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Keywords:

  • AP-3;
  • dendritic cells;
  • HIV-1;
  • infectious synapse;
  • tetraspanin;
  • virological synapse;
  • virus assembly

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Dendritic cells (DC) are crucial components of the early events of HIV infection. Dendritic cells capture and internalize HIV at mucosal surfaces and efficiently transfer the virus to CD4+ T cells in trans through infectious synapses (trans-infection pathway). Alternatively, HIV-1 replicates in DC (R5-HIV-1) (cis-infection pathway). Here, we analyzed HIV trafficking in DC during the trans-infection pathway as well as the cis-infection pathway. Confocal immunofluorescence microscopy demonstrated that after capture by DC, R5-HIV-1 and HIV-1 pseudotyped with vesicular stomatitis virus protein G colocalized in a viral compartment enriched in tetraspanins including CD81, CD82 and CD9, although at different levels, indicating a role of the viral envelope in targeting to the tetraspanin-rich compartment. Replication of R5-HIV-1 in DC (cis-infection pathway) also led to the accumulation, in an envelope-independent manner, of mature viral particles in a tetraspanin-rich compartment. A fraction of the HIV-1-containing compartments appeared directly accessible from the cell surface. In sharp contrast with the trans-infection pathway, the δ-subunit of the adaptor protein 3 (AP-3) complex was enriched on the HIV-1-containing compartment during R5-HIV-1 replication in DC (cis-infection pathway). Downregulation of AP-3 δ-adaptin reduced significantly viral particle release from HIV-1-infected DC. Together, these studies demonstrate a role for AP-3 in HIV replication in a tetraspanin-rich compartment in DC and contribute to the elucidation of the trafficking pathways required for DC–T cell transfer of HIV-1 infection, a critical step during the early events of HIV infection.

Model systems have demonstrated that dendritic cells (DC) are crucial mediators of the early events of HIV-1 infection (reviewed in 1–4). As professional antigen-presenting cells (5), DC reside in the skin and mucosae in an immature state and migrate from the periphery to the secondary lymphoid organs upon pathogen encounter.

In the absence of viral replication, DC/Langerhans cells are able to capture and internalize intact HIV-1 at mucosal surfaces and successfully transmit infection in trans to CD4+ T cells through an infectious synapse (6–12). Although the in vivo contribution of this pathway (trans-infection pathway) remains to be determined, the rapid transfer of captured infectious HIV-1 to mucosal CD4+ T cells (prior to replication) could very well establish HIV-1 replication locally, before a subsequent systemic dissemination [(13) and reviewed in 4,14].

HIV-1 infection of DC is difficult to detect both in vitro and in vivo, with HIV-1 replication in DC occurring at much lower levels than in CD4+ T cells (reviewed in 1,2). Nevertheless, DC can be preferentially infected with chemokine receptor 5 (CCR5)-using HIV-1 strains (R5-HIV-1) (15–18) and have been shown to subsequently transfer newly synthesized HIV-1 infectious particles to CD4+ T cells (7,19). This replication pathway (cis-infection pathway) is believed to have a central role in HIV-1 infection of CD4+ T cells. As DC migration to lymph nodes is a prerequisite for HIV-1 to gain access to the replication-competent site, replication in DC, even if minor, would allow the retention of an infectious HIV-1 population within DC prior to its transfer to CD4+ T cells [(13) and reviewed in (4,14)]. The preferential replication of R5-HIV-1 over chemokine receptor 4 (CXCR4)-using HIV-1 (X4-HIV-1) in DC appears to result from differential levels of coreceptors expression on the surface of immature DC (iDC) and is also partly due to a block during viral fusion with target iDC (16,17). However, HIV-1 infection of DC is still insignificant, regardless of the viral tropism, when compared with activated CD4+ T cells. The cellular HIV-1 restriction factors APOBEC3G/3F were recently shown to be responsible for the defective HIV-1 infection of DC (20). The potent host antiretroviral factors function at a post-entry level, in the same way as for resting CD4+ T cells (21).

While HIV-1 is usually considered to assemble at the plasma membrane of infected cells (reviewed in 22), several observations have hinted at the possibility that intracellular organelles could play a part in this process (23,24). Studies in primary macrophages have shown accumulation of HIV-1 components at the membranes of multivesicular bodies (MVB) as well as budding of viral particles into such organelles (23,24). Although a matter of debate (25–27), these data underline a potential cell-specific role for a cell-type-specific specialized compartment in HIV-1 assembly and infection and raise the question as to whether this could also be the situation in DC. HIV-1 budding requires the recruitment of many cellular proteins to the site of viral particle formation. Mammalian homologues of the yeast vacuolar protein sorting (VPS) proteins, such as Tsg101, have been described as essential for HIV-1 assembly and budding (28–31). Recently, however, the role of the adaptor protein complex family (AP-1 to AP-4) (reviewed in 32) on HIV-1 components trafficking has been investigated (33–35). Among those adaptors, the δ-adaptin subunit of the AP-3 complex has been shown to target HIV-1 Gag to late endosome/MVB and exerts an important role in HIV-1 assembly and production (36).

We showed previously that non-replicating X4-HIV-1 is internalized into a non-conventional, non-lysosomal, tetraspanin-rich compartment in DC prior to its subsequent presentation to T cells (trans-infection pathway) (37). Here, we describe a role for the tetraspanin-rich compartment in replication (cis-infection pathway). We show the differential recruitment of the δ-adaptin subunit of AP-3 to this compartment after HIV-1 replication in DC when compared with the trans-infection pathway. We also demonstrate a role for AP-3 δ-adaptin in viral particle release from HIV-1-infected DC.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

R5-HIV-1 targets a tetraspanin-rich compartment in DC during the trans-infection pathway

We showed previously that X4-HIV-1 is internalized into a tetraspanin-rich compartment after capture by DC (37). To further extend our knowledge of HIV-1 trafficking within DC during the trans-infection pathway, we set out to characterize the trafficking of R5-HIV-1 particles within DC. For this purpose, monocyte-derived iDC were incubated with full-length provirus HIV-1 using the coreceptor CCR5 (R5-HIV-1) for 24 h at 37°C to allow viral capture and internalization. Cells were then washed, fixed and submitted to immunofluorescence staining. Cells were labeled with classical markers of endocytic compartments, including EEA1 (early endosomes), lysobisphosphatidic acid (LBPA) and CD63 (late endosome/MVB), tetraspanins CD81, CD9, CD53 and CD82, HLA-DM and HLA-DR [major histocompatibility complex (MHC)II compartment], LAMP-1 (lysosomes) as well as TGN46 (trans-Golgi network) and caveolin-1 (caveosome). Most cellular markers analyzed did not show significant colocalization with R5-HIV-1 (data not shown) as previously observed with X4-HIV-1 (37). However, similar to X4-HIV-1, iDC clustered captured R5-HIV-1 virions in a compartment where R5-HIV-1 strongly colocalized with the tetraspanins CD81 (Figure 1A), CD9 and CD82 (data not shown). Confocal immunofluorescence microscopy showed marginal colocalization with markers of late endosome/MVB (CD63), lysosome (LAMP-1) and MHCII (HLA-DR) (Figure 1A). We quantified the amount of colocalization between R5-tropic virions and the cellular markers. Pixel analysis of single middle sections revealed that ∼65% of R5-HIV-1 colocalized with CD81, ∼20% with HLA-DR, ∼14% with LAMP-1 and ∼12% with CD63 (Figure 1B). Although the amounts of colocalization between R5-HIV-1 and the different markers analyzed here are slightly different to those observed for X4-HIV-1 [(37) and Figure 2B], the R5-HIV-1 colocalization pattern was similar to that of X4-HIV-1 and suggested that both strains target the same tetraspanin-rich compartment in DC during the trans-infection pathway.

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Figure 1. R5-HIV-1 targets a tetraspanin-rich compartment in DC during the trans-infection pathway. Immature DC were incubated 24 h at 37°C with R5-HIV-1 to allow virus internalization (trans-infection pathway). A) Dendritic cells pulsed with R5-HIV-1 were analyzed by confocal immunofluorescence microscopy. One representative single middle section is depicted here with the corresponding cellular markers (green: HIV-1 p24gag, red: cellular markers and blue: LAMP-1). Bar = 5 μm. B) Quantification of R5-HIV-1 colocalization with the cellular markers CD81, CD63, HLA-DR and LAMP-1 in DC during the trans-infection pathway. Single middle sections of a minimum of 20 cells within one representative experiment were analyzed for each marker. Error bars indicate SEM. BF, bright field images.

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Figure 2. HIV-1 targeting to the CD81-rich compartment during the trans-infection pathway is envelope dependent. Immature DC were incubated 24 h at 37°C with X4-, R5- or VSVG-HIV-1Δenv to allow virus internalization (trans-infection pathway). A) Dendritic cells were analyzed by confocal immunofluorescence microscopy. One representative single middle section of a DC pulsed with each viral strain [X4-HIV-1 (top), R5-HIV-1 (middle) and VSVG-HIV-1Δenv (bottom)] is depicted here with the corresponding cellular markers (green: HIV-1 p24gag, red: CD81 and blue: LAMP-1). Bar = 5 μm. Insets show magnification of the viral compartment. B) Quantification and comparison of X4-, R5- and VSVG-HIV-1Δenv colocalization with the cellular markers CD81 and LAMP-1 in DC during the trans-infection pathway (left). Quantification and comparison of cellular markers CD81 and LAMP-1 colocalization with X4-, R5- and VSVG-HIV-1Δenv in DC during the trans-infection pathway (right). Single middle sections of a minimum of 20 cells within one representative experiment were analyzed for each marker. Error bars indicate SEM. The extent of targeting to the CD81-rich compartment after capture is statistically dependent on HIV-1 envelope (***p < 0.001). BF, bright field images.

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HIV-1 targeting to the CD81-rich compartment during the trans-infection pathway is envelope dependent

To further characterize HIV-1 targeting to the CD81-rich compartment in DC during the trans-infection pathway, we investigated the role of the HIV-1 viral envelope. Immature DC were incubated with full-length provirus X4-HIV-1, R5-HIV-1 as well as with a HIV-1Δenv pseudotyped with vesicular stomatitis virus protein G (VSVG-HIV-1Δenv) for 24 h at 37°C to allow viral capture. Cells were then washed, fixed and stained with the appropriate antibodies. Confocal immunofluorescence microscopy showed on single middle sections that VSVG-HIV-1Δenv also colocalized with CD81 (Figure 2A). However, pixel analysis revealed different levels of colocalization with CD81 for X4-HIV-1 (∼90%), R5-HIV-1 (∼65%) and VSVG-HIV-1Δenv (∼56%) (Figure 2B, left panel). The overall HIV-1 dependency on its envelope for targeting the CD81-rich compartment was statistically significant (p < 0.001).

The extent of HIV-1 colocalization with LAMP-1 varied as well according to the HIV-1 envelope analyzed, with X4-HIV-1 exhibiting less than 10% colocalization with LAMP-1, when R5-HIV-1 and VSVG-HIV-1Δenv colocalized to ∼14% and ∼20%, respectively (Figure 2B, left panel). It is also important to notice that the total amount of CD81 colocalizing with HIV-1 also varied between these HIV-1 strains ranging from ∼63% for X4-HIV-1 to ∼38% and ∼11% for R5-HIV-1 and VSVG-HIV-1Δenv, respectively (Figure 2B, right panel).

HIV-1 also targets the tetraspanin-rich compartment in DC during the cis-infection pathway

Although at low levels when compared with CD4+ T cells, HIV-1 infects DC, both in vivo and in vitro (20). Despite its relatively low efficiency, viral replication within DC plays an important part in the later stages of HIV-1 transmission from DC to CD4+ T cells (7). In order to characterize the fate of newly synthesized HIV-1 particles in DC (cis-infection pathway), iDC were incubated with full-length replicating R5-HIV-1 overnight at 37°C, thoroughly washed and replated in DC culture medium for 6 days to allow viral replication and subsequent optimal detection of newly synthesized virions. Cells were then collected, fixed and stained with the well-defined markers of endocytic compartments described above. Most cellular markers tested, such as EEA1, TGN46, HLA-DM and caveolin-1, did not show any significant colocalization with newly synthesized HIV-1 (data not shown). However, the tetraspanins CD81, CD9 and CD82 showed significant overlap with R5-HIV-1 (Figure 3A, upper panel and data not shown). In order to discard the possible but improbable detection of input viral particles in our assay, control cells were treated with the reverse transcriptase inhibitor AZT prior to their overnight incubation with R5-HIV-1 and all along the course of the experiment. Immunofluorescence labeling and microscopy analysis of these AZT-treated DC yielded no R5-HIV-1 signal and established that the viral signal detected after 6 days of replication resulted only from newly synthesized viral particles (data not shown). This viral compartment observed after 6 days of HIV-1 replication in DC was also devoid of CD63 and LAMP-1, while containing some HLA-DR (Figure 3A, upper panel). Quantification by confocal immunofluorescence microscopy revealed, however, that ∼25% of R5-HIV-1 colocalized with HLA-DR, but that ∼70% of the newly synthesized R5-HIV-1 colocalized with CD81, when only ∼10–15% colocalized with CD63 and LAMP-1 (Figure 3B, left panel).

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Figure 3. HIV-1 targets the CD81-rich compartment in DC during the cis-infection pathway. Immature DC were infected with full-length R5-HIV-1 or VSVG-HIV-1Δenv for 6 days (cis-infection pathway). A) Dendritic cells were analyzed by confocal immunofluorescence microscopy. One representative single middle section of a HIV-1-infected DC for R5-HIV-1 (upper panel) and VSVG-HIV-1Δenv (lower panel) is depicted with the corresponding cellular markers (green: HIV-1 p24gag, red: cellular markers and blue: LAMP-1). Bar = 5 μm. B) Quantification of R5-HIV-1 (left) and VSVG-HIV-1Δenv (right) colocalization with the cellular markers in DC during the cis-infection pathway. Single middle sections of a minimum of 20 cells within one representative experiment were analyzed for each marker. Error bars indicate SEM. BF, bright field images.

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In order to assess the role of HIV-1 envelope in targeting to the DC compartment during replication (cis-infection pathway), iDC were infected with VSVG-HIV-1Δenv as described above and processed 6 days post-infection for confocal immunofluorescence microscopy (Figure 3A, lower panel). VSVG-HIV-1Δenv is deleted in the HIV envelope and can only infect cells in a single round. Therefore, it does not undergo a full replication cycle, but produces HIV-1 p24gag that accumulates in DC. Pixel analysis on single middle sections indicated that ∼70% of newly synthesized VSVG-HIV-1Δenv overlapped CD81 labeling and ∼10% colocalized with CD63 and LAMP-1 (Figure 3B, right panel), while ∼40% of VSVG-HIV-1Δenv colocalized with HLA-DR. Together, these results imply that targeting of the tetraspanin-rich compartment during HIV-1 replication (cis-infection pathway) is envelope independent (Figure 3).

Mature HIV-1 particles accumulate in the CD81-rich compartment during the cis-infection pathway

Because HIV-1 accumulation in the tetraspanin-rich compartment during the cis-infection pathway could result from defective viral particle production, we examined the maturation status of HIV-1 virions found in the compartment after replication in DC. To do so, we labeled infected DC with a specific anti-HIV-1 matrix protein (p17MA) antibody that strictly recognizes the proteolytically cleaved p17MA present in mature virions (38). Immature DC were infected with full-length replicating R5-HIV-1 as described above and processed after 6 days of replication (cis-infection pathway). AZT-treated DC did not show any staining indicating that only newly synthesized virus particles were detected (data not shown). We observed a strong colocalization between CD81 and p17MA indicating that mature R5-HIV-1 virions are present in the tetraspanin-rich compartment during R5-HIV-1 replication in iDC (Figure 4). Robust colocalization between p24gag and p17MA labeling confirmed that most HIV-1 particles detected after replication are mature virions (Figure 4).

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Figure 4. Mature HIV-1 particles accumulate in the CD81-rich compartment during the cis-infection pathway. Immature DC were infected for 6 days with full-length R5-HIV-1 (cis-infection pathway). HIV-1-infected DC were analyzed by confocal immunofluorescence microscopy. One representative single middle section of a R5-HIV-1-infected DC is depicted here with the corresponding cellular markers [(upper panel – green: CD81, red: HIV-1 p17MA and blue: LAMP-1) (lower panel – green: HIV-1 p24gag, red: HIV-1 p17MA and blue: LAMP-1)]. Bar = 5 μm. BF, bright field images.

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A fraction of HIV-1-containing compartments during the cis-infection pathway is directly accessible from the cell surface

Observations made in HIV-1-infected human primary macrophages, a cell type related to DC, demonstrated that assembling HIV-1 can bud directly into a MVB compartment that also contains CD63 and CD81 (24). Recent findings, however, underlined the link between such HIV-1-containing MVB and the plasma membrane (26,27). In order to determine if, during the cis-infection pathway, HIV-1-containing compartments in DC are accessible from the cell surface, iDC were infected with full-length replicating R5-HIV-1 as described above. After 6 days of replication, HIV-1-infected DC were cooled down on ice to shut down endocytosis and incubated for 1 h at 4°C in cold horseradish peroxidase (HRP)-containing medium. HRP-treated DC were then processed at 4°C for immunofluorescence labeling. AZT-treated control DC showed no p24gag staining (data not shown). By confocal microscopy, we observed that only 17.5% (n = 51) of HIV-1-infected DC harboring an internal HIV-1-containing compartment showed significant colocalization between p24gag and HRP labeling (Figure 5, upper left panel), a pattern similar to the one observed in HIV-1-infected human primary macrophages (26). In these experimental conditions, a majority of DC harboring an internal HIV-1-containing compartment did not show HRP access into the virus-containing compartment (82.5%) (Figure 5, upper right panel).

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Figure 5. A fraction of HIV-1-containing compartments during the cis-infection pathway is accessible from the cell surface. Immature DC were infected with full-length R5-HIV-1 for 6 days to allow viral replication (cis-infection pathway). HIV-1-infected DC were incubated at 4°C in HRP-containing medium and analyzed by confocal immunofluorescence microscopy. One representative single middle section of a R5-HIV-1-infected DC with HRP present (left upper panel) or absent (right upper panel) from the HIV-1 compartment is depicted here (green: HIV-1 p24gag and red: HRP). Orthogonal sections of the same cells are depicted above (x, z-axis) and to the right (y, z-axis) for each image. Corresponding bright field images (BF) are in the corresponding lower panels. Bar = 5 μm. White arrows show HIV-1-containing compartments connected (upper left panel) or close to the cell surface (upper right panel).

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δ-subunit of AP-3 is specifically recruited to the HIV-1-containing compartment in DC during the cis-infection pathway

In order to address the role of the tetraspanin-rich compartment during the cis-infection pathway of HIV-1 in DC, we investigated if AP-3 is required for HIV-1 replication in DC. We first analyzed the intracellular distribution of the δ-subunit of AP-3 in iDC during the trans- and cis-infection pathways. For the trans-infection pathway, iDC were incubated 24 h at 37°C with full-length R5-HIV-1, washed and processed for immunofluorescence labeling. For the cis-infection pathway, iDC were infected with full-length R5-HIV-1 and processed for immunofluorescence labeling after 6 days of viral replication. Using confocal microscopy, we observed only a minor presence of AP-3 δ-adaptin within the CD81 compartment after HIV-1 capture by DC (trans-infection pathway) as shown in Figure 6A. Pixel analysis showed that less than 5% of R5-HIV-1 present in the compartment colocalized with AP-3 δ-adaptin during the trans-infection pathway (Figure 6C). In contrast, we witnessed a strong AP-3 δ-adaptin enrichment in the tetraspanin-rich compartment after R5-HIV-1 replication (cis-infection pathway) (Figure 6A). Quantification of the colocalization revealed that ∼40% of R5-HIV-1 present in the compartment after replication colocalized with AP-3 δ-adaptin (cis-infection pathway) (Figure 6C). Differential recruitment of AP-3 δ-adaptin on the tetraspanin-rich compartment during the cis-infection pathway when compared with the trans-infection pathway was statistically significant (p < 0.001). We also analyzed the intracellular localization of the human class E proteins involved in HIV release and observed no colocalization between HIV-1 and VPS4A during the trans- and cis-infection pathways (data not shown). VPS4A is an adenosine triphosphatase that functions late in the pathway of HIV budding (39) as well as for multivesicular body formation. We did not detect endogenous Tsg101, a subunit of the endosomal sorting complex required for transport (ESCRT)-1, in iDC using several antibodies (data not shown).

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Figure 6. δ-subunit of AP-3 is specifically recruited to the HIV-1-containing compartment in DC during the cis-infection pathway. Immature DC were incubated 24 h at 37°C with full-length R5-HIV-1 to allow virus internalization (trans-infection pathway) or infected for 6 days with full-length R5-HIV-1 (cis-infection pathway). Dendritic cells were analyzed by confocal immunofluorescence microscopy. A) One representative single middle section of a DC pulsed with R5-HIV-1 (trans-infection pathway, left) and one representative single middle section of a R5-HIV-1-infected DC (cis-infection pathway, right) are depicted here with the corresponding cellular markers (green: HIV-1 p24gag, red: δ-adaptin and blue: LAMP-1). Orthogonal sections of the same cells are depicted above (x, z-axis) and to the right (y, z-axis) for each image. White arrows show p24gag/AP-3 δ-adaptin colocalization events. B) Corresponding bright field images (BF) showing the location of HIV-1 (trans-infection pathway, left and cis-infection pathway, right). Bar = 5 μm. Red, green and blue represent x-, y- and z-axis, respectively. C) Quantification of R5-HIV-1 colocalization with δ-adaptin within the virus-containing compartment in DC during the trans-infection pathway (left column) and the cis-infection pathway (right column). Difference between the trans- and cis-infection pathways is statistically significant (***p < 0.001).

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These data imply a specific recruitment of AP-3 on the HIV-containing compartment during the cis-infection pathway in DC.

HIV-1 production from DC is AP-3 dependent

To further dissect the potential role of AP-3 for HIV-1 replication in DC, we used RNA interference to target the δ-adaptin subunit of AP-3. Immature DC were transfected twice with small interfering RNA (siRNA) targeting the δ-adaptin subunit of AP-3 (siδ-adaptin) or a non-specific control (siCtrl). Transfected and non-transfected (NT) DC were subsequently infected with full-length R5-HIV-1 to allow viral replication (cis-infection pathway). At 3 days post-infection, DC were harvested. The level of δ-adaptin downregulation was monitored in HIV-1-infected DC by Western blotting followed by densitometry analysis. siδ-adaptin yielded 70–80% downregulation in DC (70.75% ± 9.7 SD; n = 4) (Figure 7B). Confocal immunofluorescence microscopy confirmed knockdown of δ-adaptin in HIV-1-infected DC (Figure 7A, right panel). Dendritic cells knocked down for δ-adaptin (siδ-adaptin-DC) showed an overall similar HIV-1 compartment morphology as that of control cells (siCtrl-DC) in these conditions (Figure 7A). AZT-treated DC yielded no p24gag signal after immunolabeling, which confirmed that the viral particles detected in the HIV-1 compartment were indeed newly synthesized virions and did not result from viral input (data not shown). Using a flow cytometry-based assay, percentage of p24gag-positive DC was approximately identical in NT-DC, siCtrl-DC and siδ-adaptin-DC 3 days post-infection, whereas AZT-treated DC had residual p24gag staining (Figure 7C). Percentage of p24gag-positive DC was normalized to siCtrl arbitrarily set to 100%. In order to investigate the effect of δ-adaptin downregulation on the production and release of HIV-1 particles in DC, DC-associated p24gag levels as well as p24gag levels in the supernatant of infected DC were determined by enzyme-linked immunosorbent assay (ELISA). Three days post-infection, siδ-adaptin-DC showed roughly twice the amount of DC-associated p24gag present in siCtrl or NT-DC (2.2-fold increase ± 0.68 SD, n = 4) (Figure 7D). In contrast, p24gag release in supernatants was clearly inhibited by ∼60% (58.9% ± 11 SD; n = 4) in siδ-adaptin-DC when compared with NT-DC and siCtrl-DC (Figure 7E). p24gag levels in infected DC and their supernatant were normalized to siCtrl arbitrarily set to 100%. Increased amounts of cell-associated p24gag as well as inhibition of HIV-1 release in AP-3 knocked-down DC were statistically significant (p < 0.03 both). Together, these results clearly demonstrate the involvement of the AP-3 complex in HIV-1 release from infected DC.

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Figure 7. HIV-1 production from DC is AP-3 dependent. Immature DC transfected with siδ-adaptin or siCtrl as well as NT-DC (treated or not with AZT) were infected for 3 days with full-length R5-HIV-1 (cis-infection pathway). A) Dendritic cells were analyzed by confocal immunofluorescence microscopy. One representative single section of a R5-HIV-1-infected DC transfected with siCtrl (upper left panel) or siδ-adaptin (upper right panel) is depicted with the corresponding cellular markers (green: HIV-1 p24gag, red: δ-adaptin and blue: LAMP-1). Bar = 5 μm. B) δ-adaptin downregulation was analyzed in infected DC by Western blotting followed by densitometry analysis. Density values were normalized to siCtrl. C) Quantification of p24gag-positive cells was determined by flow cytometry analysis. Values were normalized to siCtrl arbitrarily set to 100%. D) Quantification of DC-associated p24gag levels was determined by ELISA. Values of DC-associated p24gag were normalized to siCtrl arbitrarily set to 100%. Increase of DC-associated p24gag between the siCtrl and the siδ-adaptin conditions is statistically significant (*p < 0.03). E) Quantification of p24gag levels in the supernatant was determined by ELISA. Values of virus release were normalized to siCtrl arbitrarily set to 100%. Inhibition of viral particle release between the siCtrl and the siδ-adaptin conditions is statistically significant (*p < 0.03). In C, D and E, the mean (±SD) of four independent experiments is shown. Inf., infected; BF, bright field images.

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R5-HIV-1 and CD81 colocalize at the infectious synapse in the trans- and cis-infection pathways

Dendritic cells are known to rapidly transmit HIV-1 infection upon contact with CD4+ T cells. This occurs, in the case of the non-replicating X4-HIV-1, through the fast relocation of the whole viral-containing compartment to the infectious synapse (37). To analyze the pathway of R5-HIV-1 trafficking to the DC–T cell infectious synapse during the trans- and cis-infection pathways, we used a well-characterized infectious synapse assay (8,37). In this assay, DC were incubated with full-length R5-HIV-1 for 24 h (trans-infection pathway) or infected for 6 days (cis-infection pathway) as previously described above. Prior to fixation, R5-HIV-1-pulsed DC (trans-infection pathway) or R5-HIV-1-infected DC (cis-infection pathway) were washed twice and left to adhere at 37°C on coverslips for 30 min to allow contact with previously seeded autologous CD4+ T cells as previously reported (8). DC–T cell conjugates were fixed as described previously, stained with p24gag antibody (trans-infection pathway) or p17MA antibody (cis-infection pathway) to detect mature HIV-1 particles and processed for immunofluorescence. In the trans-infection pathway, we were able to observe R5-HIV-1 relocation from the tetraspanin-rich compartment (Figure 1A, lower panel) to the infectious synapse (Figure 8, upper panel). As described for X4-tropic HIV-1 (37), CD81 labeling relocated to the infectious synapse, strongly colocalizing with R5-HIV-1 within this structure (Figure 8, upper panel). In contrast, neither CD63 nor LAMP-1 showed any apparent redistribution to the infectious synapse. For the cis-infection pathway, HIV-1 relocalization from the viral-containing compartment to the infectious synapse was observed upon R5-HIV-1-infected DC contact with autologous CD4+ T cells (Figure 8, lower panel). We then assessed the proportion of DC–T cell conjugates showing R5-HIV-1 polarization to the infectious synapse after replication. In the cis-infection pathway, polarization of R5-HIV-1 virions to the DC–T cell zone of contact (infectious synapse) was seen in 36.9% of DC–T cell conjugates, while polarization of viral particles away from the T cell was noticed in 15.5% of DC–T cell conjugates (n = 103). Whether this last situation results from T-cell detachment or random polarization mechanisms is difficult to say. We observed that 47.6% of DC–T cell conjugates harboring an HIV-1 compartment in the DC did not show viral relocalization to the infectious synapse, but instead mainly maintained the HIV-1 compartment in DC. AZT controls confirmed that R5-HIV-1 particles present at the infectious synapse were indeed newly synthesized virions (data not shown). CD81 in the cis-infection pathway relocated to the DC–T cell interface, in contrast with CD63 (Figure 8, lower panel). As a result of the thresholds of detection, CD81 and CD63 labeling were faint in T cells when compared with DC, although both tetraspanins have been described to be present on T cells (40–42). These results demonstrate that R5-HIV-1 relocates from a tetraspanin-rich compartment to the DC–T cell infectious synapse during the trans- and cis-infection pathways.

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Figure 8. R5-HIV-1 and CD81 colocalize at the infectious synapse in the trans- and cis-infection pathways. Immature DC were incubated 24 h at 37°C with full-length R5-HIV-1 to allow virus internalization (trans-infection pathway) or infected for 6 days with full-length R5-HIV-1 (cis-infection pathway). Dendritic cells were incubated with autologous CD4+ T cells for 30 min to allow infectious synapse formation. DC–CD4+ T cell conjugates were then fixed and analyzed by confocal immunofluorescence microscopy. Large cells represent DC and small cells autologous CD4+ T cells. One representative DC–T cell conjugate for the trans-infection pathway (upper panel) and the cis-infection pathway (lower panel) is depicted here with the corresponding cellular markers [green: HIV-1 p24gag (upper panel) and HIV-1 p17MA (lower panel), red: cellular markers and blue: LAMP-1). Bar = 5 μm. BF, bright field images.

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Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

In this study, we characterize the tetraspanin-rich compartment in which HIV-1 accumulates during the trans- and cis-infection pathways, prior to the subsequent transmission of infectious viral particles to CD4+ T cells across infectious synapses (6–8,10,11,37,43,44).

We described previously that X4-HIV-1 captured by DC was internalized into a novel tetraspanin-rich compartment containing MVB (37). Here, we show using confocal immunofluorescence microscopy that R5-tropic HIV-1 is also internalized in a compartment characterized by the presence of the tetraspanin CD81, CD9 and CD82. Although this compartment contained some CD63 and HLA-DR, it was distinct from HLA-DM and LAMP-1-containing lysosomes, as previously described for X4-HIV-1 (37).

Although X4-tropic and R5-tropic HIV-1 accumulated in a CD81-rich viral compartment during the trans-infection pathway, we observed that the percentage of HIV-1 particles colocalizing with CD81 labeling was significantly different (∼90 and ∼65%, respectively). In order to evaluate the role of the viral envelope in the targeting to the compartment, we used VSVG-pseudotyped particles. Unlike HIV-1 (45), VSV requires the acidic pH-dependent triggering of its envelope protein for fusion with endosomal membranes during entry into the cells (46,47). We could demonstrate that HIV-1 targeting to the tetraspanin-rich compartment is envelope dependent during the trans-infection pathway, with a decrease in HIV-1/CD81 colocalization when we compared X4-HIV-1 with R5-HIV-1 or VSVG-HIV-1Δenv. It is possible that differences in the extent of colocalization with the tetraspanin CD81 might result from differential fusion properties of VSVG-, X4- or R5-HIV envelopes with iDC (16,17).

Infection of DC, although poor in comparison with CD4+ T cells, is likely to play a part in the systemic propagation of HIV infection in patients by transferring HIV-1 infection to CD4+ T cells after migration to lymph nodes (cis-infection pathway) (7,48). Using confocal immunofluorescence microscopy, we demonstrated that, during the cis-infection pathway, replicating R5-HIV-1 accumulated in DC in a clustered compartment containing the tetraspanins CD81, CD82 and CD9. We also observed that VSVG-HIV-1Δenv-infected DC accumulated virus in a CD81-rich compartment 6 days post-infection, indicating that targeting to the tetraspanin-rich viral compartment in DC appears envelope independent during the cis-infection pathway.

Upon or after budding, HIV-1 is known to go through a maturation stage during which p55gag is proteolytically cleaved and the capsid (CA) and nucleocapsid (NC) proteins reassembled into a conical core structure in the center of the viral particle (reviewed in 49,50). Using a monoclonal anti-p17MA antibody that specifically recognizes p17MA only, following cleavage from p55gag, we observed that HIV-1 virions present in the CD81-rich viral compartment during the cis-infection pathway are mature viral particles. Although it is difficult to completely rule out the possible recapture of newly assembled HIV-1 virions budding from the surface, the strong presence of mature HIV-1 particles in the tetraspanin-rich compartment suggests that this compartment is a major site of viral assembly in iDC.

HIV-1 Gag was shown to accumulate at membranes of MVB in human primary macrophages, directing HIV-1 subsequent assembly and budding steps to an intracellular compartment (23,51–54). This model of cell-type-dependent budding of HIV-1 in a MVB-containing compartment argues in favor of the DC tetraspanin-rich compartment as a HIV assembly site. However, this model is still a matter of vivid debate. Recent findings argue against a significant production of HIV-1 in MVB-containing late endosomes in macrophages, favoring instead HIV-1 assembly and budding from the plasma membrane, regardless of the cell type (25–27). In fact, these sites of viral budding containing MVB appear to be close or connected to the cell surface of macrophages, underlying an unexpected complexity between the plasma membrane and MVB-containing structures (26,27). In DC, our results demonstrate that only a fraction of HIV-1-containing compartments (17.5%) is accessible from the cell surface. Of note, our assay is performed at 4°C and may underestimate the number of compartments directly accessible from the extracellular milieu over a more prolonged period of time.

We showed that the δ-adaptin subunit of the AP-3 complex, a component of HIV-1 assembly machinery, is specifically enriched in the tetraspanin-rich/virus-containing compartment during HIV-1 replication in DC (cis-infection pathway) but not after HIV-1 capture by DC (trans-infection pathway). The direct interaction between HIV-1 Gag and AP-3 δ-adaptin was shown to target HIV-1 Gag to MVB and to play a functional part in HIV-1 assembly in cell lines (36), making AP-3 δ-adaptin a potential cellular factor required for HIV-1 assembly. RNA interference allowed us to demonstrate that AP-3 δ-adaptin plays a significant role in the production of HIV-1 from DC as its downregulation decreased viral particle release from HIV-1-infected cells (cis-infection pathway) (Figure 7). Concomitantly, AP-3 δ-adaptin downregulation increased cell-associated HIV-1 p24gag amounts in infected DC. Morphology of the virus-containing compartment did not show major alterations in the absence of AP-3 δ-adaptin, but we cannot rule out that the residual levels of AP-3 δ-adaptin present in DC after RNA interference are sufficient for an appropriate targeting of HIV-1 p24gag to the tetraspanin-rich HIV-1-containing compartment.

Finally, we demonstrate that HIV particles present within the tetraspanin-rich compartment in DC during the trans- and cis-infection pathways relocate to infectious synapses upon contact with autologous CD4+ T cells. As we showed previously in DC–Jurkat CD4+ T cell clusters (37), CD81, as opposed to CD63, is enriched at the DC–T cell infectious synapse, in accordance with CD81 role in the immunologic synapse (55–59).

In conclusion, our studies characterize a tetraspanin-rich compartment targeted by HIV-1 during the trans- and cis-infection pathways. Our data reveal that the DC trans-infection pathway and the DC cis-infection pathway intersect at the level of this tetraspanin compartment and share components of their respective intracellular trafficking machinery. Our study highlights the presence of HIV-1 during replication in iDC in a tetraspanin-rich compartment sharing some similarities with the MVB-containing compartment observed in macrophages during HIV-1 replication (24,26,60). Furthermore, we describe for the first time the selective recruitment of AP-3 to this tetraspanin-rich compartment in iDC during the cis-infection pathway but not during the trans-infection pathway. We also show a direct role for AP-3 δ-adaptin in viral particle release from infected DC, indicating that the tetraspanin-rich compartment is a likely site of production of viral particles. We do not rule out a contribution of direct budding from the cell surface potentially involving tetraspanins as observed in CD4+ T cells (40–42), but our data show that a significant fraction of HIV production in iDC is likely to result from production in a compartment enriched in tetraspanins as well as AP-3 δ-adaptin. Together, these studies highlight the cellular machinery required for DC–T cell transfer of HIV infection, an essential step in the early dissemination of HIV infection.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

Preparation of human primary DC

Monocytes from buffy coats were obtained according to institutional guidelines of the ethical committee of the University of Geneva. Human iDC were generated with modifications from Sallusto and Lanzavecchia (61) as previously described (37) by incubating purified monocytes in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum, 2 mml-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mm HEPES, 1% nonessential amino acids, 1 mm sodium pyruvate, 50 μmβ-mercaptoethanol solution, 50 ng/mL granulocyte–macrophage colony-stimulating factor (GM-CSF) and 50 ng/mL interleukin-4 (IL-4) (both Strathmann Biotec). On days 2 and 4, a third of the culture medium was replaced by fresh medium containing GM-CSF and IL-4. Immature DC were harvested at day 6 and analyzed by flow cytometry.

Viral stocks

These were produced by transient transfection of 293T cells as described previously (62). Viral stocks were generated by transfection of 293T cells with calcium phosphate-coprecipitated proviral plasmids pR9 and pR8Ba-L, which encode for a full-length HIV-1 X4 strain provirus and a full-length HIV-1 R5 strain provirus, respectively. pR8Ba-L is a full-length HIV-1-R5 molecular clone in which the R5-tropic envelope of strain HIV-1-Ba-L has replaced the X4-tropic envelope of R9. The plasmids R8Ba-L and R9 were provided by D. Trono (EPFL). VSVG-HIV-1Δenv was produced by cotransfection of 293T cells with pHIV-GFP, a plasmid containing the full-length genome except for a deletion in env, vif and nef where green fluorescent protein is cloned (a gift of D. Littman, New York University School of Medicine, New York, NY, USA), and pMDG, a plasmid encoding for the VSVG protein. VSVG-HIV-1Δenv was produced in the presence of Nef. Infectious titres of viral stocks were evaluated by limiting dilution on HeLa P-4.2-R5 cells (63). These cells derive from HeLa P-4.2 cells stably expressing coreceptor CCR5 and containing the β-gal gene under the control of HIV long terminal repeat. Viral titres were expressed as infectious units per milliliter. Approximately 500 ng of p24gag on 2.5 × 105 CD4+ HeLa P-4.2-R5 corresponds to a multiplicity of infection (MOI) of 1. Physical titres were evaluated by quantification of HIV-1 p24gag by ELISA kit (AIDS Vaccine Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, USA).

Antibodies and reagents

Most antibodies used in this study have been previously described (8,37). Monoclonal antibodies against CD9 (M-L13), CD81 (JS-81), CD82 (50F11), CD53 (HI29), HLA-DM (MaP.DM1), EEA1 (clone 14) and δ-adaptin (clone 18) were from BD. δ-adaptin (clone SA4) was a gift from A. Peden (64), and anti-HLA-DR (L-243) was a gift from J. Pieters (Biozentrum, University of Basel, Switzerland). Anti-CD63 (1B5, a gift from M. Marsh, London) and anti-LBPA (6C4, a gift from J. Gruenberg, Geneva) were used as supernatants from the hybridoma cells. Anti-VPS4A antibody was a gift from W. Sundquist, Salt Lake City, UT, USA. The rabbit polyclonal anti-LAMP-1 was a gift from M. Fukuda (Cancer Research Center, La Jolla, CA, USA) (65), and sheep polyclonal anti-TGN46 was purchased from Serotec. Rabbit polyclonal anti-HRP was purchased from Jackson ImmunoResearch Laboratories, Inc. Rabbit polyclonal anti-caveolin-1 (N-20) and goat polyclonal antibody anti-actin (clone C-11) were bought from Santa Cruz Biotechnology. Monoclonal anti-HIV-1 p24gag (KC57) was from Coulter and was used for immunofluorescence/confocal analysis as well as flow cytometry analysis. Anti HIV-1 p17MA (4C9) was obtained from R. B. Ferns and R. S. Tedder (through the NIBSC Centralised Facility for AIDS Reagents, supported by the EU Programme EVA and the UK Medical Research Council).

Confocal immunofluorescence microscopy

To localize HIV-1 after capture in DC (trans-infection pathway), iDC (105 cells/condition) were loaded with full-length X4-, full-length R5- or VSVG-HIV-1Δenv (∼500 ng of p24gag per 2.5 × 105 DC) for 24 h at 37°C, washed twice in PBS and left to adhere on poly-l-lysine-treated (Sigma-Aldrich) glass coverslips for 1 h at 37°C. To localize HIV-1 after replication in DC (cis-infection pathway), iDC (105 cells/condition) were incubated with either full-length R5- or VSVG-HIV-1 (∼500 ng of p24gag per 2.5 × 105 DC; an approximate MOI of 1) overnight at 37°C, washed twice in PBS and replated in fresh DC culture medium for 6 days of replication to facilitate detection of newly synthesized particles. DC pretreated for 30 min (prior to HIV-1 incubation) with AZT (50 μg/mL) served as HIV-1 new synthesis control in the replication experiments. At this point, infected DC were washed twice in PBS and left to adhere on poly-l-lysine-treated glass coverslips for 1 h at 37°C. From this point on, cells were processed in the same way. DC were fixed 20 min at room temperature in 3% paraformaldehyde, permeabilized with 0.05% saponin (Sigma-Aldrich) and washed with PBS containing 0.2% BSA (Sigma-Aldrich) and human IgG (20 μg/condition). Triple labeling of cells was performed as follows: HIV-1-loaded DC or HIV-1-infected DC were first stained with primary antibodies. After extensive washes in BSA/saponin-containing PBS, cells were stained with secondary donkey antibodies coupled to rhodamine or Cy-5 (Jackson ImmunoResearch Laboratories). In order to avoid unspecific labeling, cells were incubated 20 min at room temperature in PBS containing BSA, saponin and mouse serum (0.5 mg/mL). Finally, HIV-1 p24gag was detected using a monoclonal anti-HIV-1 p24gag (KC57) coupled to fluorescein isothiocyanate (FITC) (Coulter). When the anti-HIV-1 p17MA (4C9) was used as a primary antibody (Figures 4 and 7), CD81 and CD63 were detected using monoclonal antibodies coupled to FITC (BD Pharmingen) in the same way as the monoclonal anti-HIV-1 p24gag (KC57) coupled to FITC.

For experiments with HRP, iDC were incubated with R5-HIV-1 (∼500 ng of p24gag per 2.5 × 105 DC; an approximate MOI of 1) overnight at 37°C, washed twice in PBS and replated in fresh DC culture medium for 6 days of replication. R5-HIV-1-infected DC were then harvested and washed once in PBS. Next, cells were cooled on ice for 20–30 min and incubated for 1 h at 4°C in cold HRP-containing medium (10 mg/mL, HRP type II; Sigma-Aldrich). Cells were subsequently washed once in cold PBS, left to adhere on poly-l-lysine-treated glass coverslips for 1 h at 4°C and processed for confocal immunofluorescence microscopy as described previously. Alternatively, R5-HIV-1-infected DC were plated on poly-l-lysine-treated glass coverslips prior to incubation in the HRP-containing medium.

Infectious synapse assays were performed as previously described (8,37). Autologous CD4+ T cells (3 × 105 cells/condition) were left to adhere on poly-l-lysine-treated glass coverslips for 2 h at 37°C. For the trans-infection pathway, iDC (105 cells/condition) were incubated with R5-HIV-1 (∼500 ng of p24gag per 2.5 × 105 DC) for 24 h at 37°C, washed twice in PBS and left to adhere at 37°C on the coverslips for 30 min to allow contact with autologous CD4+ T cells. For the cis-infection pathway, iDC were incubated with R5-HIV-1 (∼500 ng of p24gag per 2.5 × 105 DC; an approximate MOI of 1) overnight at 37°C, washed twice in PBS and replated in fresh DC culture medium for 6 days of replication. Cells were subsequently washed and left to adhere at 37°C on the coverslips for 30 min to allow contact with autologous CD4+ T cells. For both pathways, conjugates were then fixed, permeabilized and stained as described above. We defined an infectious synapse as a DC–T cell conjugate where the majority of HIV (>75%) is focused at the zone of contact with the CD4+ T cells. All confocal laser scanning microscopy were performed with a LSM 510 microscope (Zeiss). All confocal images presented here are single middle sections. All single-section images were then processed using Photoshop following Traffic guidelines. Quantifications of colocalization were performed using the Metamorph software (Universal Imaging) on single middle sections of a minimum of 20 cells for each marker. One representative experiment out of three was statistically analyzed.

δ-adaptin RNA interference in DC

Immature DC were transfected with siδ-adaptin [(5′-AATCTGCAAGCTGACGTATTT-3′) siRNA sequence specific for δ-adaptin] or siCtrl [(5′-AAATGAACGTGAATTGCTCAA-3′) non-specific sequence] (Custom high-performance siRNAs; Qiagen) using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s recommendations, and 8 × 105 monocyte-derived DC were transfected with 100 nm siRNA in 600 μL of complete IMDM medium in 12-well plates. A second round of transfection was performed 24 h later. Prior to HIV infection, DC were washed twice with PBS and replated in 800 μL complete IMDM medium. Cells were incubated with R5-HIV-1 (∼1 μg of p24gag per 8 × 105 DC; an approximate MOI of 1) overnight at 37°C, washed twice in PBS and replated in fresh DC culture medium. At day 3 post-infection, cells were harvested, washed and counted for subsequent analysis. δ-adaptin gene knockdown was assessed by Western blot followed by densitometry analysis (Quantity One; Bio-Rad Laboratories). The siCtrl condition was used as control with an arbitrary value of 1. Percentage of p24gag-positive DC was determined using Cytofix/Cytoperm (BD) and phycoerythrin-coupled anti-p24gag monoclonal antibody (Coulter). Cells were then washed, fixed in 1% paraformaldehyde and analyzed by flow cytometry analysis. Percentage of p24gag-positive DC was normalized to the siCtrl condition arbitrarily set to 100%. DC-associated p24gag levels as well as p24gag levels in the supernatant of infected DC were determined by ELISA using the HIV-1 p24 Antigen Capture Assay Kit (AIDS Vaccine Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, USA) following manufacturer’s instructions. DC-associated p24gag levels as well as p24gag levels in the supernatant of infected DC were normalized to the siCtrl condition arbitrarily set to 100%.

Statistical analysis

Variance homogeneity of HIV-1 envelopes/CD81 colocalization results was tested using a Levene test. Statistical analysis was subsequently performed using anova and the corresponding non-parametric or parametric post hoc test (Tamhane and Tukey, respectively). Variance homogeneity of p24gag/AP-3 δ-adaptin colocalization results was tested using a Levene test. Statistical analysis was subsequently performed using the non-parametric Mann–Whitney U-test. Statistical relevance of AP3 δ-adaptin silencing on DC-associated p24gag levels and p24gag levels in the supernatant of infected DC was performed using the non-parametric Mann–Whitney U-test.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References

We thank W. Sundquist, M. Marsh, D. Littman, D. Trono, J. Gruenberg and M. Fukuda for the reagents and F. Leuba, R. Stalder and R. Correa for their technical assistance. We thank M. Marsh for critical reading of the article. D. S. N. is the beneficiary of a MD–PhD scholarship from the Swiss National Science Foundation. This work was supported by the Geneva Cancer League and Swiss National Science Foundation and The Human Science Frontier Program to V. P.

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