The trans-Golgi network-associated human ubiquitin-protein ligase POSH is essential for HIV type 1 production

Abstract

HIV type 1 (HIV-1) was shown to assemble either at the plasma membrane or in the membrane of late endosomes. Now, we report an essential role for human ubiquitin ligase POSH (Plenty of SH3s; hPOSH), a trans-Golgi network-associated protein, in the targeting of HIV-1 to the plasma membrane. Small inhibitory RNA-mediated silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native, but not a RING finger mutant, hPOSH restores virus release and Gag plasma membrane localization in hPOSH-depleted cells. Furthermore, expression of the RING finger mutant hPOSH inhibits virus release and induces accumulation of intracellular Gag in normal cells. Together, our results identify a previously undescribed step in HIV biogenesis and suggest a direct function for hPOSH-mediated ubiquitination in protein sorting at the trans-Golgi network. Consequently, hPOSH may be a useful host target for therapeutic intervention.

Fig. 1.

Effect of hPOSH silencing on HIV-1 release. (A) Effect of E3 silencing on HIV release. RNAi-treated cells were cotransfected with HIV-1_NL4–3 env- and VSV-G expression plasmid. Cellular and VLP detergent extracts of cells transfected with either lamin A/C RNAi (control) or RNAi targeting the indicated E3 ligases were resolved by SDS/PAGE and subjected to immunoblot analysis with anti-CA as described in Materials and Methods. (B) Effect of hPOSH inhibition on the production of infectious virus. Cells were transfected with lamin A/C (control) or hPOSH RNAi. A single cycle infectivity assay was subsequently carried out as described in Materials and Methods. The percentage of infected cells was determined by FACS analysis and was plotted against the corresponding dilution of the initial viral stock. The results are the mean value of triplicate incubations. (C) SEM of HeLa SS6 cells. Cells initially were transfected with either hPOSH scrambled RNAi (a and c) or hPOSH RNAi (b and d) and then either mock transfected (c and d) or transfected with pNLenv1 p6^ATAA (a and b). Subsequently, cells were subjected to SEM as described in Supporting Materials and Methods.

Fig. 4.

The ubiquitin ligase activity of hPOSH is required for HIV release. (A) Analysis of Gag-EGFP localization in HeLa cells. H187 cells were transfected with Gag-EGFP expression plasmid. (Upper) Then, 6 h after transfection, cells were transferred to 20°C for 2 h and then shifted back to 37°C for 5 h. (Lower) A parallel culture was maintained at 20°C for 7 h. Cells then were fixed and processed for confocal microscopy as described in Materials and Methods and in the Fig. 4 legend. (B) HIV-1 release at 37°C and 20°C. H187 cells were transfected with pNLenv1. Then, 12 h after transfection, the culture medium was replaced with fresh medium, and cells were incubated for 2 h at 20°C, after which the medium was replaced, and cells were further incubated for 5 h at 37°C. Control reactions were incubated in parallel at 20°C for 7 h. Subsequently, VLP were harvested, and aliquots were tested for reverse-transcriptase activity. The measured reverse-transcriptase activity was normalized to the relative Gag expression determined in parallel by quantitative anti-CA Western blot analysis using Cy3-conjugated secondary antibodies. (C) Cells were cotransfected with pNLenv-1 and expression plasmids for the indicated hPOSH proteins. Then, 12 h after transfection, cells were transferred to fresh medium at 20°C (2 h) and then to a fresh medium at 37°C (5 h). (Upper) Cells and VLP were processed, and reverse-transcriptase activity was determined as described in B.(Insets) Anti-V5 Western blot analysis of exogenous V5-hPOSH. (Lower) Quantitative analysis of Gag expression. Western blot analysis was performed with primary anti-CA antibody and a secondary Cy3-labeled anti-rabbit.

Fig. 2.

hPOSH is an E3 ligase. (A) Self-ubiquitination of maltose-binding protein–POSH–his tag fusion protein was assayed as described in Materials and Methods. One of the ubiquitin-conjugating enzymes was omitted in each of the control reactions. Samples were resolved by SDS/PAGE and subjected to Western blot analysis with antiubiquitin antibodies. (B) Ubiquitination of exogenous hPOSH. Cells were transfected with empty vector, V5-tagged hPOSH, or hPOSH_V14A-encoding vectors. The tagged POSH then was immunoprecipitated with an anti-V5 antibody and incubated with E1, Ubch5c, ATP, and biotinylated ubiquitin as described in Materials and Methods. The reactions were stopped by the addition of SDS sample buffer, resolved by SDS/PAGE, transferred onto nitrocellulose, and blotted with streptavidin–horseradish peroxidase to detect ubiquitinated proteins.

Fig. 3.

Intracellular localization of hPOSH. (A) hPOSH localizes at the TGN. H153 and H187 cells stably expressing RNAi targeting the hPOSH coding sequence or a scrambled sequence, respectively, were incubated with rabbit anti-hPOSH (hP^285–430) and mouse monoclonal antibodies directed to Lamp1, EEA1, or CD63 and sheep anti-human TGN46. Subsequently, cells were immunostained for confocal microscopy with Alexa Fluor 488-conjugated goat anti-rabbit (hPOSH detection), Cy3-conjugated donkey anti-sheep (TGN46 detection), and Alexa Fluor 546-conjugated goat anti-mouse secondary antibodies for detection of Lamp1, EEA1, and CD63. (Bars: 20 μM.) (B) Primary human lymphocytes and macrophages (10⁵ cells) were subjected to confocal microscopy for detection of TGN and hPOSH as described above. (Bars: 10 μM.) (C) Inhibition of VLP secretion from Jurkat cells. J3′UTR1–3 and J187 cells stably expressing RNAi targeting the hPOSH 3′ UTR or a scrambled sequence, respectively, were transfected with pNLenv1. Subsequently, VLP were quantified through measurement of reverse-transcriptase activity secreted into the culture medium as described in Supporting Materials and Methods.

Fig. 5.

POSH regulates transport of Gag to the plasma membrane. The intracellular distribution of Gag-EGFP was determined in H187 (A) and H153 (B) by confocal microscopy. Cells initially were transfected with empty plasmid (mock) or a plasmid encoding either V5-tagged hPOSH or hPOSH_V14A and subsequently with Gag-EGFP-encoding plasmid. Then, 6 h later, cells were fixed and immunostained with anti-V5 and anti-TGN46 and with Alexa Fluor 633-conjugated goat anti-mouse and Cy3-conjugated donkey anti-sheep antibodies, respectively. (Bars: 20 μM.)

Fig. 6.

Proposed pathways for HIV-1 Gag trafficking to the cell membrane. (A) Newly synthesized Gag assembles with endocytosed Env at the TGN before transport to the cell surface. (B) Newly synthesized Gag is sorted by means of the TGN to late endosomes (LE) where it assembles with Env before transport to the cell membrane. Consistent with both mechanisms, the role of hPOSH (indicated by a star) is to facilitate egress of Gag cargo vesicles from the TGN.