PMC

HLTF silencing does not affect HIV-1 replication in macrophages. MDMs were treated with siRNA Ctrl or siRNA against HLTF for 24 h. The cells were then not infected (NI) or infected with either Yu-2 wt virus (WT) or Yu-2 ∆Vpr virus (∆Vpr). A second siRNA treatment was done 48 h after infection. Cell supernatants were collected at the indicated time points, and p24 levels were measured by ELISA. Error bars represent SEM of triplicates from one donor. Same results were obtained with a second donor.

(A) Efficient silencing of DCAF1 and HLTF by siRNA in cells used for cell cycle analysis. (B) Depletion of HLTF by several siRNA does not perturb cell cycle and does not inhibit Vpr-mediated G2 arrest. Western blot was performed using antibodies directed against the indicated proteins. (C) Screening of Vpr mutants for their ability to induce HLTF degradation. HeLa cells were transfected with plasmids encoding HA-Vpr or indicated mutants. Cells were lysed 2 d after transfection, and an immunoblot against relevant proteins was done. *Interesting Vpr mutants that are still able to arrest cells in G2/M but not to induce the degradation of HLTF.

(A) MT4 cells expressing doxycycline-inducible shRNA against HLTF together with RFP were obtained under puromycine selection. Percentage of RFP-positive cells expressing shRNA was determined by flow cytometry (NTd, nontransduced cells; shCtrl, cells transduced with a control shRNA; and shHLTF, cells transduced with an shRNA against HLTF). Data show a mean of three experiments ± SD. (B) The presence of Vpr or the absence of HLTF did not alter infection by HIV-1. Cells were infected with VSV-G pseudotyped NL4-3 viruses expressing or not Vpr [NI, noninfected; NL WT, cells infected with NL4-3 (VSV) expressing Vpr; and NL ∆Vpr, cells infected with NL4-3 (VSV) lacking Vpr]. Infection levels were assessed by measuring Gag expression by flow cytometry at 24 and 48 h postinfection (pi). Data show a mean of three experiments ± SD.

HLTF degradation and G2 arrest are two independent activities of HIV-1 Vpr. (A) Mus81 depletion does not trigger HLTF degradation and, conversely, HLTF depletion does not trigger Mus81 degradation. HeLa cells were treated with siRNA control or siRNA against DCAF1, HLTF, or Mus81 for 24 h. Cells were then transduced with the VLP and harvested 24 h later. Protein expression was analyzed by Western blotting. Quantification was performed: ratios between the HLTF and the GAPDH signals were calculated relative to a 100% reference indicated in red. The Western blot is representative of three independent experiments. (B) Depletion of HLTF by siRNA does not perturb cell cycle and does not inhibit Vpr-mediated G2 arrest. HeLa cells were treated as in A, and cell cycle was analyzed 24 h after VLP treatment. (C) The Vpr G56A mutant, defective for HLTF degradation, is still able to arrest the cell cycle. HeLa cell were treated with VLP [empty VLP (R−), VLP containing WT Vpr (R+), or mutants G56A, K27M, or S79A] for 24 h. Protein expression was then assessed by Western blot and quantification performed as in A and the cell cycle analyzed (D).

HIV-1 Vpr down-regulates HLTF in a DCAF1-dependent manner. (A) HLTF expression in HeLa cells is down-regulated by VLP-encapsidated WT HIV-1 Vpr. Cells were transduced with the VLP used for SILAC. After 24 h, cells were harvested and fractioned. Protein expression was analyzed by Western blotting. (B) HLTF expression in Jurkat T cells is down-regulated by VLP-encapsidated WT HIV-1 Vpr. Cells were treated 48 h with VLP and lysed, and whole cell extracts were analyzed by Western blot. (C) Endogenous and exogenous HLTF expression levels are reduced by VLP-encapsidated WT HIV-1 Vpr. HeLa cells were cotransfected with a vector expressing Flag-HLTF or an empty vector together with a transfection control vector encoding the GFP (ratio10:1). Twenty-four hours after transfection, cells were transduced with the VLP and were harvested 24 h later. Protein expression was analyzed by Western blotting. (D) HLTF depletion induced by HIV-1 Vpr is proteasome-dependent. HeLa cells were treated with or without MG132 during 6 h after VLP incubation. Cells were transduced with the different VLP: empty VLP (R−), VLP containing wt Vpr (R+), or VLP containing Vpr K27M or Vpr S79A. (E) Vpr hijacks DCAF1 to mediate HLTF degradation. HeLa cells were treated with siRNA Control or siRNA against DCAF1 for 24 h. Cells were then transduced with the VLP and harvested 24 h later. Protein expression was analyzed by Western blotting. In each panel, quantification was performed: ratios between signals were calculated relative to a 100% reference indicated in red. All Western blots are representative of three independent experiments except B, which corresponds to a direct analysis of cells used for the SILAC.

HLTF degradation induced by HIV-1 Vpr precedes G2/M arrest. (A) Kinetic of HLTF disappearance. HeLa cells were transduced with empty VLP or WT Vpr VLP for 2 h. Cells were harvested 6, 12, 24, and 48 h after VLP treatment, and whole cell lysates were analyzed by Western blot. Quantification was performed: ratios between the HLTF and the GAPDH signals were calculated relative to a 100% reference indicated in red. The Western blot is representative of two independent experiments. (B) Kinetic of Vpr-mediated G2 arrest. Cell cycle analysis after VLP treatment, same time points as in A. (C) Short time kinetic of HLTF disappearance. HeLa cells were transduced with empty VLP (R−), VLP containing WT Vpr (R+), or Vpr mutants K27M or S79A. Cells were harvested at time 0 min, 30 min, 2 h, and 4 h after the 2-h VLP treatment, and whole cell lysates were analyzed by Western blot as in A. Quantification was performed as in A. (D) HLTF degradation precedes Vpr-mediated cell cycle arrest. Cell cycle analysis performed after VLP treatment at the indicated time points (same as in C) for the 6-h time point: 6(−) indicates with no MG132 treatment and 6(+) with MG132 all along the kinetic.

HLTF degradation is induced by HIV-1 Vpr in infected T cells and HeLa cells. (A) HLTF is degraded in MT4 cells by HIV-1 viruses expressing WT Vpr (Left), in Jurkat T cells (Center), and in HeLa cells (Right). MT4 cells were transduced with lentiviruses expressing shRNA against HLTF and cultured in the presence of doxycycline for 3 d. Cells were then either not infected or infected with HIV-1 NL4.3 WT or Vpr-deleted viruses (∆Vpr) for 48 h. Jurkat and HeLa cells were either not infected or infected with NL4.3 WT or Vpr- deleted viruses (∆Vpr). Cells were lysed 48 h after infection, and cell lysates were analyzed by Western blot. (B) Vpr from HIV-1 VLP triggers HLTF degradation in macrophages. Monocyte-derived macrophages were differentiated for 4 or 7 d and then exposed to HIV-1 VLP containing or not Vpr for 24 h [empty VLP (R−), VLP containing WT Vpr (R+)]. Cells were then lysed, and HLTF expression was analyzed by Western blot. In each panel, quantification was performed: ratios between signals were calculated relative to a 100% reference indicated in red. All Western blots are representative of three independent experiments. (C) HLTF levels are reduced in macrophages following infection with the YU2 WT virus, in comparison with the ∆Vpr virus. Monocyte-derived macrophages (MDMs) were treated with siRNA control or siRNA against HLTF for 24 h. Cells were then infected with either YU2 WT virus or YU2 ∆Vpr virus (∆Vpr). Analysis of HLTF and GADPH expression was done in extracts collected at day 16 after infection. The kinetic of replication is shown .

Setup and results of the SILAC experiment. (A) Experimental procedure for SILAC-based strategy. HeLa cells were cultured in medium containing light, medium, or heavy isotopes for 30 d. Cells were then transduced for 2 h with empty VLP, VLP containing Vpr S79A or Wt Vpr. Two independent biological replicates were performed named Silac (S1) and Switch (S2), in which isotopic conditions were inverted. At 12 h, treated cells were mixed in 1:1:1 ratio, separated into cytoplasmic and nuclear fractions, and subjected to proteomics experiments. (B) Vpr expression in HeLa cells treated with the VLP used for SILAC. HA-Vpr expression level was checked by Western blot. (C) WT Vpr-containing VLP used for SILAC trigger a G2 arrest. An aliquot of cells used for SILAC was fixed and then stained with propidium iodide, and DNA content was monitored by flow cytometry. The histogram displays the ratio between cells in G2/M and cells in G1 phases. (D) Identification of a new Vpr target by SILAC. The values were normalized taking as 100% the mock condition treated with empty VLPs. Two experiments were performed: Silac (S1) and Switch (S2), the values displayed for down- and up-regulated proteins by HIV-1 Vpr are the means of S1 and S2 ± SD. Only proteins with at least 20% variation are shown. *Group of peptides shared by several proteins. (E) Representation of significantly down- and up-regulated proteins by HIV-1 VprS79A compared with the mock condition (empty VLP). The values displayed for down- and up-regulated proteins by HIV-1 Vpr are the means of S1 and S2 ± SD. Only proteins with at least 20% variation are shown.