Models for <i>trans</i>-Infection of CD4 T cells by DCs.

<p>After capture by DCs, HIV-1 virions are either internalized or remain at the cell surface, possibly at the tips of dendrites or within extensively folded invaginations of the plasma membrane. A. In the prevailing Trojan horse model of HIV <i>trans</i>-infection, <i>trans</i>-infection is primarily mediated by internalized virions. B. In the new model, surface-bound HIV-1 virions are mainly responsible for <i>trans</i>-infection. Virions possibly surf the surface of the DCs on lipid rafts that collect at the infectious synapse to promote effective delivery to interacting CD4 T cells.</p

Variable Effects of Protease Treatment on HIV <i>trans</i>-Infection.

a<p>Controls used to assess the effectiveness of proteolytic digestion in removing surface-bound virions.</p

PKC/IKK inhibition strongly reduces prostratin/ionomycin-induced latent HIV transcription in primary CD4+ T cells.

<p><i>A</i> and <i>B</i>, evaluating inhibitors of relevant pathway in suppressing NL4-3 luciferase reporter activities in latently infected primary CD4+ T cells. <i>A,</i> latently infected cells pretreated with DMSO (white and black bars), calcineurin inhibitors CsA (dark gray bar) or FK-506 (light gray bar) were incubated in medium that contained no or suboptimal dosages of prostratin (0 to 100 nM, white bars), or contained both prostratin and 1.5 µM ionomycin (black, dark and light gray bars) for 48 h. Either calcineurin inhibitors inhibited the dose-dependent synergistic effect between suboptimal dosages of prostratin and ionomycin on luciferase reporter activities. <i>B,</i> latently infected cells pretreated with DMSO (white and black bars) or various kinase/phosphatase inhibitors (dark, medium and light gray bars) were incubated in media alone (white bar), or media containing anti-CD3/anti-CD28 Dynabeads (1∶1 ratio), or prostratin, in the presence or absence of 1.5 µM ionomycin as indicated for 30 h. All inhibitors suppressed reporter activities induced by TCR crosslinking or prostratin/ionomycin, which involved induction of calcium/calcineurin signaling, but CsA was ineffective against reporter activities induced by prostratin alone. In both <i>A</i> and <i>B</i>, the RLU were normalized based on total protein present in the various cell lysates. All stimulations were performed in triplicate with error bars representing ± standard deviation. Results are representative of experiments performed with cells from three independent donors. <i>C</i>, analysis of prostratin/ionomycin-induced IκBα degradation in primary CD4 cells. PBMC-purified CD4 cells were treated with DMSO or 500 nM CsA for 2 h, stimulated with a suboptimal dose of prostratin at 100 nM in the presence or absence of 1.5 µM ionomycin for 10 min and whole-cell lysate was prepared. Immunoblotting analyses revealed that CsA effectively reduced the effect of stimulus-coupled degradation of cytoplasmic IκBα.</p

Calcium/calcineurin signaling synergizes with prostratin to antagonize latent HIV-1 by targeting RelA to LTR.

<p><i>A and B,</i> dose-response analyses of LTR-driven GFP and κB-driven DsRed2 reporter activities. 5A8 cells were pretreated with DMSO (black and white bars) or 500 nM CsA (gray bars) for 2 h, followed by stimulation with PMA or prostratin only (black bars), or together with 2 µM ionomycin (white and gray bars) for 24 h at the concentration of the phorbol ester as indicated before FACS analysis. Values are mean ± standard deviation from one representative experiment. CsA potently suppressed the synergistic effect of ionomycin with PMA or prostratin and reverted back the activities of DsRed2 and GFP reporters to PMA- or prostratin-only levels. <i>C,</i> ChIP analysis of RelA recruitment to HIV-1 LTR. 5A8 cells were pretreated with DMSO or 500 nM CsA, followed by stimulation with 200 nM prostratin alone or together with 2 µM ionomycin for times indicated. Cells were fixed and subjected to chromatin immunoprecipitation with anti-RelA antibodies. Enrichment of LTR-chromatin in anti-RelA immunoprecipitates was expressed as a percentage of input chromatin. Ionomycin synergized with suboptimal dosage of prostratin to promote robust to substantial RelA occupancy at HIV-1 LTR at 30 min and 2 h, respectively. CsA effectively diminished RelA recruitment.</p

Analyses of reactivation profiles in latently infected transitional memory and central memory CD4 T cells.

<p>(A) CD4+CD45RO+ cells were purified by single step negative selection and HIV latency was established in these cells as described above. Cells were plated in 96-well plates and stimulated with 200 nM PMA with 1.5 µM ionomycin, anti-CD3+anti-CD28 beads (ratio 1∶1), 62.5 ng/ml IL-7, 10 µM prostratin, or left unstimulated for 24 h. Cells were stained with CD45RA-APC-H7, CCR7-PE-Cy7, CD27-APC, and CD45RO-FITC (NL4-3 mCherry:Luc) or CD45RO-PE (NL4-3 GFP), and analyzed for receptor expression and viral reporter expression. To obtain fold stimulation ratios, data were normalized as the percentage of cells expressing the viral reporter with the indicated stimulation divided by the % cells expressing the viral reporter in the absence of stimulation. Data shown represent an average of results obtained from four independent donors for each viral construct. Error bars represent +/− SEM. (B) CD4+CD45RO+ cells (upper panel) were sorted for CCR7+CD27+ central memory cells (T_CM) and CCR7-CD27+ transitional memory cells (T_TM). Cells were cultured for 2 days and then infected by spinoculation of NL4-3 mCherry:Luc. At the time of infection, cells were analyzed by flow cytometry for receptor expression to determine the relative levels of CCR7 expression in each sorted population (lower panel). (C) Latently infected cells were either left unstimulated or stimulated for 30 h with the indicated inducers. Two independent donors are shown, and fold change was determined as described above for luciferase levels.</p

Establishing postintegration HIV-1 latency in primary CD4 T cells and reactivating virus.

<p>(A) Production of a primary cell model of HIV-1 latency. Total CD4 T cells were isolated from PBMC with a single-step negative-selection procedure with magnetic beads to remove unwanted cell subpopulations. Within 24 h, isolated cells were spinoculated at 1200× <i>g</i> for 2 h at room temperature with viral supernatants corresponding to NL4-3 GFP IRES Nef, NL4-3 Luciferase, or NL4-3 mCherry:Luciferase viruses as schematically depicted in (B). After spinoculation, cells were placed in medium containing 5 µM saquinavir and cultured for 3 days. Cells are then counted and plated in 96-well plates in medium containing 30 µM raltegravir and various stimulators. (C) Reactivation profiles of cells latently infected with NL4-3 GFP IRES Nef, NL4-3 Luciferase, or NL4-3 mCherry:Luciferase. Latently infected cells were cultured with medium alone or medium containing 200 nM PMA and 1.5 µM ionomycin and harvested after 24 or 48 hours of culture. GFP- or mCherry-expressing cells were quantified by flow cytometry, and the percentage of GFP⁺ or mCherry cells was calculated based on uninfected controls. Luciferase levels are reported as relative light units (RLU) and have been normalized to total protein content in cell lysates to control for different cell proliferation rates. All samples were analyzed in triplicate with error bars representing +/− SD. Results are representative of those obtained in analyses of at least 10 independent donors with each virus. (D) Flow cytometric gating and analysis of cells latently infected with NL4-3 GFP IRES Nef or NL4-3 mCherry:Luciferase 24 or 48 hours after stimulation with PMA and ionomycin. Forward scatter versus side scatter plots show cells infected with virus and left unstimulated or stimulated for 24 h.</p

Summary of synergistic activity of inducer combinations.

<p>Values are the calculated synergistic index of the inducers when used in combination versus when used as single agents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030176#pone.0030176-Blazkova1" target="_blank">[44]</a>. Each value represents the highest synergistic index value obtained for a given donor and time period of simulation over a range of six dose combinations (prostratin) or four dose combinations (HMBA). Combinations of the following dose concentrations were used: prostratin (0.1, 1, 10 µM); HMBA (0.5, 5 mM); TSA (0.1, 1 µM); SAHA (1, 10 µM); VPA (0.1, 1 mM). The index of synergism was calculated with the following formula: the luciferase value from cells after stimulation with the indicated combination of inducers divided by the sum of the luciferase values from cells after stimulation with each inducer separately. Background luciferase values from unstimulated samples were subtracted prior to synergistic index calculation. Combinations of given inducers that gave a synergistic index >1 are considered synergistic and shown in bolded text.</p

CsA reduces PKC-induced NF-κB/RelA activation and LTR-transcription in 5A8 cells.

<p><i>A, in vitro</i> kinase assay of IκBα phosphorylation by IKK. 5A8 cells were pretreated with DMSO or 500 nM CsA for 2 h, stimulated with 20 nM PMA and 2 µM ionomycin for the indicated times, lysed, and <i>in vitro</i> kinase assays was performed using glutathione-S-transferase IκBα (1–62) as the substrate as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077749#pone.0077749-Williams1" target="_blank">[10]</a>. CsA delayed the degradation of endogenous IκBα induced by PMA/ionomycin, which correlated with reduced phosphorylation of GST-IκBα by immunoprecipitated IKK-α. <i>B</i>, analysis of PMA/ionomycin-induced IκBα degradation and RelA nuclear translocation. 5A8 cells were treated with DMSO or 500 nM CsA for 2 h, stimulated with 20 nM PMA and 2 µM ionomycin, and fractionated into nuclear and cytoplasmic extracts. Immunoblotting analyses revealed that CsA interfered with complete degradation of cytoplasmic IκBα at 30 min and reduced its reappearance at 60 min. CsA also reduced the first and second rounds of nuclear NF-κB/RelA expression at 30 min and 120 min. <i>C,</i> characterization of 5A8-κB-DsRed2 cells. Cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077749#pone-0077749-g002" target="_blank">Figure 2C</a>. Unlike 5A8-NFAT-DsRed2 cells, PMA alone induced both κB-dependent DsRed2 and LTR-driven GFP reporter activities (<i>panel 2</i>). Combined PMA/ionomycin stimulation further enhanced the activities of both reporters (<i>panel 3</i>), which were partially suppressed by CsA to levels similar to those after stimulation with PMA only (<i>panel 4</i>). <i>D,</i> effects of RelA knockdown on expression of κB-dependent DsRed2 and LTR-driven GFP. 5A8-κB-DsRed2 cells were nucleofected with negative control siRNA (<i>panels 1 and 3</i>) or siRNA against RelA (<i>panels 2 and 4</i>) twice within 48 h, followed by drug and antibody treatments as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077749#pone-0077749-g002" target="_blank">Figure 2d</a>. RelA knockdown suppressed both κB-DsRed2 and LTR-driven GFP reporter activities (<i>panels 2 and 4</i>).</p

Latently infected cells harbor integrated proviruses.

<p>(A) Cells were cultured in the presence (pre-treatment) or absence (no pre-treatment) of 30 µM raltegravir that was added immediately after spinoculation. After 3 days, cells were washed and stimulated with PMA+ionomycin in the presence or absence of 30 µM raltegravir (pre-activation). Results are representative of data obtained using three independent donors with each reporter virus. Error bars represent +/−SD of triplicate experiments. (B) CD4 T cells were isolated and pre-treated with 30 µM raltegravir, 250 nM AMD3100, 100 nM Efavirenz, or medium alone for 30 min before spinoculation. Cells were spinoculated and then cultured in the presence or absence of each antiretroviral drug. Three days after spinoculation, total DNA was isolated from the cells, and levels of HIV integration were determined by Alu-gag qPCR (left panel) or levels of total HIV DNA were determined by gag qPCR (right panel). Viral integration levels were compared in cultures incubated in medium alone versus in the presence of raltegravir (RTGR), AMD3100 (AMD), or Efavirenz (EFV) antiviral drugs to confirm the specificity of the assay. Data shown represent an average of six replicate PCR samples +/− SD. Data are presented as the number of copies of HIV DNA per 100 cells. (C) Three days after spinoculation, cells were either lysed for DNA isolation or stimulated with PMA+ionomycin for 24–72 hours. Peak GFP expression data are shown as the mean of three replicate samples. HIV integration was analyzed by Alu-gag qPCR to specifically detect integrated proviral DNA, and levels were normalized to levels obtained for the single copy RNaseP gene. Data shown are average of six replicate PCR samples +/− SD. Data are presented as copies of integrated HIV DNA/100 cells and the number of GFP+ cells/100 cells.</p

Multiplex screening of inducing compounds on the reactivation of HIV-1 latency.

<p>(A) Latently infected cells were stimulated with 10-fold increasing concentrations of SAHA, TSA, HMBA, VPA, or prostratin. Cells were treated with 200 nM PMA+1.5 µM ionomycin as a positive control. The highest and lowest concentrations of the 10-fold dilution series are indicated for each compound tested. Stimulations were performed in triplicate reactions and error bars represent +/− SD. (B) Cells were treated for 24 or 48 h with the indicated concentration of compounds. Results are representative of independent experiments performed with at least three independent donors.</p