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. 2019 Mar 12;8:e41431. doi: 10.7554/eLife.41431

Figure 5. Vif-dependent cellular targets in primary T cells.

Figure 5.

(A) Protein abundances in WT HIV-infected vs ΔVif HIV-infected cells from single time point proteomic experiment (Figure 3A). Statistical significance (y axis) vs fold change (x axis) is shown for 8795 cellular and 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%). Highlighted groups of differentially regulated proteins are summarised in the key, including two quantitated isoforms of PP2R5C (Q13362 and Q13362-4) and FMR1 (Q06787 and Q06787-2). (B) Abundances of Vif-dependent PPP2R5 family and related proteins highlighted in a) in mock-infected (grey), WT HIV-infected (red) and ΔVif HIV-infected (green) cells from single time point proteomic experiment (Figure 3A). Mean abundances (fraction of maximum) with 95% CIs are shown. Only the canonical isoform of PPP2R5C (Q13362) is shown. (C) Activation of AURKB by Vif. CEM-T4s were mock-transduced or transduced with control (pCtrl) or Vif-expressing (pVif) lentiviruses for 48 hr (62–78% GFP+), lysed in 2% SDS and analysed by immunoblot with anti-phospho-AURK (T232), anti-total AURKB, anti-Vif and anti-α-tubulin antibodies. (D) Vif-dependent activation of AURKB. AFMACS-selected (LNGFR+) HIV-infected cells from Figure 3A (donor A) were lysed in 2% SDS and analysed by immunoblot with anti-PPP2R5D, anti-phospho-AURK (T232), anti-total AURKB and anti-Vif antibodies. Same lysates as Figure 3E. (E) Abundances of DPH7 and FMR1 in mock-infected (grey), WT HIV-infected (red) and ΔVif HIV-infected (green) primary T cells from single time point proteomic experiment (Figure 3A) and CEM-T4s (Greenwood et al., 2016). Mean abundances (fraction of maximum) with 95% CIs intervals are shown. Only the canonical isoform of FMR1 (Q06787) is shown. (F) Vif-dependent depletion of DPH7 and FMR1. CEM-T4s were mock-infected or infected with WT or ΔVif HIV-AFMACS viruses for 48 hr (77–82% LNGFR +cells), lysed in 2% SDS, and analysed by immunoblot with anti-DPH7, ant-FMR1, anti-Vif and anti-α-tubulin antibodies. (G) Depletion of DPH7, FMR1 and PPP2R5D by Vif. CEM-T4s were mock-transduced or transduced with control (pCtrl) or Vif-expressing (pVif) lentiviruses for 48 hr (86–88% GFP +cells), lysed in 2% SDS and analysed by immunoblot with anti-phospho-AURK, anti-total AURKB, anti-Vif and anti- α-tubulin (loading control) antibodies.