag is gated through endosomal membranes, irrespective of whether HIV-1 is released from the plasma membrane or whether it accumulates intracellularly. The goal of the present study was to characterize the membrane microdomains where HIV-1 components converge, before or simultaneously with particle formation. We opened our investigation by tracking the overall subcellular localization of HIV-1 Gag in different cell types in parallel. Gag from T-cell tropic virus was expressed either in the context of full-length provirus or tagged with fluorescent protein. In both cases the newly synthesized Gag accumulated either at the plasma membrane or at intracellular membranes, depending on the cell-type in which it was produced (Figure 1). We thus conclude that targeting of Gag and in particular Gag accumulation at endomembranes is primarily a function of the host cell. Our next goal was to determine which subcellular membranes viral Gag was attaching to for particle formation. The human melanoma cell line Mel JuSo was instrumental for this part of the investigation because Gag was sorted both to endomembranes and to the surface of these cells, whether it was expressed in the context of full-length virus or alone (Figure 1 and supplemental data for Figure 1 available at http://www.traffic.dk/Suppmat/4_12.asp). Size and distribution of membrane patches where Gag localized immediately led us to hypothesize that late endosomes/ MVBs may be involved in anchoring Gag for subsequent assembly and release of virus. Intriguingly, it had been shown previously that late endosomes/MVBs in Mel JuSo cells can serve as transport organelles, which guide MHC class II to the cell surface (15). This was of particular significance because it is well known that MHC class II belongs to a select group of host cell proteins which are enriched in HIV-1 particles [for a recent review see (34)]. The finding that Gag was sorted to late endosomes was corroborated in experiments where this protein was coexpressed with MHC class II-GFP (Figure 2A), and also when Gag-expressing cells were stained with markers for various other endomembranes (Figure 3A and data not shown). We assume that, analogous to the fate of MHC class II, the virus was not trapped in late endosomes for degradation because Mel JuSo cells released newly synthesized Gag very efficiently (Figure 1A, last panel). Together, our data thus suggest that Gag, analogous to MHC class II, can accumulate at late endosomes for subsequent transport to the cell surface (15). Part of this transport occurs along microtubules, as evidenced both by the characteristics of vesicle motion and the finding that expression of wild-type and mutant RILP, a rab7 effector known to recruit the dynein-dynactin motor complexes to late endosomes/MVBs thus stabilizing their association with microtubules, severely affects intracellular distribution of Gag (Figure 2C). These effects exactly mimic Traffic 2003; 4: 902–910

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those observed for MHC class II (20). Of note, overexpression of RILP affected only Gag expressed in the context of the whole virus. Thus, while Gag clearly possesses the basic information for its targeting to late endosomal membranes, other viral components, the most likely candidate being Env, affect the association of newly synthesized Gag with these membranes (also compare Figure 1A,B). An ultrastructural investigation then revealed that Gag aggregates at the cytosolic face of the limiting membrane of late endosomes/MVBs and also accumulates as VLPs within the lumen of this compartment (Figure 4A,B). Our finding that MVBs typically carry either LBPA-containing internal vesicles or Gag-VLPs, but not both, supports the notion that there is significant microheterogeneity within late endosomal membranes (35). Budding into MVBs is seen much less frequently in HeLa cells than in K562 cells though, and virus is less densely packed. In HeLa cells, as in T cells, most of Gag thus appears sorted directly to the plasma membrane (see also Figure 1). But where at the plasma membrane would Gag anchor? Late endosomes, lysosomes and even the endoplasmic reticulum can nurture the plasma membrane through insertion of their membranes into the latter (22,23). Such focal exocytosis temporarily creates microdomains whose composition differs from that of the surrounding area. Colocalization at the cell surface of Mel JuSo cells and T cells (Figure 3) of viral Gag and CD63, a membrane protein known to shuttle between late endosomes and the plasma membrane [e.g. (36)], is in agreement with the hypothesis that such late endosome-derived islands within the plasma membrane serve as anchors for Gag and thus as platforms for subsequent virus egress. CD63 is enriched in HIV-1 particles produced in acutely infected T lymphocytes, though this protein is largely excluded from the surface of these cells (37), (see also Figure 3B, middle panel). Most importantly, it was previously demonstrated that CD63 is associated with infective virions released from either transformed T cells or from primary cells (4,34,38). Thus, colocalization during viral assembly of HIV-1 Gag and CD63 followed by subsequent viral exit is not an irrelevant side-road in the egress process but gives rise to infectious virus. Strong support for the proposal that late endosome-derived membrane contributes to the generation of platforms for HIV-1 egress at the plasma membrane also comes from the analysis by conventional electron microscopy of Mel JuSo cells (Figure 4C). In perfect agreement with fluorescence microscopy/colocalization data shown in Figures 1 and 3 for this cell type, discrete zones of particle budding are interspersed within larger areas where absolutely no budding takes place. Whether such distinct areas of retrovirus generation, observed previously by fluorescence microscopy also for HIV-1 [e.g. (12,14,39)] and reminiscent of the clusters of nascent Rous sarcoma virus viral particles produced in cells treated with a proteasome inhibitor (40), are pre-existing loci or if they are newly created at random sites by retroviral Gag is unclear. Results presented in this study clearly favor the Traffic 2003; 4: 902–910

idea that Gag is targeted to pre-existing terminals, i.e. to the segments of late endosomal/MVB limiting membrane before or after their insertion into the plasma membrane. The events leading to the formation of such viral exit gateways are currently under investigation. Besides acquiring specific membrane proteins such as MHC class II, HIV-1 also displays a distinct lipid profile (41) and it is well documented now that this virus buds from membrane microdomains enriched in sphingomyelin, glycosphingolipids and cholesterol (42–46). The ideas that viral morphogenesis occurs at so-called rafts or barges or at late endosome-derived platforms, as postulated in this report, are not mutually exclusive. In fact, it is well established that late endosomes play an important role in cholesterol transport, and the existence of rafts in late endosomes has recently been reported (47,48). Also, MHC class II, an important marker for late endosomes in this study, is known to segregate into detergent-resistant lipid rafts [e.g. (49)]. In addition, recent studies showed that VP40, the matrix protein of Ebola and Marburg virus, associates with so far undefined endomembranes and with late endosomal membranes, respectively (50,51), but also that these filoviruses, which may utilize the same budding machinery as HIV-1 [(28,52), reviewed in (53)], exit via lipid rafts (54). In summary, our data suggest that HIV-1 exits through endosome-derived membranes independently of whether budding takes place intracellularly or at the cell cortex. The endosomal/lysosomal compartment, besides having degradative functions, can also supply membrane to the cell periphery, thus allowing for rapid remodeling of the plasma membrane in response to external cues. Hence, gating of viral Gag through preassembled terminals recruited to the cell cortex from the endocytic compartment may be critical for viral spread, as it would allow infected cells to rapidly direct particle shedding towards target cells, thus increasing the chances of cell-to-cell transmission at the virological synapse.

Materials and Methods Cell culture, plasmids and transfections HeLa and Mel JuSo cells were grown in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 units of penicillin/ml and 100 mg of streptomycin/ml (Invitrogen). Mel JuSo cells stably transfected with a GFP-tagged version of MHC class II expressor (15) were cultured in the same medium with addition of 0.5 mg G418/ml (Invitrogen). The human cell lines Jurkat, THP-1 and K562 were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS and 100 units of penicillin/ml and 100 mg of streptomycin/ml. 907

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The following plasmids were used: pNL4-3, pGag-EGFP/CFP, pRILP-myc, pDN-vsv. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol, by electroporation and by nucleofection (Amaxa, Cologne, Germany). Analysis of cells was performed 24 h after transfection with pGag-EGFP or after 48 h when transfected with pNL4-3 alone or when cotransfected with pNL4-3, pGag-EGFP/CFP, pRILP-myc, pDN-vsv. Quantification of virus release Cells were plated in 6-cm cell culture dishes and transfected with expressor plasmids. Culture medium was exchanged 12 h before collection (48 h post transfection), in order to ensure that only freshly produced virus was quantified and to exclude contribution by non-virion-associated p24 released from dead cells. The supernatant was cleared by centrifugation (800 g) for 10 min. Cells were washed twice with PBS and then lysed on ice with NP-40 buffer (50 mM Tris HCl; 150 mM NaCl; 1% NP40) supplemented with protease inhibitors (Sigma, St. Louis, MO, USA). Viral Gag in supernatant and lysate was quantified by ELISA (Alliance HIV-1 p24 ELISA Kit, Perkin Elmer, Wellesley, MA, USA) according to the manufacturer’s instruction. Immunocytochemistry, live and fixed cell imaging analysis by fluorescence microscopy Adherent cells were plated in chambered coverglasses (Laboratory-Tek, Nunc, Naperville, IL, USA). Transfection and staining was performed directly in these chambers. Heated chambers together with heated objectives (both Bioptechs Inc., Butler, PA, USA) were utilized for long-term analysis of live cells. Suspension cells were immobilized onto coverglasses treated with Cell-Tak (BD Bioscience, San Jose, CA, USA) before staining. Cells were washed with PBS and fixed with 3.7% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA) for 10 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 for 10 min, washed again and blocked with 1% BSA in PBS for 10 min, followed by 1 h incubation with primary antibody at 37 °C. After extensive washing with PBS, cells were blocked again with 1% BSA for 10 min and then incubated with the appropriate secondary antibody for 30 min. After three more washes with PBS, cells were overlaid with PBS/1% BSA and examined by fluorescence microscopy. The following antibodies were used: Serum of HIV-positive patients (NIH AIDS Reagent Program, Rockville, MD, USA and Department of Infectious Diseases, UVM, Burlington, VT, USA), anti-CD63 (H5C6, Hybridoma Bank, Iowa City, IA, USA), anti-EEA-1, antiLamp-2 (Transduction Laboratories, San Diego, CA, USA), anti-vsv-g (Roche Diagnostic, Indianapolis, IN, USA), antic-myc (Molecular Probes, Eugene, OR, USA), anti-mouse or anti-human TexasRed-conjugated antibodies and antimouse or anti-human FITC-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA), anti-mouse or anti-human Alexa Fluor 594-conjugated antibodies (Molecular Probes). The slides were examined on a Delta Vision 908

Workstation (DV base 3, Nikon Eclipse TE200 epifluorescence microscope fitted with an automated stage, Applied Precision Inc., Issaquah, WA, USA) and images were captured in z-series with a CCD digital camera (CH350E). Out of focus light was digitally removed using Softworks deconvolution software (Applied Precision Inc.). Quantification of colocalization was computed using the Classifier Module of the Volocity Software 2.5 (Improvision, Lexington, MA, USA). Sections of 20 representative cells were evaluated. Fluorescence thresholds were selected individually for the red and green channel by eye and the fluorescence intensity of each pixel was measured. The same thresholds were applied for all three cellular markers (EEA1, Lamp-2 and CD63) and also for Gag. The extent of overlap of the two channels was calculated. The numbers represent the percentage of pixel intensity of the green channel overlapping with the red channel divided by the total pixel intensity of the green channel.

Electron microscopy For conventional EM, cells were fixed in 2.5% glutaraldehyde (EMS), dehydrated, embedded in epon resin and processed for electron microscopy. For immunogold labeling of ultrathin cryosections, cells were fixed for 15 min in cell culture medium containing 4% paraformaldehyde (EMS) and 0.1% glutaraldehyde (EMS). Medium was aspirated and replaced with phosphate buffer (100 mM NaPO4, pH 7.4) containing the same fixative and incubated further for 1 h. Thereafter, cells were detached and pelleted, the fixative was rinsed out three times with phosphate buffer and the cells were processed for cryosectioning as described (54). Briefly, the cell pellet was infiltrated with sucrose and frozen in liquid nitrogen. Frozen sections (45 nm thickness) were cut with a Leica FCS cryotome, transferred to grids, and incubated with antibodies against HIV-1 Gag (rabbit-anti-Gag polyclonal serum), GFP, LBPA (clone 4CE of mouse-anti-LBPA IgG1) and CD63 (clone 1B5 of mouse anti-CD63 IgG2b). Grids were examined with a Philips CM10 transmission electron microscope.

Acknowledgments We thank Genevie`ve Porcheron-Berthet for excellent technical assistance in the electron microscopy analysis, Mary Ramundo, Jean Gruenberg, Mark Marsh, Hans-Georg Kraeusslich, the Hybridoma Bank, and the NIH AIDS reagent program for patient serum and antibodies. Jacques Neefjes, Vineet KewalRamani and David Ott are acknowledged for their contribution of cells and plasmids, for discussions and for critical reading of the manuscript. This work was supported by grants RO1 AI 47727 to M.T., RO1 AI 40338 to P.S., SNSF 31–55170.98 to M.F. and fellowships to S.N. by the Swiss National Science Foundation and the Novartis Foundation.

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