Human Ubc9 contributes to production of fully infectious human immunodeficiency virus type 1 virions

Abstract

Ubc9 was identified as a cellular protein that interacts with the Gag protein of Mason-Pfizer monkey virus. We show here that Ubc9 also interacts with the human immunodeficiency virus type 1 (HIV-1) Gag protein and that their interaction is important for virus replication. Gag was found to colocalize with Ubc9 predominantly at perinuclear puncta. While cells in which Ubc9 expression was suppressed with RNA interference produced normal numbers of virions, these particles were 8- to 10-fold less infectious than those produced in the presence of Ubc9. The nature of this defect was assayed for dependence on Ubc9 during viral assembly, trafficking, and Env incorporation. The Gag-mediated assembly of virus particles and protease-mediated processing of Gag and Gag-Pol were unchanged in the absence of Ubc9. However, the stability of the cell-associated Env glycoprotein was decreased and Env incorporation into released virions was altered. Interestingly, overexpression of the Ubc9 trans-dominant-negative mutant C93A, which is a defective E2-SUMO-1 conjugase, suggests that this activity may not be required for interaction with Gag, virion assembly, or infectivity. This finding demonstrates that Ubc9 plays an important role in the production of infectious HIV-1 virions.

FIG. 1.

HIV-1 Gag proteins interact with Ubc9 in vitro. (a) GST-Ubc9 pull-down assay. [³⁵S]methionine-labeled in vitro-translated Pr55 was incubated in GTN buffer with GST-bound glutathione beads or GST-Ubc9-bound beads. As a loading control, 1/10 of the amount of Pr55 added to the GST pull-down reaction mixtures was loaded directly. Bound proteins were eluted with PSB and separated by SDS-PAGE. The radiolabeled proteins were visualized by phosphorimaging. (b) Mapping the Ubc9 binding domain within HIV-1 Gag. (c) GST-Ubc9 pull-down assays using wt Gag and the Gag deletion mutants were done as described above.

FIG. 2.

HIV-1 Gag proteins interact with Ubc9 in vivo. (a) Coimmunoprecipitation of endogenous Ubc9 and HIV-1 Gag. HeLa cells were either mock transfected (Untrans.) (lanes 1 and 5); cotransfected with pCMVgagpol-RRE-R and pCMVrev (lane 2 and lane 4); cotransfected with pCMVgagpol-RRE-R, pCMVrev, and pCMV Myc-Ubc9 (lane 3); or transfected with pNL4-3 (lane 6). The cells were lysed with GTN buffer and immunoprecipitated with anti-Ubc9 (lanes 1, 2, 3, 5, and 6) or preimmune sera (lane 4), and analyzed by immunoblotting using anti-p24 antibodies. (b to j) Gag and Ubc9 colocalize at distinct perinuclear puncta and areas near the plasma membrane. 293T cells were transfected with Myc-Ubc9 (b to d) or cotransfected with pNL4-3 and Myc-Ubc9 expression plasmid (e to j). The cells were fixed and processed for immunofluorescence with anti-Ubc9 PAb (b to d), anti-p24 MAb and anti-Ubc9 PAb (e to g), or anti-Ubc9 PAb and 10E9 anti-Env MAb (h to j). Cy5-conjugated donkey anti-mouse and Cy2-conjugated donkey anti-rabbit antibodies were used as secondary antibodies. Colocalization was examined by confocal microscopy using 0.3-μm optical sections. Representative medial sections are shown. The yellow arrowheads indicate the perinuclear sites where Ubc9 and Gag should colocalize. The white arrowheads indicate areas where Ubc9 and Gag should colocalize near the plasma membrane. Scale bars, 10 μm.

FIG. 3.

Ubc9 is important for the production of infectious HIV-1 virions. (a) Ubc9 expression can be suppressed by RNA silencing. 293T cells were transfected with either Ctr. RNA or Ubc9 siRNA. A second transfection with the RNAs was performed 24 h later. The cells were lysed in PSB and separated by SDS-PAGE, and protein expression levels were analyzed by immunoblotting using antibodies against Ubc9, actin, and p53 72 h after the initial siRNA transfection. (b) Suppression of Ubc9 does not affect cell viability. The viability of Ctr. RNA- and/or Ubc9 RNAi-transfected 293T cells was analyzed using the BD ApoAlert Annexin V and Apo 2.7-PE kit following the manufacturer's suggested protocol. The percentages of cells undergoing apoptosis were measured by flow cytometry. (c) Ubc9 is not required for budding. 293T cells were either left untreated or transfected twice with Ubc9 siRNA or Ctr. RNA. Wt HIV-1 proviral DNA (pNL4-3) was transfected 24 h following the second siRNA transfection. Twenty-four hours later (72 h after the initial siRNA transfection), the culture medium was collected and clarified. Virions in the culture medium were pelleted through a 20% sucrose cushion and lysed with PSB. Equal volumes of the lysates were separated by SDS-PAGE, and p24 levels were measured by immunoblotting using anti-p24 antibodies. (d) Ubc9 in virus-producing cell is required for HIV-1 infectivity. An amount of virus normalized to pelletable p24 present in the medium from transfected 293T cells was used to infect TZM-bl indicator cells for 36 h. The numbers of infectious events were analyzed by counting the cells that were positive for X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining. The error bars indicate standard deviations.

FIG. 4.

Biochemical composition of viral particles. The relative levels of vRNAs and proteins packaged into equivalent numbers of defective and infectious virions (normalized to the p24 content) were analyzed. (a) VRNA packaging. RNA from pelleted virions was extracted, reverse transcribed using oligo(dT) 12-18, and quantified by real-time PCR using vRNA-specific primers. Real-time PCR data from three independent experiments is shown as the amount of vRNA packaged into virions relative to that of vRNA packaged into virions produced from cells transfected with pNL4-3 only. The error bars indicate standard deviations. (b) Packaging of cellular and HIV-1 regulatory proteins. The levels of cellular (cyclophilin A [Cyp A]) and viral (Vif, Vpr, and Vpu) regulatory proteins packaged into virions were analyzed by immunoblotting. The bands were quantified using Discovery Series Quantity One software. (c) Glycoprotein packaging into virions. Envelope packaging into virions was analyzed by immunoblotting using polyclonal anti-HIV-1 and monoclonal anti-gp41 antibodies. (d) Glycoprotein production in cell lysates. Envelope production inside transfected cells was analyzed by immunoblotting using polyclonal anti-HIV-1 and monoclonal anti-gp41 antibodies. (e) Decreases in virion infectivity are not due to defects in early postentry events. Cells were transfected with RNAs as in previous experiments, followed by DNA transfections with pNL4-3-ΔE-EGFP and pHyg-VSV-G or pNL4-3 ΔEnv alone. Media containing pseudotyped virions were harvested and clarified 24 h after DNA transfections. Virion infectivity assays were carried out as before using TZM-bl target cells. Ubc9 expression in cell lysates. No RNA (lanes 1 to 4), control RNA (lanes 5 to 7), Ubc9 RNAi (lanes 8 to 10).

FIG. 5.

Gag and Env processing in the absence of Ubc9. (a and b) Viral-protein assembly kinetics. 293T cells were transfected with NL4-3, NL4-3 plus Ctr. RNA, or NL4-3 plus Ubc9 RNAi or left untransfected (U). The cells were pulsed (P) with [³⁵S]methionine for 45 min and then chased for 2 and 4 h. HIV-1 proteins in the cell lysates (a) and the culture supernatants (b) were immunoprecipitated with pooled AIDS patient sera, separated by SDS-PAGE, and detected by fluorography. The viral-protein bands from multiple pulse-chase experiments were quantified and analyzed using Discovery Series Quantity One software. (c) Ratio of cellular gp120/Pr55. (d) Ratio of gp120/p24 released into the media. The error bars indicate standard deviations.

FIG. 6.

Env processing in the absence of Gag and Ubc9 expression. (a) Ubc9 does not directly affect Env processing. 293T cells were transfected with LTR-Env, LTR-Env plus Ctr. RNA, or LTR-Env plus Ubc9 RNAi or left untransfected (U). The cells were pulsed (P) with [³⁵S]methionine for 45 min and then chased for 2 and 4 h. HIV-1 proteins were immunoprecipitated from cell lysates with AIDS patient sera. (b) Immunoprecipitated gp160 and gp120 bands were quantitated using Discovery Series Quantity One software, and the percentages of gp160 processed were calculated at 2 and 4 h. The error bars indicate standard deviations. (c) Summary of proteins involved in Env stability.

FIG. 7.

Ubc9 may function independently of its E2 SUMO-conjugating activity during HIV-1 assembly. (a) Myc-C93A is a trans-dominant-negative active-site mutant and suppressed SUMOylation of cellular proteins. 293T cells were cotransfected with pEGFP-SUMO-1 and either pCMV-Tag3A (lane 2) or Myc-C93A (lane 3), with pCMV-Tag3A alone (lane 4), or left untransfected (lane 1). The cells were washed with PBS, lysed in SUMO lysis buffer, and boiled for 10 min. The lysates were assayed by immunoblotting using antibodies against SUMO-1 and Ubc9. (b) Overexpression of Myc-Ubc9 or Myc-C93A does not affect virion budding. 293T cells were cotransfected with pNL4-3 and either PCMV-Tag3A (sample set 1), Myc-Ubc9 (sample set 2), or Myc-C93A (sample set 3) or left untreated (Un). The media were harvested, and virions were pelleted through a 20% sucrose cushion. Virion budding and release were assayed by immunoblotting using antibodies against p24. The transfected cells were lysed directly in PSB, and protein expression was analyzed by immunoblotting using antibodies against Ubc9 and actin. (c) Overexpression of Myc-Ubc9 or Myc-C93A does not affect HIV-1 infectivity. Medium from transfected cells was harvested, and equal amounts of virus were used to infect TZM-bl indicator cells, as in previous experiments. The error bars indicate standard deviations. (d) HIV-1 Gag and Myc-C93A do not affect Gag trafficking or colocalization with Ubc9 at perinuclear sites. Transfected 293T cells were fixed and processed for immunofluorescence analysis by confocal microscopy, using anti-Ubc9 PAb and anti-p24 MAb. Cy5-conjugated donkey anti-mouse and Cy2-conjugated donkey anti-rabbit antibodies were used as secondary antibodies. Colocalization was analyzed using 0.3-μm optical sections. Representative medial sections are shown. The yellow and white arrowheads indicate areas of Ubc9 and Gag colocalization at perinuclear puncta and near the plasma membrane, respectively. Scale bars, 10 μm.