Pseudotyped HIV-1 infects epithelial cells derived from vagina.

<p>Immunofluorescence analysis of HIV-1 infection in VK-2. VK-2 cells were infected with progeny virus from primary CD4+ T cells or CEMX174 and were stained for HIV-1 Gag expression 5 days post-infection. The input virus used to infect producer T cells are as indicated. An enlarged view of epithelial cells exposed to progeny virus from HIV-1/XMRV co-infected CEMX174 cells is shown (second panel). HIV-1 Gag expression is indicated by green fluorescence (FITC); green fluorescence merged to the corresponding bright field image is shown in the bottom panels. B. Target epithelial cells were exposed to progeny viruses in the presence or absence of AZT as indicated on the panels. Data shown are representative of six independent experiments. Bar = 10 µm. <b>C</b> and <b>D</b>, HIV-1 and XMRV viral RNA in the supernatants of the same infected VK-2 cells as in (A) and (B) were quantified by qRT-PCR. The viruses used to infect producer cells are shown on the X axis. The data shown represent the mean ± standard deviation from three independent experiments.</p

Pseudotyped HIV-1 infects primary endocervical epithelial cells.

<p>(<b>A, B</b>) Visualization of HIV-1 infection in primary endocervical epithelial cells by dual immunofluorescence staining with FITC-anti-HIV-1 Gag and anti-CK19 MAbs. Primary endocervical epithelial cells were exposed to progeny viruses from infected CEMX174 cells and immunofluorescence staining was performed 5 days post-infection. Epithelial cells exposed to virus in presence of AZT or anti-MLV polyclonal sera diluted 1∶300 are shown as indicated (<b>B</b>, left two panels). HIV-1 Gag fluorescence is shown as green and CK19 as red. The corresponding bright field images are shown on the right (<b>A</b>) or at the bottom (<b>B</b>). HIV-1 Gag staining merged with CK19 staining and enlarged views of HIV-1 infected cells are shown in (A). <b>C and D:</b> XMRV RNA (<b>C</b>) and HIV-1 RNA (<b>D</b>) in supernatants from the same infected primary endocervical epithelial cells from (<b>A</b>) and (B) above were quantified by qRT-PCR. The input virus used to infect producer CEMX174 cells and the treatments are indicated on the X-axis of the graphs (pAb 1∶300 = anti-MLV polyclonal sera at dilution 1∶300; Control serum = normal goat serum diluted 1∶300; No serum = culture medium control). **, p<0.001, control serum vs indicated treatments. The data shown represent the mean ± standard deviation from three independent experiments. <b>^a</b>, HIV-1 and XMRV from CEMX174 cells infected with each virus alone were quantified and mixed at a ratio equal to the ratio of the two viruses in the progeny virus from HIV-1 and XMRV co-infected CEMX174 cells. The virus mixture was then inoculated onto primary endocervical epithelial cells and immunostaining and qPCR was performed exactly as described for progeny virus from co-infected CEMx174 cells.</p

LNcap epithelial cell line is susceptible to infection by HIV-1 co-produced with XMRV.

<p><b>A.</b> Visualization of HIV-1 infection in epithelial cells using immunofluorescence staining with anti-HIV Gag monoclonal antibody. LNcap cells were exposed to culture supernatants from CEMX174 cells infected with indicated viruses. Cells were fixed and stained for HIV-1 Gag expression 5 days after exposure to the viral supernatants. Green fluorescence (FITC) indicates HIV-1 Gag expression. Green fluorescence merged with corresponding bright field images are shown in the bottom panels. An enlarged view of cells exposed to HIV-1 co-produced with XMRV is shown in the second panel from the left. <b>B.</b> AZT inhibits HIV-1 infection of epithelial cells. LNcap cells and progeny viruses from HIV-1/XMRV co-infected CEMX174 cells were pretreated with AZT or with medium alone. The virus preparations were then added to the cells and the input virus was removed after 24 hrs. The cells were then cultured for an additional four days in medium alone or medium containing AZT as outlined in Materials and Methods. The cells were then fixed and stained for HIV-1 Gag expression. Cells infected in the absence of AZT are shown on the left. The corresponding bright field image is shown in the bottom panels. Bar = 10 µm. Data are representative of 5 independent experiments. <b>C.</b> HIV-1 RNA in the supernatants of the same LNcap cells from (<b>B.</b>) infected in the presence or absence of AZT was quantified by qRT-PCR. <b>D.</b> XMRV RNA in the supernatants of the infected LNcap cells was quantified by qPCR. The input viruses were supernatants from CEMx174 cells infected as indicated. For (C) and (D) the data shown represent the mean ± standard deviation from five independent experiments. **p<0.001, infection in the absence vs presence of AZT.</p

CEMx174 cells support both XMRV and HIV-1 replication.

<p><b>A.</b> Supernatants from CEMx174 cells infected with the viruses indicated were collected 3 days post-infection and subjected to Western blot analysis with goat anti-serum against MLV Gag (top panel) or with an anti-HIV-1 Gag mAb (bottom panel). Data are representative of 5 independent experiments. <b>B.</b> Supernatants from CEMx174 cells infected with the viruses indicated were subjected qPCR to quantify XMRV and HIV-1 RNA genomes. Values represent an average of 5 experiments.</p

Inhibition of HIV-1 infection of epithelial cells by neutralizing anti-MLV polyclonal antiserum.

<p><b>A.</b> Anti-MLV polyclonal antibody neutralizes XMRV infection in a dose-dependent manner. Progeny viruses from CEMX174 cells infected with HIV-1 and/or XMRV were pre-incubated with the indicated dilutions of anti-MLV polyclonal serum at 37°C for 1 hr before addition to LNcap epithelial cells. XMRV released into the supernatants of infected LNcap cells was quantified by qRT-PCR. pAb = Anti-MLV polyclonal antibody; Con. Serum = normal goat serum diluted 1∶100. The input viruses used to infect producer CEMx174 cells are indicated at the bottom. The data shown represent the mean ± standard deviation from three independent experiments. <b>B.</b> Immunofluorescence analysis of HIV-1 infection in LNcap cells in the presence or absence of anti-MLV neutralizing antibody. The same LNcap cells infected with progeny viruses from HIV-1/XMRV co-infected cells (shown in A) in the presence of indicated dilutions of anti-MLV neutralizing pAb or control serum were stained for HIV-1 Gag (green). The corresponding bright field image for each panel is shown on the right. Bar = 10 µm. Images are representative of three independent experiments. <b>C.</b> HIV-1 viral RNA in supernatants of from the same infected LNcap cells in (B) was quantified by qRT-PCR. The input virus used to infect producer cells and the dilutions of neutralizing or control sera are indicated. The data shown represent the mean ± standard deviation from three independent experiments. *, p<0.05; **, p<0.001, control serum vs anti-MLV pAb.</p

Pseudotyped HIV-1 infects primary vaginal squamous epithelial cells.

<p>(<b>A, B</b>) Dual immunofluorescence staining with FITC-anti-HIV-1 Gag and anti-CK19 MAbs was performed on primary vaginal squamous epithelial cells which were exposed to progeny virus from infected CEMX174 cells. HIV-1 Gag is shown as green and CK19 is shown as red. Corresponding bright field images are also shown as indicated. The input viruses used to infect producer CEMX174 cells is indicated on the panels. Epithelial cells exposed to progeny virus in the presence of AZT or anti-MLV polyclonal sera diluted 1∶300 are shown in the left two columns in <b>B</b>.</p

Pseudotyped HIV-1 infection of primary endocervical cells via cell-associated virus.

<p>Primary endocervical epithelial cells were co-cultured for two days with an equal number of mitomycin C-treated CEMX174 cells infected with HIV-1 alone, XMRV alone or co-infected with both viruses. After washing to remove non-adherent cells, dual immunofluorescence staining with FITC-anti-Gag and anti-CK19 MAbs was performed on the adherent primary endocervical epithelial cells on day 5. Merged images confirmed that HIV-1 infected cells were epithelial cells. Primary endocervical epithelial cells were co-cultured with (A) HIV-1/XMRV co-infected CEMX174; (B) same as (A) in presence of AZT; (C) HIV-1 infected CEMx174; (D) XMRV infected CEMx174; (E) mock infected CEMx174.</p

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

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