Fig 2: Vif-mediated depletion of PP2A-B56 family members PPP2R5A-E.(A) Proteomic analysis of CEM-T4 T cells infected with WT and ?Vif HIV. Cells were infected with NL4-3-?E-EGFP viruses at an MOI of 1.5, and harvested 48 hr post-infection. Scatterplots display pairwise comparisons between WT, ?Vif 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 ?Vif 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

Fig 3: Peptide phosphorylation and phenotype analysis of proteins with altered phosphorylation site occupancy in PPP2R5D E420K-variant cells.A, Icelogo displaying enrichment and deselection of amino acids surrounding the phosphorylated residue (position 0) and (B) RegPhos analysis of kinase substrates enriched in significantly regulated, localized, single phosphorylation sites compared with all localized, single phosphorylation sites identified in the analysis. C, significantly regulated, localized, single phosphorylation sites in E420K-variant cells known or predicted to be phosphorylated by the indicated kinase. Node color (blue increased, green decreased, yellow both increased and decreased) indicates type of phosphorylation site regulation. Edges indicate known or predicted upstream kinase. D, overrepresentation analysis of human phenotype ontology data base. E, Gene ontology analysis of enriched biological processes. F, overrepresentation analysis of human KEGG pathways data.

Fig 4: PPP2R5D E420K-variant signaling. Diagram of pathway regulation by PPP2R5D containing PP2A-holoenzymes altered in E420K variant HEK-293 cells based on the data presented. Phosphorylation sites shown in blue (hyperphosphorylated) and green (hypophosphorylated) differ in the E420K variant cells as compared with wild-type cells.

Fig 5: Temporal proteomic analysis of HIV infection in primary T cells.(A) Overview of time course proteomic experiment. Control (pale grey, resting/activated/mock) and experimental (dark grey, resting/activated; red, LNGFR+, HIV-infected, selected; blue, LNGFR-, uninfected, flow-through) cells are indicated for each condition/time point. (B) Magnetic sorting of HIV-infected (red, LNGFR+, selected) cells used for (A). Corresponding uninfected (LNGFR-, flow-through) cells are shown in Figure 2—figure supplement 1A. Cells were separated using AFMACS at the indicated time points post-infection with HIV-AFMACS, stained with anti-LNGFR and anti-CD4 antibodies and analysed by flow cytometry. Mock-infected cells are shown in grey. (C) Expression profiles of illustrative restriction factors regulated by T cell activation and HIV infection (tetherin) or T cell activation alone (SAMHD1) in cells from (A–B). Relative abundances (bars, fraction of maximum) and log2(ratio)s of abundances (lines) in experimental (Expt):control (Ctrl) cells are shown for each condition/time point and coloured as in (A) (summarised in the key). (D) Expression profiles of illustrative accessory protein targets (CD4, Nef/Vpu; SERINC5, Nef; SNAT1, Vpu; APOBEC3G, Vif; PPP2R5D, Vif; UNG, Vpr) in cells from (A–B). Axes, scales and colours are as in (C). Expression profiles of other accessory protein targets are shown in Figure 2—figure supplement 1B. (E) Patterns of temporal regulation of Vpr vs other accessory protein (Vif/Nef/Vpu) targets in cells from (A–B). Log2(ratio)s of abundances in experimental (Expt):control (Ctrl) cells are shown for 45 accessory protein targets (as in Figure 2—figure supplement 3A). Colours are as in (C), and average profiles (mean, black lozenges/dotted lines) are highlighted for each group of targets. 10.7554/eLife.41431.006Figure 2—source data 1.Functional proteomic atlas of HIV-infection in primary human CD4+ T cells.Interactive spreadsheet enabling generation of temporal profiles of protein abundance for any quantitated genes of interest (‘Gene search and plots’ worksheet). Time course data (cells from Figure 2A) are presented as in Figure 2C, with relative protein abundances (fraction of maximum) for each condition depicted by bars, and log2(ratio)s of protein abundances in paired experimental/control cells from each condition/time point depicted by lines (grey, resting/activated; red, LNGFR+, infected; blue, LNGFR-, uninfected). Single time point data (cells from Figure 3A) are presented as in Figure 3D, with relative protein abundances (fraction of maximum, mean plus 95% CIs) for each condition depicted by bars (grey, mock; red, WT HIV; green, ?Vif HIV). The number of unique peptides is shown for each protein/experiment, with most confidence reserved for proteins with values > 1. For the single time point experiment, p values (unadjusted) and q values (Benjamini-Hochberg FDR-adjusted) are shown (highlighted in gold if <0.05). Complete (unfiltered) proteomic datasets (‘Time course dataset’ and ‘Single time point dataset’ worksheets) are also included.