IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production

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Hepatitis C Virus Egress and Release Depend on Endosomal Trafficking of Core Protein

Hepatitis C virus (HCV) assembly is known to occur in juxtaposition to lipid droplets, but the mechanisms of nascent virion transport and release remain poorly understood. Here we demonstrate that HCV core protein targets to early and late endosomes but not to mitochondria or peroxisomes. Further, by employing inhibitors of early and late endosome motility in HCV-infected cells, we demonstrate that the movement of core protein to the early and late endosomes and virus production require an endosome-based secretory pathway. We also observed that this way is independent of that of the internalization of endocytosed virus particles during virus entry.Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV usually infects host cells via receptor-mediated endocytosis (6, 21), followed by the release of genomic RNA after uncoating of the nucleocapsid in the endosome. HCV core protein constitutes the viral nucleocapsid and may possess multiple functions. Intracellular HCV core protein is localized mainly in lipid droplets (LDs) (23, 29). Recent studies have indicated that core protein promotes the accumulation of LDs to facilitate virus assembly (1, 10) and recruits viral replication complexes to LD-associated membranes, where virus assembly takes place (23). However, the precise mechanisms of HCV assembly, budding, and release remain largely unclear. Most recently, HCV virion release has been shown to require the functional endosomal sorting complex required for transport III (ESCRT-III) and Vps4 (an AAA ATPase) (13), which are required for the biogenesis of the multivesicular body (MVB), a late endosomal compartment (12). Late endosomes have been implicated in the budding of several other viruses, including retroviruses (8, 17, 24, 25, 27), rhabdoviruses (14), filoviruses (18, 20), arenaviruses (26, 32), and hepatitis B virus (35). However, little is known about the roles of late endosomes in the HCV life cycle.Since LDs are associated with the endoplasmic reticulum membrane, endosomes, peroxisomes, and mitochondria (16, 37), we investigated what subcellular compartments may be involved in HCV assembly and release. We first compared the intracellular distribution of HCV core protein with that of early endosome markers Rab5a and early endosome antigen 1 (EEA1), as well as the late endosome marker CD63 in the HCV Jc1-infected Huh7.5 cells at day 10 postinfection (p.i.). In immunofluorescence studies, we demonstrated that the core protein partially colocalized with Rab5a (Fig. ​(Fig.1A,1A, left panel) or EEA1 (Fig. ​(Fig.1A,1A, right panel). This finding was confirmed by the expression of enhanced green fluorescent protein (EGFP)-tagged Rab5a (Fig. ​(Fig.1A,1A, middle panel). Similarly, core protein also showed partial colocalization with CD63 (Fig. ​(Fig.1B).1B). In particular, core protein showed numerous vesicle-like structures of homogeneous size that partially colocalized with CD63 at the cell periphery (Fig. ​(Fig.1B,1B, right panel inset and drawing). This result contrasts with that of Ai et al. (2), who observed, by confocal microscopy, that core protein did not interact with markers of early and late endosomes. Ai et al. did find, however, that multimeric core complexes cofractionated with ER/late endosomal membranes in HCV-infected cells.Open in a separate windowFIG. 1.HCV core protein colocalized with early and late endosomes but not mitochondria and peroxisomes. HCV-infected cells were costained with anti-core protein (red) and anti-Rab5a (A, left panel), -EEA1 (A, right panel), or -CD63 antibodies (green) (B) or transfected with plasmids expressing enhanced green fluorescent protein (EGFP)-Rab5a (A, middle panel), enhanced yellow fluorescent protein (EYFP) (C, left panel), EYFP-mitochondria (C, middle panel), or EYFP-peroxisome (C, right panel). Cellular DNA was labeled with DAPI (4′,6-diamidine-2-phenylindole) (blue). Images shown were collected sequentially with a confocal laser scanning microscope and merged to demonstrate colocalization (yellow merge fluorescence). Enlarged views of parts of every image are shown (insets). The cartoon in panel B illustrates the core protein-containing vesicle-like structures (depicted as red circles), which partially colocalized with CD63 at the cell periphery in HCV-infected cells. PM, plasma membrane.To demonstrate the specificity of the association of core protein with endosomes, we transfected HCV-infected cells (at day 10 p.i.) with pEYFP, pEYFP-mito, and pEYFP-peroxi (Clontech) (Fig. ​(Fig.1C),1C), which label the cytoplasm/nucleus, mitochondria, and peroxisomes, respectively. The results showed that HCV core protein did not colocalize with mitochondria or peroxisomes. Taken together, these results indicate that core protein is partially associated with early and late endosomes.To investigate the functional involvement of the endosomes in HCV release, we employed HCV-infected cells (at day 10 p.i.). In our observation, at day 10 p.i., not all the cells were infected with HCV, as revealed by immunofluorescence staining against core protein (data not shown), suggesting that these cells are a mixture of infected and noninfected cells. We examined the effects of inhibitors of endosome movement, including 10 μM nocodazole (which induces microtubule depolymerization), 100 nM wortmannin (which inhibits early endosomes), 20 nM Baf-A1 (which blocks early endosomes from fusing with late endosomes) (Sigma), and 10 μg/ml U18666A (which arrests late endosome movement) (Biomol), on the release of HCV in the HCV-infected cells. We first determined the possible cytotoxicity of these drugs. We found that within 20 h of the drug application, no significant effect on cell viability, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay, was observed (Fig. ​(Fig.2C).2C). We therefore treated the cells with the various drugs for 20 h. This protocol focuses only on virus release, not on virus entry, as the reinfection of Huh7.5 cells could not account for the effects on virus release, as one round of HCV replication requires about 24 h (11).Open in a separate windowFIG. 2.HCV virion release requires endosome motility. HCV-infected cells (at day 10 p.i.) were treated with DMSO, nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM) for 20 h, and then the levels of extracellular (A) and intracellular (B) HCV RNA and HCV core proteins (F) in cells were analyzed by RT-qPCR and Western blotting, respectively. Results of Western blotting were quantified by PhosphorImager counting. (C) Analysis of cellular proliferation and survival by MTS assay. (D) Assay of extracellular viral infectivity. The culture supernatants from the cells treated with the various drugs as indicated were used to infect naïve Huh7.5 cells. The cells were stained with anti-core protein antibody (green) and DAPI (blue). The images were analyzed by using Metamorph, and the proportion of cells (of 5,000 counted) expressing core protein was counted (E). Results are presented as percentages and are averages and standard deviations from results of triplicate experiments. (G) In parallel, the HCV-infected cells were costained with anti-Rab5a (green) and -NS5A (red) antibodies. Cellular DNA was labeled with DAPI (blue). Enlarged views of parts of every image are shown (insets). (H) Colocalization efficiency between NS5A and early endosomes was analyzed by using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments. (I) Assay of intracellular HCV titers. Intracellular HCV particles were prepared from the HCV-infected cells (at day 10 p.i.) treated with U18666A at concentrations varying from 2.5 to 10 μg/ml for 20 h. The titers of intracellular HCV particles were determined by immunofluorescence staining for core-positive cell foci and are reported in focus-forming units (FFU)/ml. (J) HCV-infected cells (at day 10 p.i.) were treated with U18666A at concentrations varying from 2.5 to 20 μg/ml for 8 h, and then the levels of extracellular HCV RNA were analyzed by RT-qPCR. (K) Effects of nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM) on HCV production in single-cycle HCV growth assays. Huh7.5 cells were infected with HCV at an MOI of 1 and then incubated with DMEM containing the various drugs. At 24 h p.i., the cells and their culture supernatants were collected and used to determine the levels of intracellular and extracellular HCV RNA, which were converted to percentages of the control levels (DMSO) as 100%. Noc, nocodazole; U18, U18666A; Baf-A1, Bafilomycin A1; Wortman, wortmannin.After treatments for 20 h, the cells and their culture supernatants were collected. Intracellular RNA was isolated from cell lysates using a High Pure RNA isolation kit (Roche), and viral RNA was isolated from cell culture supernatants using a QIAamp viral RNA kit (Qiagen). Equivalent RNA volumes were subsequently analyzed on a LightCycler 1.5 real-time PCR system (Roche) for quantitative PCR (qPCR), with a primer-probe set specific for the 5′ untranslated region (UTR) sequence of HCV Jc1 and a second set specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene to quantitate the RNA amount. To calculate the percentage of HCV RNA remaining after the inhibitor treatments, the mean HCV RNA levels from triplicate wells of each sample type were standardized to the mean GAPDH RNA level in the dimethyl sulfoxide (DMSO) control wells. The relative levels of HCV RNA (percentage of control) were then analyzed with LightCycler software (version 3.53) and calculated using the relative quantification method as described in Roche Applied Science, Relative Quantification, Technical Note no. LC 13/2001 (http://www.gene-quantification.de/roche-rel-quant.pdf).As indicated in Fig. ​Fig.2A,2A, the extracellular HCV RNA levels were reduced to 32.1%, 21.7%, 76.2%, and 53.8% of the DMSO control after treatments with nocodazole, U18666A, Baf-A1, and wortmannin, respectively. We further determined the intracellular levels of HCV RNA to test whether these drugs have an effect on HCV RNA replication. Intracellular HCV RNA was reduced to 64% by nocodazole treatment (Fig. ​(Fig.2B),2B), confirming previous reports that microtubules are required for HCV RNA synthesis (9, 28). Treatments with Baf-A1 and wortmannin, however, did not affect HCV RNA replication. Interestingly, the intracellular HCV RNA level was increased to 167% by U18666A treatment (Fig. ​(Fig.2B),2B), suggesting that U18666A blocks HCV particle release (Fig. ​(Fig.2A),2A), thereby causing accumulation of HCV RNA in the late endosomes. We further determined the extracellular viral infectivity after treatments with these drugs by using the culture supernatant to infect naïve Huh7.5 cells. The infectivity was checked by counting core protein-expressing cells. Raw acquired 8-bit images (Fig. ​(Fig.2D)2D) were converted to 16-bit and analyzed with the Multi Wavelength Cell Scoring application module in Metamorph (Molecular Device). All of these treatments significantly inhibited the production of infectious HCV particles, as shown by the reduced infectivity in the supernatant of the infected cells (Fig. 2D and E). Determination of the amounts of core protein in the lysates showed that the levels of core protein in cells were not significantly affected by the various drug treatments (Fig. ​(Fig.2F).2F). Taken together, these results suggest that the inhibitors of endosome movement, including U18666A, Baf-A1, and wortmannin, reduced the secretion of HCV particles but not the HCV RNA replication. On the other hand, nocodazole-induced microtubule depolymerization reduced both HCV RNA synthesis (Fig. ​(Fig.2B)2B) (9, 28) and virus release (Fig. 2A and E). Since nocodazole also blocks the movement of endosomes between pericentriolar regions and the cell periphery (3, 7), it therefore should perturb particle release. Our present results show that nocodazole had a greater effect on the reduction in extracellular HCV RNA levels (to 32%) than intracellular HCV RNA levels (to 64%) (Fig. 2A and B), and this effect may be due to blocking of endosome movement and reduced HCV RNA replication. These results can be explained on the premise that nocodazole treatments may affect both HCV RNA replication and virus release; it led us to conclude that microtubules may simultaneously play a key role in both HCV RNA replication and virus egress. Thus, both microtubules and the movement of endosomes are required for HCV particle egress.Earlier reports indicated that Rab5, an early endosomal protein, interacts with NS4B (31) and is required for HCV RNA replication. This effect was demonstrated in Rab5 small interfering RNA (siRNA)-transfected replicon cells (5, 31). However, in our current studies (Fig. ​(Fig.2B)2B) treatments with endosome inhibitors did not reduce the levels of intracellular HCV RNA. In order to investigate the discrepancy between our results and the earlier studies, we examined the effects of endosome inhibitors on the colocalization of NS5A with Rab5a in the HCV-infected cells (at day 10 p.i.) by calculating the percentage of colocalization in the cells. Colocalization scatter diagrams were generated using the colocalization function of the Zeiss LSM Zen software. The weighted colocalization coefficient, defined as the sum of intensities of colocalizing pixels for NS5A with Rab5a in comparison to the overall sum of pixel intensities (above threshold) for NS5A, was determined. Under control conditions (DMSO), NS5A was colocalized with Rab5a throughout the cytoplasm and perinuclear region (Fig. ​(Fig.2G,2G, upper left panel). The proportion of NS5A that colocalized with Rab5a was 41% (Fig. ​(Fig.2H).2H). After treatment with U18666A, the dispersed Rab5a compartments were found only in the perinuclear region (Fig. ​(Fig.2G,2G, upper right panel). Importantly, the colocalization of NS5A with Rab5a was increased to 57% by U18666A treatment. Treatments with Baf-A1 and wortmannin, however, had no significant effect. In contrast, nocodazole treatment reduced the colocalization of NS5A with Rab5a to 12% (Fig. ​(Fig.2G2G and ​and2H).2H). Previous studies have reported that the expression levels of Rab5 and its colocalization with HCV NS4B (or NS5A) play a functional role in HCV RNA replication (31). In addition, Rab5 remained in an early endosome fraction and the expression levels of Rab5 showed no significant difference between wortmannin (100 nM)-treated or untreated cells (22). More importantly, the total levels of early endosome proteins, including EEA1 (Fig. ​(Fig.3F)3F) and Rab5a (data not shown), are not altered by the endosome inhibitors. The colocalization efficiency of NS5A with Rab5a was not reduced by U18666A, Baf-A1, or wortmannin treatments, suggesting that these drugs cannot decrease HCV RNA replication. This finding is consistent with the previous results (Fig. ​(Fig.2B).2B). These findings suggest that the observed discrepancy between our results in Fig. ​Fig.2B2B and those of the other studies (5, 31) is most likely due to differences in the expression levels of Rab5a.Open in a separate windowFIG. 3.HCV particles formed are transported from early to late endosomes. HCV-infected cells (at day 10 p.i.) were treated with the various drugs and 14 h later labeled with antibodies specific for core protein (red) and CD63 (green) (A) or EEA1 (green) (D). At the right is an enlarged area from the merged image. Nuclei were stained with DAPI (blue). (B) (E) Colocalization efficiency between core protein and early or late endosomes was analyzed by using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments. In parallel, the cell lysates were collected and then immunoblotted with antibodies against CD63 (C) and EEA1 (F). Results were quantified by PhosphorImager counting. The HCV-infected cells (at day 10 p.i.) were fixed either for immunofluorescence microscopy (G) or for thin-section electron microscopy (H and I). Cells were costained with anti-Rab5a (green) (G, left panel), -CD63 (green) (G, right panel) and -core protein (red). Lipid droplets (LDs) and nuclei were stained with BODYPI 493/503 (blue) and DAPI (white), respectively (G). Enlarged views of parts of every image are shown (insets). (H, left panel) Early endosome (EE) (white arrow) containing particles resembling HCV adjacent to the LDs. (I, left panel) MVB/late endosome (LE) containing particles resembling HCV and internal vesicles (white arrow). High-magnification images of the early endosome (H, right panel) and MVB/late endosome (I, right panel) harboring particles resembling HCV (black arrow).To further confirm that U18666A suppresses HCV release and/or virus assembly, we determined the titer of the accumulated infectious virus particles inside the cells. The HCV-infected cells (at day 10 p.i.) were treated with U18666A at various concentrations between 2.5 and 10 μg/ml for 20 h, and then intracellular HCV particles were isolated from the cells by repeated freezing and thawing (15). The infectivity was assayed on naïve Huh7.5 cells. The results showed that the titers of infectious intracellular HCV particles were increased in a dose-dependent manner by the U18666A treatments (Fig. ​(Fig.2I).2I). These data, combined with our previous results (Fig. 2A, B, D, and E), indicate that U18666A blocks HCV particle release. Further, we determined the 50% effective dose (ED₅₀) and 50% cytotoxic concentration (CC₅₀), which are defined as the concentration of U18666A that reduced the levels of extracellular HCV RNA by 50% and the concentration of U18666A that produced 50% cytotoxicity in an MTS assay, respectively. We observed that the HCV-infected cells (at day 10 p.i.) treated with U18666A at concentrations varying from 2.5 to 20 μg/ml for 8 h showed a dose-dependent reduction in extracellular HCV RNA levels (Fig. ​(Fig.2J).2J). The ED₅₀ and CC₅₀ were calculated by polynomial regression analysis. An ED₅₀ of 8.18 μg/ml (19.2 μM) and a CC₅₀ of 40.26 μg/ml (94.9 μM) were observed for U18666A for the reduction of extracellular HCV RNA and the cytotoxicity of HCV-infected cells, respectively. These results indicate that U18666A acts as a specific inhibitor of HCV release.In our previous results in Fig. 2A and D, we used a multiple-cycle virus growth assay, which could not discriminate the role of endosome movement in infection from that in virus assembly or egress. We therefore used a single-cycle HCV growth assay to further confirm that these endosome inhibitors could suppress HCV release. Previous studies have suggested that one round of HCV replication requires about 24 h (11). Therefore, Huh7.5 cells were infected with HCV JC1 at a multiplicity of infection (MOI) of 1 for 3 h. The HCV-infected cells were washed with phosphate-buffered saline (PBS) and then incubated with Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and nocodazole (10 μM), U18666A (10 μg/ml), Baf-A1 (20 nM), or wortmannin (100 nM). Therefore, the experiment will focus on virus egress and RNA replication instead of virus entry because the cells were first inoculated with HCV and then treated with the various drugs. At 24 h p.i., the cells and their culture supernatants were collected. Intracellular RNA and viral RNA were isolated from cell lysates and cell culture supernatants, respectively. The percentage of HCV RNA remaining after the inhibitor treatments was determined in the same way as for Fig. 2A and B. As indicated in Fig. ​Fig.2K,2K, the levels of extracellular HCV RNA (or viral particles) were reduced to 61%, 48.8%, 59.7%, and 60.4% after treatments with nocodazole, U18666A, Baf-A1, and wortmannin, respectively, compared to the control DMSO treatment. We further determined the intracellular levels of HCV RNA to test whether these drugs have an effect on HCV RNA replication. Intracellular HCV RNA was reduced to 69% by nocodazole treatment (Fig. ​(Fig.2K),2K), but not by U18666A, Baf-A1, and wortmannin. Overall, these results of single-cycle HCV growth assay are similar to those of multiple-cycle virus growth assay (Fig. 2A and B), again suggesting that the endosome movement inhibitors reduced the secretion of HCV particles.To further understand the roles of the early and late endosomes in the HCV life cycle, we next examined the effects of endosome inhibitors on the colocalization of core protein with CD63 or EEA1 in the HCV-infected cells (at day 10 p.i.) by calculating the percentage of their colocalization in the cells. The percentage of colocalization of core protein with CD63 or EEA1 was determined in the same way as for Fig. ​Fig.2H.2H. Under control conditions (DMSO), core protein was colocalized with CD63 throughout the cytoplasm, perinuclear region, and cell periphery (Fig. ​(Fig.3A,3A, top row). The proportion of core protein that colocalized with CD63 was 17% (Fig. ​(Fig.3B).3B). After treatment with U18666A, a characteristic collapse of dispersed CD63 compartments to the perinuclear region of the cells was revealed (Fig. ​(Fig.3A,3A, second row). Importantly, the colocalization of core protein with CD63 increased to 30% when the movement of late endosome was arrested by U18666A, whereas it was reduced to 2% and 7% by Baf-A1 and wortmannin treatments (Fig. 3A and B), respectively, suggesting that the movement of core protein was blocked by U18666A and accumulated in the juxtanuclear region. Additionally, EEA1-labeled distinct puncta, which are dispersed throughout the cytoplasm and partially colocalized with core protein in DMSO treatment (Fig. ​(Fig.3D,3D, top row), were found clustered in the perinuclear region following treatments with U18666A and Baf-A1 (Fig. ​(Fig.3D,3D, second and third rows). Correspondingly, the colocalization of core protein and EEA1 was increased after these treatments. In contrast, very little colocalization between core protein and EEA1 was seen after treatment with wortmannin (Fig. ​(Fig.3D,3D, bottom row). The colocalization of core protein and EEA1 was 10% in the DMSO control, in contrast with 35% and 20% after treatments with U18666A and Baf-A1, respectively, and 4% after treatment with wortmannin (Fig. ​(Fig.3E).3E). These data suggest that core protein was blocked by U18666A and accumulated in the juxtanuclear region. In parallel, the levels of CD63 and EEA1 protein in the lysates were determined by Western blotting. The results indicated that the total levels of CD63 and EEA1 were not altered by the various drug treatments (Fig. 3C and F, respectively). These results collectively indicate that the colocalization of core protein with CD63 or EEA1 and the release of virus particles depend on the motility of endosomes. Thus, we suggest that HCV core and/or the viral particles formed are transported from early to late endosomes.The above results prompted us to characterize the location of the early and late endosomes in relation to the site of core-LD colocalization, where HCV assembly takes place (23). In HCV-infected cells (at day 10 p.i.), early endosomes (Fig. ​(Fig.3G,3G, left panel) were colocalized with core protein and were located in juxtaposition to LDs, whereas the late endosomes were located far away from LDs (Fig. ​(Fig.3G,3G, right panel). These data suggest that following the assembly of viral particles in juxtaposition to LDs, the HCV particles are transported through early to late endosomes. To gain further insight into the trafficking patterns of HCV particles in Huh7.5 cells, we performed electron microscopy of the HCV-infected cells (at day 10 p.i.). Particles resembling HCV were present in both the early endosomes adjacent to the LDs (Fig. ​(Fig.3H)3H) and MVBs (late endosomes) (Fig. ​(Fig.3I).3I). The morphology of these endosomal compartments is similar to that reported previously (34, 36). These results again suggest that the HCV particles formed are transported from early to late endosomes.In order to rule out the possibility that the reduction in HCV particle secretion by endosome inhibitors (Fig. ​(Fig.2)2) was caused by the inhibition of endocytosis-mediated virus entry, we determined the percentage of cells that could be infected by HCV in the presence of endosome inhibitors. Cells were first treated with inhibitors of endosome movement, followed by HCV inoculation for 3 days. This analysis revealed that the same percentage of cells was infected and produced core protein following DMSO and U18666A treatments, 52% and 54%, respectively (Fig. 4A and B), demonstrating that U18666A did not affect HCV entry, and HCV could proceed normally to RNA replication. In contrast, Baf-A1 and wortmannin treatments yielded 7% and 20% (Fig. 4A and B), respectively, of infected cells, indicating that they blocked HCV entry, as previously reported (6, 21). Overall, these findings indicate that late endosome motility is dispensable for HCV entry and subsequent RNA replication and translation but is required for viral egress.Open in a separate windowFIG. 4.HCV entry and RNA replication are not affected by inhibiting late endosome movement, and endosomal localization of core protein is not affected by inhibiting endocytosis. Huh7.5 cells were treated with the various drugs and 14 h later washed and inoculated with HCV Jc1. At 3 days p.i., cells were stained with anti-core protein antibody (green) and DAPI (blue) (A). The images were analyzed by using Metamorph and the proportion of cells (of 5,000 counted) expressing core protein was counted (B). (C) Colocalization of HCV core protein and late endosomes is not affected by DN mutants of Esp15 or Rab5a. HCV-infected cells (at day 5 p.i.) were transfected with a control plasmid pCMV-IE (C, left panel) or with dominant negative mutants of Eps15 (pEGFP-Eps15-DN) (C, middle panel) or Rab5a (pEGFP-Rab5a-DN) (C, right panel). At day 2 posttransfection, cells were labeled with antibodies specific for core protein (blue) and CD63 (red). Cells expressed EGFP-Eps15-DN or -Rab5-DN proteins (green). Nuclei were stained with DAPI (white). Enlarged views of parts of every image (insets) are shown. Colocalization of core protein and CD63 is depicted in magenta. (D) Results from colocalization analysis are shown using Zeiss LSM Zen software. Error bars represent standard deviations of the mean result from 20 cells of two experiments.Moreover, we observed an almost complete block in virus infection after expression of either an Eps15 dominant negative (DN) mutant, EGFP-Eps15D95/295 (EGFP-Eps15-DN) (4), or a Rab5a dominant negative mutant, EGFP-Rab5a-S34N (EGFP-Rab5a-DN) (30), in naïve Huh7.5 cells (data not shown). This finding is consistent with a previous report (21). We further studied the effects of the DN mutants of Eps15 and Rab5a on the colocalization of core protein and the late endosomes to confirm that this colocalization was not due to the process of virus entry. Since the DN mutants of endocytosis will block HCV infection, we first performed virus infection followed by expression of the DN mutants. This strategy has been used to demonstrate that the trafficking of HIV-1 RNA and Gag protein to late endosome is independent of the endocytosed virus (19). EGFP-Eps15-DN and EGFP-Rab5-DN were transfected into HCV-infected cells (at day 5 p.i.). As shown in Fig. ​Fig.4C,4C, the localization of core protein with CD63 in these cells was not affected by the expression of these DN mutants; core protein remained well colocalized with CD63 in both the cell periphery and juxtanuclear positions in the multiple-cycle HCV growth assays. The calculated efficiency of colocalization of core protein with CD63 (19 to ∼20%; Fig. ​Fig.4D)4D) was not altered. These results indicate that core protein localization with late endosomes is not the result of the accumulation of endocytosed viruses but rather represents the trafficking intermediates of the core protein during the late viral replication stages. Thus, we conclude that the late endosome-based secretory pathways are not involved in virus entry but rather deliver the assembled virions to the extracellular milieu.Taken together, our results indicate that HCV egress requires the motility of early to late endosomes, which is microtubule dependent, and that this pathway is independent of the one required for virus entry. Thus, we postulate that following the assembly of virus particles in juxtaposition to LDs, the HCV particles are transported through early to late endosomes to the plasma membrane, where the membrane of late endosomes is fused with plasma membrane to release virions into the extracellular milieu. This transport appears to be important for HCV egress, but it is not clear how endosomes adapt in these processes. It is possible that HCV particles are transported into endosomes after their synthesis near LD. The endosome may facilitate transport of the virus particles to the plasma membrane or even to specialized cell surface domains, such as cell junctions. Notably, the tight junction protein claudin 1 has been reported to be required for HCV cell-to-cell transmission (33), which may help viruses to sequester away from the immune system.     

Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.     

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55^Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4⁺ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.     

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4⁺ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel

Ubiquitination plays a key role in trafficking of the epithelial Na⁺ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na⁺ absorption.     

HIV-1 Infection of Bone Marrow Hematopoietic Progenitor Cells and Their Role in Trafficking and Viral Dissemination

Patients with HIV-1 often present with a wide range of hematopoietic abnormalities, some of which may be due to the presence of opportunistic infections and to therapeutic drug treatments. However, many of these abnormalities are directly related to HIV-1 replication in the bone marrow (BM). Although the most primitive hematopoietic progenitor cells (HPCs) are resistant to HIV-1 infection, once these cells begin to differentiate and become committed HPCs they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and infectious virus production. Trafficking of BM-derived HIV-1-infected monocytes has been shown to be involved in the dissemination of HIV-1 into the central nervous system (CNS), and it is possible that HIV-1 replication in the BM and infection of BM HPCs may be involved in the early steps leading to the development of HIV-1-associated dementia (HAD) as an end result of this cellular trafficking process. In addition, the growth and development of HPCs in the BM of patients with HIV-1 has also been shown to be impaired due to the presence of HIV-1 proteins and changes in the cytokine milieu, potentially leading to an altered maturation process and to increased cell death within one or more BM cell lineages. Changes in the growth and differentiation process of HPCs may be involved in the generation of monocyte populations that are more susceptible and/or permissive to HIV-1, and have potentially altered trafficking profiles to several organs, including the CNS. A monocyte subpopulation with these features has been shown to expand during the course of HIV-1 disease, particularly in HAD patients, and is characterized by low CD14 expression and the presence of cell surface CD16.     

Human Endogenous Retrovirus K Gag Coassembles with HIV-1 Gag and Reduces the Release Efficiency and Infectivity of HIV-1

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.     

Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5′end of the viral gRNA, known as the packaging signal (ψ), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to ψ, as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Rev-responsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16, 35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17, 18, 37, 41). Simple retroviruses do not encode Rev-like regulatory proteins, so other strategies for the export of unspliced viral RNAs are needed. For Mason-Pfizer monkey virus, a cis-acting constitutive transport element induces nuclear export of the unspliced viral RNA in a process mediated by the cellular mRNA nuclear export factor TAP (5, 25, 46, 63). In RSV, an RNA element composed of either of the two direct repeats flanking the src gene mediates the cytoplasmic accumulation of unspliced viral RNA by using host export proteins TAP and Dpb5 (29, 42, 44).The findings of recent studies suggest that specific RNA export pathways direct viral gRNA to sites of virion assembly (56); for example, HIV-1 gRNA export out of the nucleus by the Rev-RRE-CRM1 complex is required for the proper subcellular localization of Gag and efficient virus particle production (26, 57). In the case of RSV, little is known about the trafficking of the viral RNA destined for virion encapsidation or the effects of the gRNA nuclear export pathway on Gag trafficking and virus particle production. However, we do know that RSV Gag enters the nucleus during infection, owing to nuclear localization signals (NLSs) in the matrix (MA) and NC domains. The nuclear localization of Gag is transient, and export is mediated by a CRM1-dependent NES in the p10 region (6, 52, 53). Thus, it is feasible that Gag may facilitate the nuclear export of the gRNA, either directly or indirectly, to promote particle assembly (53).In support of this idea, Gag mutants engineered to be more efficiently directed to the plasma membrane than wild-type Gag by the addition of the Src membrane-binding domain (in Myr1E virus) or by the insertion of extra basic residues (in SuperM virus) are not concentrated in nuclei when cells are treated with the CRM1 inhibitor leptomycin B (LMB) (8, 20, 53). Moreover, Myr1E and SuperM virus particles incorporate reduced levels of viral gRNA compared to the levels incorporated by wild-type particles. Thus, there is a correlation between the nuclear transit of Gag and gRNA packaging, although the Myr1E and SuperM viruses may be deficient in gRNA encapsidation because they are transported to the plasma membrane too rapidly (8). To test the hypothesis that the loss of Gag nuclear trafficking is responsible for the gRNA packaging defect, we used a gain-of-function genetic approach whereby a heterologous NLS was inserted into Myr1E Gag, yielding mutant virus Myr1E.NLS. Our results revealed that restoring the nuclear trafficking of Myr1E Gag also restored the incorporation of gRNA into mutant virus particles.     

Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses

Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.     

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z_eff) and nonelectrostatic (i.e., specific) component of binding, K_d(1M). Gag binds to Psi RNA with a dramatically reduced K_d(1M) and lower Z_eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z_eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z_eff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.     

The stoichiometry of Gag protein in HIV-1

The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.