Comparison of the viral DNA profiles in HIV-1_NLAD8 and HIV-1_NLAD8ΔVpr infected cells.

<p>Quantitative PCR analysis was performed to determine levels of late reverse transcription (Late-RT) products, 2-LTR circles, and integrated proviral copies over a period of 7 days following infection of activated PBMCs (A-C) and MDDCs (D-F) following infection with HIV-1_NLAD8 and HIV-1_NLAD8ΔVpr. Real-time PCR amplification of the <i>glyceraldehyde-3-phosphate dehydrogenase</i> gene was performed for each sample to normalize for the amount of input DNA in each of the amplification reactions. Error bars represent standard error of the mean of duplicate samples. Statistically significant differences are indicated by the asterisks (<i>P<</i>0.05) and <i>P</i> values. The data shown represents one of three independent experiments carried out for each cell type from three different donors.</p

Vpr enhances single-cycle HIV-1 infection of activated PBMCs, primary CD4⁺ T cells, and MDDCs.

<p>(A) PHA-activated peripheral blood mononucleocytes (PBMCs), (B) PHA-activated CD4⁺ T cells, and (C) Monocyte-derived dendritic cells (MDDCs) were infected at an MOI of 1.0 with single-cycle HIV-1 Vpr⁺/VSV-G and HIV-1 Vpr⁻/VSV-G to test the role of Vpr in HIV-1 infection. Luciferase expression from the integrated provirus in the infected cells was assessed at the indicated time and normalized to protein content (10 µg/sample). The data shown represents one of three independent experiments carried out for each cell type from three different donors. Error bars represent the standard deviation of the mean of triplicate samples. Statistically significant differences are indicated by the asterisks (<i>P</i><0.05).</p

Vpr significantly enhances replication-competent HIV-1_NLAD8 infection in MDDCs.

<p>(A) The HIV-1_NLAD8 and HIV-1_NLAD8ΔVpr virus stocks produced from HEK293T cells were analyzed by immunoblotting for the presence of Vpr. (B) PHA-activated PBMCs and (C) MDDCs were infected with 5 ng and 20 ng of p24, respectively, from HIV-1_NLAD8 and HIV-1_NLAD8ΔVpr virus and levels of p24 capsid released into the media during virus replication were assayed over a period of 10 days and 7 days post infection for PBMCs and MDDCs, respectively. The data shown represents one of three independent experiments carried out with three individual donors, and error bars represent standard deviation of triplicate infections. Statistically significant differences are indicated by the asterisks (<i>P<</i>0.05).</p

Vpr complementation does not affect Vpr-defective single-cycle HIV-1 infection of MDDCs.

<p>(A) Vpr incorporation in VSV-G-pseudotyped, single-cycle HIV-Vpr+ and Vpr-complemented HIV-Vpr-. Virion pellets were analyzed by immunoblotting with anti-Vpr and anti-p24, respectively. (B) Vpr complementation does not affect single-cycle HIV-Vpr- infection of MDDCs. Infected cells were lysed at indicated times post infection for the detection of HIV-1 infection by measuring luciferase activity and normalized to protein content (20 µg/sample). cps, counts per second. The data shown represents one of three independent experiments carried out with three individual donors.</p

Vpr-mediated enhancement of single-cycle HIV-1 infection is independent of VSV-G and Ampho envelopes used for virus pseudotyping.

<p>(A) HuT/CCR5 cells were infected at an MOI of 1.0 with single-cycle HIV-1 Vpr⁺/VSV-G and HIV-1 Vpr⁻/VSV-G. Luciferase expression from the integrated provirus in the infected cells was assessed at the indicated time and normalized to protein content (10 µg/sample). The data shown represents one of three independent experiments, and error bars represent standard deviation of the mean of triplicate samples. (B) The MLV amphotrophic (Ampho) envelope pseudotyped, HIV-1 Vpr⁺ and HIV-1 Vpr⁻ stocks generated from pNL-Luc-E⁻R⁺ and pNL-Luc-E⁻R⁻ proviral constructs were analyzed by immunoblotting for the presence of Vpr. (C) HuT/CCR5 cells were infected at an MOI of 1.0 with HIV-1 Vpr⁺/Ampho and HIV-1 Vpr⁻/Ampho. Luciferase expression from the integrated provirus in the infected cells was assessed 3 days post infection and normalized to protein content. Error bars represent standard deviation of the mean of triplicate samples. Statistically significant differences are indicated by the asterisks (<i>P<</i>0.05) and the <i>P</i> value.</p

DCAF1 knockdown in HuT/CCR5 cells does not affect single-cycle HIV-1 infection.

<p>HuT/CCR5 cells were transduced with concentrated lentivirus expressing either shRNA targeting DCAF1 or a scrambled shRNA (control). Three days following transduction a fraction of cells were analyzed by immunoblotting to confirm DCAF1 knockdown (A), and cells each were infected at an MOI of 0.5 with either HIV-1 Vpr⁺/VSV-G or HIV-1 Vpr⁻/VSV-G to test whether DCAF1 was involved in the Vpr-mediated enhancement of HIV-1 infection (B). Luciferase expression in the infected cells was assessed at 3 days post infection.</p

Characterization of Vpr⁺ and Vpr⁻ single-cycle, VSV-G-pseudotyped HIV-1 stocks produced from HEK293T cells.

<p>(A) Quantification of luciferase (Luc) expression from the pNL-Luc-E⁻R⁺ (HIV-1 Vpr⁺) and pNL-Luc-E⁻R⁻ (HIV-1 Vpr⁻) proviral DNA constructs in HEK293T cells. Luciferase activity was determined 48 h following plasmid transfection and normalized to protein content. Error bars represent the standard deviation of the mean of three independent experiments. (B) The VSV-G pseudotyped, HIV-1 Vpr⁺ and HIV-1 Vpr⁻ stocks generated from pNL-Luc-E⁻R⁺ and pNL-Luc-E⁻R⁻ were analyzed by immunoblotting for the incorporation of Vpr into virion particles.</p

Titration of infectivity of single-cycle and replication-competent HIV-1 stocks.

a<p>Single-cycle, VSV-G-pseudotyped luciferase reporter HIV-1 Vpr⁺, HIV-1 Vpr⁻, and Vpr complemented HIV-1 Vpr⁻ stocks. The data represent average results of duplicated samples from two independent experiments.</p>b<p>Single-cycle, MLV amphotrophic (Ampho) envelope-pseudotyped luciferase reporter HIV-1 Vpr⁺ and HIV-1 Vpr⁻ virus stocks.</p>c<p>Replication-competent HIV-1_NLAD8(WT) and HIV-1_NLAD8(ΔVpr) stocks. All viral stocks were prepared from HEK293T cells and analyzed for p24 concentration by ELISA. The infectivity of each virus stock was evaluated on HIV-1 indicator GHOST/R5 cells by a limiting dilution assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035385#pone.0035385-Janas1" target="_blank">[35]</a>. The relative infectivity of each virus stock is presented as the number of infectious units (IU) per 1 ng of p24.</p

[Lola] Albright... [Oleg] Vidov

Additional file 3: Figure 2. Ingenols do not induce pro-inflammatory cytokine release in purified resting CD4+ T cell cultures. Concentrations of pro-inflammatory cytokines TNFα, IFNγ, IL-1β and IL-6 were not significantly elevated in the presence of ingenol-3,20-dibenzoate (ingenol DB) or ingenol B compared to media alone control at 72 h in purified resting CD4+ T cell cultures. Mean values and standard deviation of seven independent experiments using resting CD4+ T cells from aviremic ART-treated HIV positive participants are shown. Pro-inflammatory cytokine concentrations were significantly elevated in positive control cultures (T cell receptor stimulation via CD3 and CD28 antibodies). ** P value <0.01; *** P value <0.001

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy-3

Rease cell survival by preventing PTEN from binding to p53. Binding of HIV-1 Tat to p53 may result in reduced levels of both p53 by destabilization and PTEN by downregulation of PTEN expression. In vitro binding assay: Lysates containing p53-FLAG were incubated with BSA (control) or HIV-1 Tat protein and then mixed with lysate containing PTEN V5-tag. Proteins bound to p53 were immunoprecipitated using anti-FLAG immobilized antibody and analyzed for PTEN-V5 tag levels by Western blotting. Ratios of PTEN-V5 levels normalized by p53-FLAG levels are shown.<p><b>Copyright information:</b></p><p>Taken from "Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy"</p><p>http://www.retrovirology.com/content/5/1/11</p><p>Retrovirology 2008;5():11-11.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2265748.</p><p></p