Figure 5. Mechanism of Vif-mediated degradation of PPP2R5A-E subunits.

(A) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. (B) CUL5-dependent degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype cullin-5 (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. (C) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP (A and C) or mCherry (B). MFI values are shown for transfected (red/green) and untransfected (blue) cells.

DOI: http://dx.doi.org/10.7554/eLife.18296.015

Figure 5—figure supplement 1. Co-immunoprecipitation of Vif and PPP2R5D.

(A) Co-immunoprecipitation in 293Ts. WT 293T cells or 293T cells stably expressing HA-tagged PPP2R5D were transfected with FLAG-tagged NL4-3 Vif, pre-treated with bortezomib (10 nM) for 16 hr, and analysed by immunoblot (IB) for HA-PPP2R5D and FLAG-Vif 48 hr post-infection (left panels). Lysates were subjected to immunoprecipitation (IP) with anti-HA (middle panels) or anti-PPP2R5D (right panels, WT 293Ts only) and re-analysed by immunoblot. 293T cells transfected with empty vector or FLAG-tagged K5 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) were included as controls. (B) Co-immunoprecipitation during HIV infection of T cells. CEM-T4 T cells were infected with NL4-3-dE-EGFP HIV at an MOI of 1.5, pre-treated with bortezomib (10 nM) for 16 hr, and analysed by immunoblot (IB) for PPP2R5D and Vif 48 hr post-infection (left panels). Lysates were subjected to immunoprecipitation (IP) with anti-PPP2R5D (right panels) and re-analysed by immunoblot. Uninfected CEM-T4 T cells and infected CEM-T4 T cells without bortezomib pre-treatment were included as controls.

DOI: http://dx.doi.org/10.7554/eLife.18296.016

Figure 5—figure supplement 2. Time course analysis of endogenous PPP2R5D during HIV infection of T cells.

(A–B) Rescue by proteasome inhibition. CEM-T4 T cells were infected with NL4-3-dE-EGFP HIV at an MOI of 1.5 and analysed by flow cytometry (A) at the indicated timepoints in the presence (bottom panels) or absence (top panels) of bortezomib (BZB) added 24 hr post-infection (+0 hr). MFI values (PPP2R5D staining) for GFP positive (infected) cells are shown, normalized to values at +0 hr (B). (C–D) Cycloheximide chase. CEM-T4 T cells were infected with NL4-3-dE-EGFP WT (top panels) and ΔVif (bottom panels) viruses at an MOI of 1.5 and analysed by flow cytometry (C) at the indicated timepoints in the presence of cycloheximide (CHX) added 24 hr post-infection (+0 hr). MFI values (PPP2R5D staining) for GFP positive (infected) cells are shown, normalized to values at +0 hr (D). As predicted, the presence of CHX inhibited the production of new Env-EGFP protein.

DOI: http://dx.doi.org/10.7554/eLife.18296.017

Figure 5—figure supplement 3. Pulse-chase analysis of endogenous PPP2R5D during HIV infection of T cells.

CEM-T4 T cells were infected with NL4-3-dE-EGFP WT or ΔVif viruses at an MOI of 1. 5 and pulsed with [³⁵S]methionine/[³⁵S]cysteine 48 hr post-infection. Cells were chased until the indicated timepoints, subjected to immunoprecipitation (IP) with anti-PPP2R5D and analysed by autoradiography.

DOI: http://dx.doi.org/10.7554/eLife.18296.018