(
A) Remodelling of the cellular phosphoproteome by HIV infection. CEM-T4 T cells from
Figure 4A and
Figure 4—figure supplement 1A were subjected to TMT-based phosphoproteomic analysis. The scatterplot displays differences in phosphopeptide abundance between WT HIV and mock-infected cells. Each point represents a single phosphopeptide, 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. Phosphopeptides downregulated (red) or upregulated (green) with a fold change > 2 and q value < 0.01 are highlighted. (
B) Functional annotation clusters enriched amongst proteins hyperphosphorylated in the presence of HIV infection. Proteins containing phosphopeptides significantly upregulated (q values < 0.01) in cells infected with WT HIV compared with mock-infected cells were analysed. Enrichment of Gene Ontology Molecular Function and Biological Process terms against a background of all identified phosphoproteins was determined using DAVID. Functional annotation clusters with enrichment scores > 1.3 (equivalent to a geometric mean of all included enrichment p values<0.05) were considered significant. Representative Gene Ontology terms are indicated. (
C) PhosFate analysis of kinase activity in HIV-infected versus mock-infected cells. Data are shown for WT HIV (upper panel) and ΔVif HIV (lower panel). A positive activity score indicates enhanced phosphorylation of kinase-specific phosphosites in infected cells. Aurora kinases
A and
B (AurA/AurB; red) and other control mitotic/checkpoint kinases (PLK1, ATR and ATM; blue) are highlighted. Kinases represented in the dataset by a single target phosphosite were excluded. (
D–
E) Comparison of phosphoproteomic dataset with previously published data for (
D) okadaic acid-treated (
Kauko et al., 2015) and (
E) kinase inhibitor-treated (
Kettenbach et al., 2011) HeLa cells. Each row shows a different pairwise comparison: top row, HIV WT versus mock; middle row, HIV ΔVif vs mock; bottom row, HIV WT vs HIV ΔVif. Each column shows a different inhibitor treatment. At low concentrations, MLN8054 is a selective AURKA inhibitor, but at 5 μM (as shown) reduced activity of AURKB and PLK1 is also observed. AZDZM indicates a combined analysis of selective AURKB inhibitors AZD1152 and ZM447439. BI2536 is a selective PLK1-3 inhibitor. Each scatterplot compares log
2 (fold change) in WT/ ΔVif/mock-infected cells (y axis) with log
2 (fold change) in inhibitor treated cells (x axis). Lines indicate linear correlation with 95% confidence areas, r
2 values and p values of a non-zero correlation. For each column, the most significant correlation is highlighted (red).