Fig 1.
Rapamycin causes a reduction in HIV-1 NL4-3 Gag, Vif and Vpr.
The effect of mTOR inhibition by Rapamycin on HIV-1 was examined in Jurkat CD4+ T cells (A-C) and primary CD4+ T cells (D-F) for NL4-3 and NL4-3 Δnef. The levels of HIV-1 proteins were measured by densitometry assays for NL4-3 (A and D) and NL4-3 Δnef (B and E) infected cells. Protein levels were normalized to ACTB/β-actin, and their expression relative to the DMSO treatment (D on the x axis) was calculated. Data correspond to the mean and the SEM of 3 independent experiments. Dotted lines represent 2-fold threshold. Representative western blots from infections in Jurkat CD4+ T (C) and primary CD4+ T cells (F). Membranes were probed for gp120, Gag/p55, Vif, Nef, Vpr, Vpu and LC3. ACTB was used as a loading control. Red arrows indicate defects in HIV-1 proteins.
Fig 2.
The inhibition of mTOR causes the proteasomal degradation of Vif.
(A) HEK293T cells were transfected with 2,000 ng of an empty vector (V) or a NL4-3 Flag-Vif construct. 44 h later, cells were treated with Torin2 (5-10 nM) with or without BafA1 (500 nM) for 4 h. Next, cells were harvested, analyzed by western blot, and autophagy flux was measured by determining the LC3-II/I ratios and SQSTM1 levels relative to the vector-transfected and DMSO-treated cells. Data correspond to the mean and the SEM of 4 independent experiments. Blots are representative of 3 independent experiments. (B) Parental (wild type; WT) and ATG5KO HEK293T cells were transfected with 2,000 ng of a NL4-3 Flag-Vif construct. 44 h later, cells were treated with DMSO or increasing concentrations of Rapamycin (1.3-6 μM) or Torin2 (2.5-10 nM) at two-fold increments. 4 h later, cells were harvested for their analysis by western blotting. Blots are representative of 3 independent experiments confirming ATG5 depletion and its effects on SQSTM1 levels, the lipidation of LC3 as well as on Flag-Vif. ACTB was included as a loading control. (C) The LC3-II/I ratios, the SQSTM1 and Vif levels across the Rapamycin treatment range relative to DMSO were determined by densitometry analyses of western blots from 3 independent assays. SQSTM1 and Vif levels were normalized over ACTB prior to normalization to DMSO. Data represent the mean and the SEM of 3 independent experiments. Dotted lines represent 2-fold threshold. (D) The LC3-II/LC3-I ratios, the SQSTM1 and Vif levels across the Torin2 treatment range relative to DMSO were determined by densitometry analyses of western blots from 3 independent assays. Data represent the mean and the SEM of 3 independent experiments. Dotted lines represent 2-fold threshold. (E) ATG5KO HEK293T cells were transfected with NL4-3 Flag-Vif. 44 h later, cells were treated with DMSO, Torin2 (2.5 nM), Torin2 (2.5 nM) with Chloroquine (60 μM), or Torin2 (2.5 nM) with MG132 (20 μM) for 4 h. Cells were then harvested for their analysis by western blot for Flag-Vif, LC3, ATG5 and SQSTM1. ACTB was included as a loading control. Blots are representative of 3 independent experiments.
Fig 3.
NL4-3 Vpr levels are rescued in ATG5KO cells despite mTOR inhibition.
(A) HEK293T cells were transfected with 2,000 ng of an empty vector (V) or a NL4-3 Vpr-myc construct. 44 h later, cells were treated with Torin2 (5-10 nM) with or without BafA1 (500 nM) for 4 h. Next, cells were harvested, analyzed by western blot, and autophagy flux was measured by determining the LC3-II/I ratios and SQSTM1 levels relative to the vector-transfected and DMSO-treated cells. Data represent the mean and the SEM of 3 independent experiments. Blots are representative of 3 independent experiments. (B) Parental (WT) and ATG5KO HEK293T cells were transfected with 2,000 ng of a NL4-3 Vpr-myc construct. 44 h later, cells were treated with DMSO or increasing concentrations of Rapamycin (1.3-6 μM) or Torin2 (2.5-10 nM) at two-fold increments. 4 h later, cells were harvested for their analysis by western blotting. Blots are representative of 3 independent experiments from parental and ATG5KO cells confirming ATG5 depletion and its effects on SQSTM1 levels and the lipidation of LC3 as well as its impact on Vpr-myc. ACTB was included as a loading control. (C, D) Vpr and SQSTM1 levels were normalized to ACTB and their expression relative to DMSO (D on the x axis) was calculated. LC3-II and LC3-I levels were determined and provided as the LC3-II/I ratio relative to DMSO. Data represent the mean and the SEM of 3 independent experiments. Dotted lines represent 2-fold threshold.
Fig 4.
Vpr levels are rescued by autophagy inhibitors in primary cells.
(A) Primary CD4+ T cells were infected with NL4-3 Δnef in the presence of DMSO or 10 μM VPS34IN. 18 h later, cells were treated with increasing amounts of Torin2 (2.5-20 nM) for 4 h. Cells were then harvested and analyzed by western blot for LC3, SQSTM1 and Vpr. ACTB was added as a loading control. Blots are representative of 3 independent experiments. (B) Vpr and SQSTM1 levels were normalized to ACTB and their expression relative to DMSO (D on the x axis) was calculated. LC3-II and LC3-I levels were determined and provided as the LC3-II/I ratio relative to DMSO. Data represent the mean and the SEM of 3 independent experiments. Dotted lines represent 2-fold threshold.
Fig 5.
Vpr localizes to autophagosomes, and its autophagy-mediated elimination is counteracted by Nef.
(A) The subcellular distribution of Vpr relative to autophagosomes (LC3) was examined by fluorescence microscopy in HEK293T cells constitutively expressing EGFP-LC3B and treated with Torin2 (10 nM) 4 h before staining. Images are representative of 3 independent experiments. White scale bar: 10 μm. (B) HEK293T cells were co-transfected with 2,000 ng NL4-3 Vpr-myc and increasing amounts of a plasmid encoding NL4-3 Nef-HA (0-2,000 ng). 44 h later, DMSO or increasing concentrations of Torin2 (5-10 nM) were added. 4 h later, cells were harvested and analyzed by western blot. Membranes were probed for Vpr, Nef, LC3, and SQSTM1. ACTB was included as a loading control. Blots are representative of 3 independent experiments. (C) Vpr protein levels were measured by densitometry, normalized to ACTB and their expression relative to the DMSO and no Nef treatment was calculated. Data correspond to the mean and the SEM of 3 independent experiments. Dotted lines represent 2-fold threshold. (D) The degree of co-localization between Vpr and LC3 puncta was examined by fluorescence microscopy in HEK293T cells constitutively expressing EGFP-LC3B in the presence of Nef and treated with Torin2 (10 nM) 4 h before staining. Images are representative of 3 independent experiments. White scale bar: 10 μm. Graph: The Pearson’s correlation coefficient (R) for the co-localization of Vpr and EGFP-LC3B in the presence and absence of Nef was calculated from 17 randomly selected fields. Data correspond to the raw values, the mean and SEM. *: p < 0.05; **: p < 0.01; ****: p < 0.0001. ns: not significant.
Table 1.
Group M HIV-1 transmitted/founder viruses used in this study.
Fig 6.
Vpr proteins from group M HIV-1 TFVs are resistant to autophagy.
(A) HEK293T cells were transfected with 2,000 ng of constructs coding for the indicated Vpr proteins. 44 h later, either DMSO or Torin2 (at 5 and 10 nM) were added. 4 h later, cells were harvested and analyzed by western blotting. Membranes were probed for Vpr, LC3 and ACTB (as a loading control). Blots are representative of 3 independent experiments. (B) The levels of Vpr relative to the DMSO treatment (D on the x axis) were calculated by densitometry analyses after normalizing with ACTB. Data correspond to the mean and the SEM of 3 independent experiments. Dotted line represents 2-fold threshold. (C) The degree of co-localization between one of the TFV Vpr proteins (CH077) and LC3 puncta was examined by fluorescence microscopy in HEK293T cells constitutively expressing EGFP-LC3B and treated with Torin2 (10 nM) 4 h before staining. Images are representative of 3 independent experiments. White scale bar: 10 μm. Graph: The Pearson’s correlation coefficient (R) for the co-localization of CH077 Vpr and EGFP-LC3B was calculated from 25 randomly selected fields. Data correspond to the raw values, the mean and SEM.
Fig 7.
Residues responsible for Vpr’s susceptibility to autophagy cluster in the second alpha helix and C-terminus of the protein.
(A) Protein alignment of the Vpr proteins of the lab-adapted clone NL4-3 and the TFV clone CH077. Consensus sequence is shown underneath. Red squares indicate the residues that account for their different susceptibility to autophagy. (B) Left panel. Design of chimeras I and II and the I61T point mutant. Red dots are representative of amino acids that differ between NL4-3 (green background) and CH077 (orange background). Right panel: HEK293T cells were transfected with the chimeras and point mutant. As a control, cells were transfected with NL4-3 Vpr-myc. The susceptibility of these Vpr proteins to increasing amounts of Torin2 (5-10 nM) was examined by western blot. DMSO was added as a control. Membranes were probed for Myc, LC3 and ACTB. Data are representative of 3 independent experiments. The Vpr levels were normalized to ACTB and their expression relative to DMSO was calculated. The mean values from the 3 replicates are provided underneath the blots. (C-D) Design of the subsequent Vpr mutants is shown in the left panels. The right panels show the susceptibility of these Vpr mutants to autophagy. The levels of Vpr-myc over ACTB relative to the DMSO treatment is shown underneath the blots. Data are representative of 3 independent experiments. (E) Different views of the NL4-3 Vpr NMR structure where positions 37, 45, 77, 83-86 and 93-94 are highlighted in red. NTD: N-terminal domain. CTD: C-terminal domain. (F) The degree of co-localization between the autophagy-resistant VprP37Y45 mutant and LC3 puncta was examined by fluorescence microscopy in HEK293T cells constitutively expressing EGFP-LC3B and treated with Torin2 (10 nM) 4 h before staining. Images are representative of 3 independent experiments. White scale bar: 10 μm. White arrowhead shows area of co-localization. Graph: The Pearson’s correlation coefficient (R) for the co-localization of Vpr and EGFP-LC3B was calculated from 25 randomly selected fields. Data correspond to the raw values, the mean and SEM.
Table 2.
Antibody sources and conditions.
Fig 8.
Lab-Vpr but not TFV-Vpr associates with NDP52, SQSTM1 and TAX1BP1 autophagy-cargo receptors.
HEK293T cells were transfected with 2,000 ng of either Lab-Vpr or TFV-Vpr. 48 h later, cells were harvested, and lysates were immunoprecipitated for BNIP3L (A), OPNT (B), NBR1 (C), SQSTM1 (D), TAX1BP1 (E), or NDP52 (F). The pulldown fraction was analyzed for each of these autophagy receptors and Vpr. The whole cell lysates were also analyzed for these autophagy factors, Vpr and ACTB. Blots are representative of 3 independent experiments. (G) Lab-Vpr was transfected in parental, SQSTM1KO/TAX1BP1KO and SQSTM1KO/TAX1BP1KO/NDP52KO knockout cells. 44 h later, cells were treated with increasing amounts of Torin2 (5-10 nM) for 4 h. Cells were then harvested and analyzed by western blot for Vpr, SQSTM1, TAX1BP1, NDP52 and ACTB. Blots are representative of 3 independent experiments. Graph: Vpr levels were quantified by normalizing to ACTB, and their relative expression compared to WT cells treated with DMSO (D on the x axis) were calculated from 3 independent experiments. Data correspond to the mean and the SEM of 3 independent experiments. *: p < 0.05.
Fig 9.
Lab-Vpr but not TFV-Vpr co-localizes with autophagy receptors SQSTM1, TAX1BP1 and NDP52.
HEK293T cells were transfected with 2,000 ng of either Lab-Vpr (A) or TFV-Vpr (B). 44 h later, cells were treated with Torin2 (10 nM) for 4 h. Next, cells were analyzed by immunofluorescence for the subcellular distribution of Vpr (green) relative to SQSTM1, TAX1BP1 and NDP52 (red). Hoechst was used to stain the nuclei (blue). White scale bar: 10 μm. White arrowheads indicate co-localization between Vpr and the selected autophagy markers. Images are representative of 3 independent experiments. Graphs: The Pearson’s correlation coefficient (R) for the co-localization of Vpr and these receptors was calculated from 8 randomly selected fields. Data correspond to the raw values, their mean and SEM.
Fig 10.
Role of ubiquitination in targeting Vpr for autophagy-mediated degradation.
(A) HEK293T cells were transfected with 2,000 ng of Lab-Vpr. 24 h later, DMSO or TAK-243 (200 nM) was added. 44 h later, cells were treated with increasing amounts of Torin2 (5-10 nM). 4 h later, cells were harvested and analyzed by western blot. Membranes were probed for Myc, LC3 and ACTB. The LC3-II/I ratio as well as the Vpr levels relative to DMSO (D on the x axis) are provided over the Torin2 treatment range for both DMSO- and TAK-243-treated cells. Dotted line represents 2-fold threshold. Data correspond to the mean and the SEM of 3 independent experiments. (B) HEK293T cells were transfected with Lab-Vpr or the Lab-Vpr K27M mutant. 44 h later, cells were treated with DMSO or Torin2 (10 nM). 4 h later, cells were processed for western blot analyses as in panel A. Blots are representative of 3 independent experiments. (C) HEK293T cells were transfected with either Lab-Vpr or TFV-Vpr. 48 h later, cells were harvested, and lysates were immunoprecipitated (IP) for Vpr. The IP samples were analyzed by western blot by probing membranes for ubiquitin and Vpr. The whole cell lysates were analyzed for Vpr and ACTB. Blots are representative of 3 independent experiments. (D) Diagram of SQSTM1 and truncation mutants. (E) HEK293T cells were co-transfected with Lab-Vpr and the mCherry-SQSTM1 constructs. 48 h later, cells were harvested, and lysates were immunoprecipitated (IP) for Vpr. The IP samples were analyzed by western blot by probing membranes for mCherry and Myc. The whole cell lysates were analyzed for mCherry, Vpr-myc and ACTB. Blots are representative of 3 independent experiments. (F) HEK293T cells were co-transfected with Lab-Vpr and the SQSTM1 constructs shown in panel D. 44 h later, cells were treated with Torin2 (10 nM) for 4 h and were subsequently stained for the SQSTM1 constructs (mCherry, red), Vpr (green), and the nuclei (blue). White scale bar: 10 μm. Images are representative of 3 independent experiments.
Fig 11.
Vpr proteins from TFVs provide an advantage in virus spread.
(A) Experimental design for the 2D total (cell-to-cell plus cell-free) and cell-free infection assays. (B) Left panel: 2D total infection was examined by co-culturing infected MT4 cells with Jurkat LTR-GFP CCR5+ reporter cells in the presence of DMSO or Torin2 (10 nM). Right panel: 2D cell-free infection was measured in a similar manner. However, here donor (infected MT4) and recipient (Jurkat LTR-GFP) cells were separated by transwells. (C) Experimental design to measure 3D infection using the cervicovaginal epithelium model. (D) Left panel: 3D total infection was measured by infiltrating infected MT4 cells and recipient Jurkat LTR-GFP CCR5+ cells into the 3D epithelium. Right panel: 3D cell-free infection was determined by infiltrating Jurkat LTR-GFP CCR5+ into the epithelium and inoculating mCherry recombinant viruses onto the apical part of the cervicovaginal epithelium. %Infection efficacy was measured by determining the amount of mCherry+ and GFP+ cells in the Torin2 condition over the DMSO condition for each recombinant virus. Data correspond to the mean and the SEM of 3 biological replicates. *: p < 0.05; **: p < 0.01; ****: p < 0.0001; ns: not significant by One-way ANOVA. Experimental pipelines were created in BioRender (License: Serra-moreno, R. (2026) https://BioRender.com/fp56lo4).