Inhibition of PLD1 activity in activated T cells restricts HIV-1 infection in a dNTP-dependent fashion.

<p>(A) Resting primary CD4⁺ T cells were treated with 10μM PLD1i for 24 h then stimulated for 48 h with 5μg/ml PHA and 20 U/ml IL2. Cells were subsequently infected with a X4-envelope-pseudotyped HIV-1 expressing GFP. Cells were cultured in the continued presence of inhibitor. Three days post-infection, cells were analyzed for GFP expression by FACS. Where indicated, cells were treated with exogenous 50μM deoxyribonucleosides (dN) 6 h before infection. Means and SDs of triplicate samples for four independent donors are shown. Statistical significance was determined by two-tailed Student’s <i>t</i> test. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001 when PLD1i-treated samples are compared with DMSO or PLD1i + dN-treated compared to PLD1i. (B) Primary CD4+ T cells were treated and infected with X4-envelope-pseudotyped GFP-reporter-expressing HIV-1(HIV-1-GFP) as in (A) and total DNA harvested 24 h after infection. Viral cDNA (ERT, LRT, or 2-LTR circles) was quantified by qPCR. Means and SDs of triplicate samples are shown. Data are representative of three independent donors. (C) Kinetics of reverse transcription and 2-LTR formation was analyzed in CD4+ T cells pre-treated with 10μM PLD1i, 50μM Myci, or DMSO vehicle for 24 h then stimulated and infected with HIV-1-GFP as in (A). Where indicated, cells were treated with 50μM deoxyribonucleosides (dN) or 10μM AZT 6 h before infection. Total DNA was harvested at 8, 16, and 24 h after infection and viral cDNA was quantified as outlined in (B). Means and SDs of triplicate samples are shown. Data are representative of two independent donors. (D) Resting primary CD4⁺ T cells were treated with 10μM PLD1i or DMSO vehicle for 24 h then stimulated and infected with HIV-1-GFP as in (Fig 5A). Where indicated, cells were treated with 50μM deoxyribonucleosides (dN) or 10μM AZT 6 h before infection. Total DNA was harvested at 24 h after infection and viral cDNA was quantified as outlined in (Fig 5B). Means and SDs of triplicate samples are shown. Data are representative of two independent donors.