Fig. 4.

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 Fig. S4.