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. 2021 Aug 10;95(17):e0055421.
doi: 10.1128/JVI.00554-21. Epub 2021 Aug 10.

HIV-1 Gag Recruits Oligomeric Vpr via Two Binding Sites in p6, but Both Mature p6 and Vpr Are Rapidly Lost upon Target Cell Entry

Madushi Wanaguru  1 , Kate N Bishop  1
Affiliations

HIV-1 Gag Recruits Oligomeric Vpr via Two Binding Sites in p6, but Both Mature p6 and Vpr Are Rapidly Lost upon Target Cell Entry

Madushi Wanaguru et al. J Virol. .

Abstract

The p12 region of murine leukemia virus (MLV) Gag and the p6 region of HIV-1 Gag contain late domains required for virus budding. Additionally, the accessory protein Vpr is recruited into HIV particles via p6. Mature p12 is essential for early viral replication events, but the role of mature p6 in early replication is unknown. Using a proviral vector in which the gag and pol reading frames are uncoupled, we have performed the first alanine-scanning mutagenesis screens across p6 to probe its importance for early HIV-1 replication and to further understand its interaction with Vpr. The infectivity of our mutants suggests that, unlike p12, p6 is not important for early viral replication. Consistent with this, we observed that p6 is rapidly lost upon target cell entry in time course immunoblot experiments. By analyzing Vpr incorporation into p6 mutant virions, we identified that the 15-FRFG-18 and 41-LXXLF-45 motifs previously identified as putative Vpr-binding sites are important for Vpr recruitment but that the 34-ELY-36 motif also suggested to be a Vpr-binding site is dispensable. Additionally, disrupting Vpr oligomerization together with removing either binding motif in p6 reduced Vpr incorporation ∼25- to 50-fold more than inhibiting Vpr oligomerization alone and ∼10- to 25-fold more than deleting each p6 motif alone, implying that multivalency/avidity is important for the interaction. Interestingly, using immunoblotting and immunofluorescence, we observed that most Vpr is lost concomitantly with p6 during infection but that a small fraction remains associated with the viral capsid for several hours. This has implications for the function of Vpr in early replication. IMPORTANCE The p12 protein of MLV and the p6 protein of HIV-1 are both supplementary Gag cleavage products that carry proline-rich motifs that facilitate virus budding. Importantly, p12 has also been found to be essential for early viral replication events. However, while Vpr, the only accessory protein packaged into HIV-1 virions, is recruited via the p6 region of Gag, the function of both mature p6 and Vpr in early replication is unclear. Here, we have systematically mutated the p6 region of Gag and have studied the effects on HIV infectivity and Vpr packaging. We have also investigated what happens to p6 and Vpr during early infection. We show that, unlike p12, mature p6 is not required for early replication and that most of the mature p6 and the Vpr that it recruits are lost rapidly upon target cell entry. This has implications for the role of Vpr in target cells.

Keywords: Gag; Vpr; human immunodeficiency virus; p6; retroviruses.

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Figures

FIG 1
FIG 1
HIV-1 p6 has no essential functional roles in early infection. (A) Schematic representation of the gag, pol, vpr, and env open reading frames in WT pNL4-3 and the pNL4-3unc proviral plasmid (27) used in this study. In pNL4-3unc, the pol frame no longer overlaps the p6 region of gag. The arrow indicates the frameshift signal at the 3′ end of gag. The p6 amino acid sequence from pNL4-3unc is also shown, with the alanine-scanning mutants 1 to 13 used in this study annotated. Colored residues indicate those reported to recruit ESCRT proteins (yellow), the viral accessory protein Vpr (green), or both (purple). (B) Measurement of virus release and single-cycle infectivity of p6 mutants, including the combinatorial L-domain mutant (mutant 2+9). 293T cells were transfected with equal volumes of WT pNL4-3unc or mutant HIV-1 pNL4-3unc constructs. Virus-containing culture supernatants were harvested at 48 h posttransfection, and titers were estimated using an RT ELISA as a proxy for virus release. HeLa TZM-bl cells were then challenged with equivalent RT units of WT or mutant virus. Infectivity was measured at 48 h postchallenge by the detection of beta-galactosidase activity in a chemiluminescence reporter assay. The data are plotted as percentages of WT virus release and infectivity (means ± standard errors of the means [SEM] from 3 biological replicates). (C) Immunoblot showing the CA-Sp1 processing efficiencies of the WT and selected p6 mutants. Virus particles were concentrated by ultracentrifugation through a sucrose cushion, and equivalent RT units were then analyzed by SDS-PAGE and immunoblotting using an anti-CA antibody.
FIG 2
FIG 2
HIV-1 p6 recruits Vpr via two binding sites during late infection. (A) Representative immunoblots demonstrating Vpr incorporation levels of WT and mutant viruses, including combinatorial p6 mutants 4+11 and 4+11+F45A. Virus particles were concentrated by ultracentrifugation through a sucrose cushion, and equivalent RT units were then separated by SDS-PAGE and analyzed by immunoblotting using anti-CA and anti-Vpr antibodies. (B) Vpr/CA ratios were estimated by quantifying the relative band intensities compared to WT virions on immunoblots such as those shown in panel A using a Li-Cor imager. (C) Sequence of p6 showing the positions of insertions of polyalanine sequences. Vpr-binding motifs are highlighted in green. (D) Virus release efficiency and single-cycle infectivity of the p6 insertional mutants were analyzed as described in the legend of Fig. 1. Histograms show means ± SEM from 3 biological replicates. (E and F) Representative immunoblot and quantification of Vpr incorporation (Vpr/CA ratio) for the p6 insertion mutants, analyzed as described above for panels A and B.
FIG 3
FIG 3
Vpr oligomerization is necessary for incorporation if one p6-binding site is removed. (A) Immunoblot showing the presence or absence of Vpr oligomers in virus carrying WT or L67A mutant Vpr. Vpr oligomers were cross-linked for analysis by fixing equivalent RT units of virus in 1% formaldehyde prior to concentration by ultracentrifugation and analysis by SDS-PAGE and immunoblotting with the anti-Vpr antibody. Possible Vpr monomers, dimers, and trimers are indicated by black arrows. (B) Virus release efficiency and single-cycle infectivity of virus carrying mutations in both p6 and Vpr were analyzed as described in the legend of Fig. 1. Histograms show means ± SEM from 3 biological replicates. (C and D) Representative immunoblot and quantification of Vpr incorporation (Vpr/CA ratio) in p6/Vpr dual mutants, analyzed as described in the legend of Fig. 2.
FIG 4
FIG 4
Avidity-based multivalent binding model for the HIV-1 p6-Vpr interaction. WT p6 (green bars) recruits oligomeric Vpr (red bars with purple interaction sites) via two binding sites, A and B (scenario 1). Inhibiting Vpr oligomerization decreases Vpr incorporation by ∼2-fold (scenario 2). Virion-associated Vpr levels decrease by ∼4- to 5-fold when either of the binding sites in p6 is removed (scenario 3). However, preventing Vpr oligomerization, together with deleting one Vpr-binding site in p6, decreases Vpr packaging by ∼50- to 100-fold (scenario 4), and deleting both binding sites in p6 decreases Vpr incorporation >100-fold (scenario 5).
FIG 5
FIG 5
There is a rapid loss of HIV-1 p6 and Vpr compared to CA during early infection. Time course assays were performed to monitor changes in CA, Vpr, and p6 levels in infected cells. (A and G) After infection by spinoculation with virus carrying WT (A) or myc-tagged (G) p6, HeLa TZM-bl cells were incubated at 37°C for 30 min before replacement of medium with fresh DMEM. Infected cells were subsequently harvested at different time points, lysed, and analyzed by immunoblotting with anti-CA and anti-Vpr (A) and anti-CA, anti-Vpr, and anti-myc (G) antibodies. Cell lysates were analyzed alongside the viral lysate prepared from the input virus stock for comparison. Relative viral protein levels were estimated by quantifying immunoblot band intensities and are plotted as a percentage of the input. (B) Sequence of p6 showing the positions of myc tag insertion sites. Vpr-binding motifs are highlighted in green. (C to E) Virus release efficiency, single-cycle infectivity, and Vpr incorporation efficiency (Vpr/CA ratio) of virus carrying myc-tagged p6 were analyzed as described in the legend of Fig. 2. The data in histograms are plotted as a percentage of the WT virus (means ± SEM from 3 biological replicates). (F) Immunoblot analysis with anti-myc antibody of viral lysates from viruses carrying C-terminally myc-tagged p6.
FIG 6
FIG 6
Residual Vpr remains associated with CA during early infection. Time course assays were performed to monitor the localization of Vpr and CA in infected cells. After infection by spinoculation with virus carrying myc-tagged p6, HeLa TZM-bl cells were either fixed immediately or incubated at 37°C for 30 min before replacement of medium with fresh DMEM and subsequent fixation at different time points. Cells were washed twice with PBS prior to fixation using 4% paraformaldehyde and methanol, permeabilized with 0.5% saponin, and stained for CA (anti-CA) (green) and Vpr (anti-Vpr) (red) before confocal microscopy analysis.
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