Phospholipase D1 Couples CD4⁺ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication

<div><p>Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their <i>de novo</i> synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for <i>de novo</i> dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.</p></div

PLD1 inhibition decreases the c-Myc-dependent dNTP biosynthetic transcriptional program.

<p>(A) Western blot analysis of protein expression in whole cell lysates prepared from resting primary human CD4+ T cells that were pretreated for 24h with DMSO vehicle, 10 or 5 μM of PLD1i, 50 or 25 μM of c-Myci, 10 μM U0126, or 100 nM of rapamycin. Cells were either left resting or stimulated with 5μg/ml PHA and 20 U/ml IL2 in the continued presence of DMSO or inhibitors for 48h before cell harvest. (B) RRM2 mRNA levels in resting primary CD4+ T cells pretreated for 24h with DMSO vehicle, 100 μM c-Myci, or 10 μM PLDi, then left resting or stimulated as in (A) as determined by quantitative real-time PCR. mRNA abundance in mock controls was set to1. Error bars represent standard error from the mean of triplicate samples. Data are representative of experiments from three independent donors. UD; undetectable levels of mRNA. (C) Resting primary human CD4+ T cells were transfected via nucleofection with siRNAs targeting PLD1 or c-Myc. Cells were allowed to recover for 24 h and then stimulated for 48 h with anti-CD3/anti-CD28 beads, harvested and western blot analysis performed as in (A). (D) In activated CD4+ T cells, PLD1 is shown to regulate c-Myc through ERK or mTOR-dependent mechanisms. This results in the increased expression of genes essential for uptake of amino acids (box 1), glucose (box 2), and synthesis of nucleotides (box 3).</p

PLD1 activity is required for normal expression of nutrient transporters in activated T cells.

<p>(A) Cell surface expression of glucose transporter Glut1 and activation marker CD25 as determined by flow cytometry on resting primary CD4+ T cells (shaded histogram) or after stimulation with anti-CD3/anti-CD28 beads for 48h in the absence (solid line) or presence of PLD1i (dashed line). (B) Western blot analysis of protein expression in whole cell lysates from primary CD4+ T cells treated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1A</a>. (C) Q PCR analysis of expression of target genes in primary CD4+ T cells nucleofected with siRNA and then stimulated for 24 h as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1C</a>. T cells were nucleofected with non-targeting (NT) (black bars), c-Myc (grey bars), or PLD1 (open bars)-specific siRNAs. mRNA abundance in NT siRNA sample was set to1. Error bars represent standard error from the mean of triplicate samples. Data are representative of experiments from three independent donors.</p

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.</p

Inhibition of PLD1 activity limits proliferation of activated CD4+ T cells.

<p>(A) Schematic showing the different cell cycle phases detected by staining DNA and RNA with 7-AAD and Pyronin Y, respectively. (B) Cell cycle analysis of primary CD4+ T cells after 72h stimulation with anti-CD3/anti-CD28 beads in the presence or absence of 10 μM PLDi by staining with Pyronin Y and 7-AAD followed by flow cytometry. (C) Flow cytometric proliferation analysis of CellTRACE Violet-labeled CD4+ T cells pretreated with 50 μM of Myci, 5 or 10μM of PLDi after stimulation with anti-CD3/anti-CD28 beads for 72h in the presence or absence of inhibitors. The number of cellular divisions present in treated cultures is indicated above each peak.</p

PLD activity is essential for dNTP pool expansion in activated CD4+ T cells.

<p>(A-C) The levels of intracellular dATP (A), dCTP (B), and dTTP (C) were determined by mass spectrometry in extracts of primary resting cells or those stimulated by PHA/IL2 as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004864#ppat.1004864.g001" target="_blank">Fig 1</a> in the presence or absence of 10μM of PLD1i or 1mM HU for 48h. Error bars represent standard deviation from the mean of triplicate samples. Data are representative of experiments from three independent donors. Statistical significance was determined by two-tailed Student’s <i>t</i> tests. *, <i>P</i> < 0.05.</p