Figure 4. Vif-mediated depletion of PP2A-B56 family members PPP2R5A-E.

(A) Proteomic analysis of CEM-T4 T cells infected with WT and ΔVif HIV. Cells were infected with NL4-3-ΔE-EGFP viruses at an MOI of 1.5, and harvested 48 hr post-infection. Scatterplots display pairwise comparisons between WT, ΔVif and mock-infected cells. Each point represents a single protein, plotted by its log₂ (fold change in abundance) versus the statistical significance of that change. q values were determined using Limma with Benjamini-Hochberg adjustment for multiple testing, with increasing −log2 (q value) indicating increasing significance. Points above the dotted line change with a q value < 0.01. HIV proteins and host proteins of interest are highlighted with different symbols (see key). (B) Depletion of PPP2R5A and PPP2R5D during HIV infection. CEM-T4 T-cells were infected with NL4-3-dE-EGFP WT and ΔVif viruses at an MOI of 1 or 10 and analysed by immunoblot (IB) 48 hr post-infection. p24 (capsid), Vif and calreticulin are included as controls. (C) Depletion of exogenous PPP2R5A by Vif. 293T cells stably expressing HA-PPP2R5A were co-transfected with GFP plus empty vector, NL4-3 Vif or NL4-3 Vif with a single amino acid mutation C114S and analysed by intracellular flow cytometry for HA 36 hr post-transfection. Histograms show GFP positive (transfected, red shading) and negative (untransfected, blue line) cells. Median fluorescence intensity (MFI) values are shown for GFP positive (red) and negative (blue) cells. (D) Depletion of PPP2R5A-E family members by Vif. 293T cells stably expressing HA-tagged PPP2R5A-E or APOBEC3G were co-transfected with GFP plus NL4-3 Vif expression vectors, and intracellular HA staining quantitated by flow cytometry 36 hr post transfection. Histograms show GFP positive (transfected, red shading) and negative (untransfected, blue line) cells. MFI values are shown for GFP positive (red) and negative (blue) cells.

DOI: http://dx.doi.org/10.7554/eLife.18296.012

Figure 4—figure supplement 1. Workflow and controls for single timepoint proteomic/phosphoproteomic experiment.

(A) Workflow of TMT-based single timepoint whole cell proteomic and phosphoproteomic experiment. CEM-T4 T cells were mock-infected or infected with NL4-3-dE-EGFP WT or ΔVif HIV at an MOI of 1.5. Cells were harvested for proteomic analysis 48 hr post-infection. Samples were subjected to (1) whole cell proteome and (2) phosphopeptide analysis. Each condition was carried out in triplicate. (B) Quantitation of infected cells from experiment described in (A). Cells were analysed by flow cytometry for CD4 and GFP expression, with infected cells losing CD4 and gaining GFP. Example flow cytometric analysis of one replicate for each condition is shown, demonstrating the % infected cells. Across all replicates, cells infected with WT or ΔVif viruses were 74–78 % infected.

DOI: http://dx.doi.org/10.7554/eLife.18296.013

Figure 4—figure supplement 2. Depletion of endogenous PPP2R5D during HIV infection of primary cells.

(A–B) CEM-T4 T cells (A) or activated primary human CD4⁺ T cells (B) were infected with NL4-3-dE-EGFP WT and ΔVif viruses at an MOI of 1 and analysed by intracellular flow cytometry for PPP2R5D 48 hr post-infection. ICAM3 is included as a control. MFI values are shown for ΔVif (blue) and WT (red) viruses.

DOI: http://dx.doi.org/10.7554/eLife.18296.014