Fig 1: Proteins regulated by HIV in primary T cells.(A) Overview of single time point proteomic experiment. HIV-infected (LNGFR+) primary T cells were isolated using AFMACS 48 hr post-infection with WT (red) or ΔVif (blue) HIV-AFMACS. (B) AFMACS-based enrichment of WT (red circles) and ΔVif (blue triangles) HIV-infected (LNGFR+) cells used for (A), with means and 95% CIs. Corresponding cells pre-selection are included for each donor/virus (WT, grey circles; ΔVif, grey triangles). Cells were stained with anti-LNGFR and anti-CD4 antibodies and analysed by flow cytometry, with representative data in Figure 3—figure supplement 1A. (C) Protein abundances in WT HIV-infected vs mock-infected cells from (A). Volcano plots show statistical significance (y axis) vs fold change (x axis) for 8789 cellular and six viral proteins quantitated in cells from all three donors (no missing values). Proteins with Benjamini-Hochberg FDR-adjusted p values (q values) < 0.05 or > 0.05 are indicated (FDR threshold of 5%). Proteins highlighted in each plot are summarised in the key. Vpr/Vif/Nef/Vpu substrates (green circles) comprise proteins from Figure 2C–D and Figure 2—figure supplement 1B, excluding negative controls (SAMHD1, APOBEC3B, SERINC1, HLA-C) and HLA-A/B alleles (different in each donor) but including SMUG1 (not identified in time course proteomic experiment) (Schröfelbauer et al., 2005) and both quantitated isoforms of PP2R5C (Q13362 and Q13362-4) and ZGPAT (Q8N5A5 and Q8N5A5-2). Additional Vpr substrates (gold circles) and Vpr-dependent changes (gold crosses) comprise recently described direct and indirect Vpr targets (Greenwood et al., 2019). HIV-dependent changes only identified in primary T cells (red circles and crosses) comprise proteins with q < 0.05 either not identified or not concordantly regulated by HIV in CEM-T4s (Greenwood et al., 2016) (and exclude known accessory protein-dependent changes). Further details on comparator datasets used in this figure are provided in the Materials and methods. (D–E) Abundances of ARID5A and PTPN22 in mock-infected (grey), WT HIV-infected (red) and ΔVif HIV-infected (green) primary T cells from (A). Mean abundances (fraction of maximum) with 95% CIs are shown (D). As well as proteomic analysis, cells from donor A were lysed in 2% SDS and analysed by immunoblot with anti-ARID5A, anti-PTPN22, anti-Nef and anti-α-tubulin antibodies (E). Same lysates as Figure 5D. 10.7554/eLife.41431.011Figure 3—source data 1.Proteins regulated by HIV and/or control lentivectors.Interactive filter table summarising proteomic data for proteins significantly regulated by HIV (‘q < 0.05_WT HIV (n = 650)’ worksheet) and/or control lentivectors (‘q < 0.05_ctrl lentivectors (n = 37)’ worksheet). Log2(ratio)s and q values (Benjamini-Hochberg FDR-adjusted) from the single time point proteomic experiment (Figure 3A) and SBP-ΔLNGFR control proteomic experiment (Figure 3—figure supplement 4A) are included, with q values < 0.05 highlighted in red. Where known, mechanisms underlying HIV-dependent proteins changes are shown, with proteins colour-coded to match the volcano plots in Figure 3C and pie chart in Figure 3—figure supplement 3B (green, controls/known accessory protein targets; gold, novel Vpr targets/Vpr-dependent changes [Greenwood et al., 2019]); red, novel/uncharacterised changes). NaN, protein not detected.