PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-ĸB signaling.

<p><b>(A)</b> PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCθ/δ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. <b>(B)</b> NF-ĸB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11–7082, an NF-ĸB inhibitor, and were evaluated for GFP expression by RT-qPCR. <b>(C)</b> J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-ĸB to the HIV LTR was determined using ChIP-qPCR.</p

PEP005 causes minimal cytotoxicity and T cell proliferation.

<p>To evaluate the impact of PEP005 on the cell viability, U1 cells <b>(A)</b>, J-Lat A1 cells <b>(B)</b> and CD4+ T cells <b>(C)</b> were treated with 12 nM of PEP005 for 24 hours, the cell viability was examined with the MTT assay and the S-phase cell cycle progression was evaluated after BrdU incorporation using BrdU ELISA. 25–50 μM Etoposide or 100–150 μM Doxorubicin (Doxo) served as positive controls for MTT and BrdU assays respectively [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005066#ppat.1005066.ref048" target="_blank">48</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005066#ppat.1005066.ref050" target="_blank">50</a>]. Statistical analysis was performed in comparison with controls. **, p<0.01.</p

PEP005 does not induce expression of pro-inflammatory cytokines in primary CD4+ T cells from peripheral blood of HIV-negative donors.

<p>CD4+ T cells were isolated from healthy donors and treated with 6 or 12 nM of PEP005 for 24 or 72 hours, and the relative expression of TNF-α <b>(A)</b>, IFN-γ <b>(B)</b>, IL-2 <b>(C)</b>, and IL-6 <b>(D)</b> was quantified using RT-qPCR and normalized to GAPDH internal control.</p

PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-ĸB signaling.

<p><b>(A)</b> PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCθ/δ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. <b>(B)</b> NF-ĸB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11–7082, an NF-ĸB inhibitor, and were evaluated for GFP expression by RT-qPCR. <b>(C)</b> J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-ĸB to the HIV LTR was determined using ChIP-qPCR.</p

PEP005 synergizes with other latency reversing agents in reactivating latent HIV.

<p><b>(A)</b> J-Lat A1 cells were treated with 5 ng/ml PMA, 6 nM PEP005, 500 nM SAHA, 2 μM JQ1, 2 μM GSK343, or 10 μM Prostratin alone or in combination with 6 nM PEP005 for 24 hours and the percentage of GFP expressing cells was determined using flow cytometry. **, p<0.01; *, p<0.05 compared with control treatment; **, p<0.01; *, p<0.05 compared with PEP005 treatment alone. <b>(B)</b> The U1 cells were treated with 5 ng/ml PMA, 6 nM PEP005, 500 nM SAHA, 2 μM JQ1, or 2 μM GSK343, alone or in combination with 6 nM PEP005 for 24 hours and the HIV transcription was evaluated using RT-qPCR. Numbers indicate fold-increase over the control. **, p<0.01; *, p<0.05 compared with control treatment; **, p<0.01; *, p<0.05 compared with PEP005 treatment alone. <b>(C</b> and <b>D)</b> PEP005 synergizes with other LRAs to significantly increase GFP or HIV-1 mRNA expression in J-Lat A1 <b>(C)</b> or U1 <b>(D)</b> cell lines. The Bliss independence model was utilized for calculation of synergy for LRA combinations [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005066#ppat.1005066.ref035" target="_blank">35</a>] (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005066#sec002" target="_blank">materials and method</a>). Dotted horizontal line signifies pure additive effect (∆f_axy = 0). Synergy is defined as ∆f_axy>0 while ∆f_axy<0 indicates antagonism. Statistical significance was determined using a one tailed ratio t-test comparing predicted and observed drug combination effects. *p < 0.05; **p < 0.01.</p

PEP005 down-modulates the expression of CD4, CCR5 and CXCR4 and inhibits HIV infection of primary CD4+ T cells <i>ex vivo</i>.

<p><b>(A)</b> PEP005 inhibits expression of HIV receptors/co-receptors. The CD4+ T cells were isolated from the peripheral blood of HIV-negative uninfected controls and treated with 12 nM PEP005 for 72 hours. The expression of CD4, CCR5 and CXCR4 genes was evaluated using RT-qPCR after normalizing for GAPDH. <b>(B)</b> and <b>(C)</b> PEP005 inhibits HIV infection of primary CD4+ T cells <i>ex vivo</i>. Primary CD4+ T cells were pre-treated with 12 or 24 nM PEP005 overnight and infected with the virus. The CD4+ T cells were incubated for 5 days without PEP005. The cells were collected for RT-qPCR targeting the HIV LTR region (<b>B</b>), or supernatants were collected for p24 ELISA (<b>C</b>). *, p<0.05; **, p<0.01.</p

PEP005 and JQ1 synergistically induce HIV transcription 6 hrs after treatment in primary CD4+ T cells from HIV infected individuals on suppressive ART.

<p>Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 hours, and HIV transcription was quantified using RT-qPCR for the 5’ LTR region or Poly A region of the virus. The amount of cDNA was only enough to amplify either Poly A region or LTR region during PCR in donor 1 and donor 9.</p

PEP005 activates PKCδ/θ-IκBα/ε-NF-ĸB signaling.

<p><b>(A)</b> J-Lat A1 cells or PBMCs isolated from peripheral blood of healthy HIV-negative individuals were treated with 12 nM of PEP005 for up to 6 hours. Western blot analysis was performed to detect the expression of PKC isoforms, IκB isoforms, as well as expression of NF-κB/p65. <b>(B)</b> Quantitation of phosphorylation of IκBα or IκBε in J-Lat A1 cells after 2hr treatment with PEP005 in panel A. Relative band intensities from three independent experiments in J-Lat A1 cells as determined using ImageJ (NIH) are shown in the bar graph. * p<0.05. <b>(C)</b> Quantitation of PKCδ/θ S643/S676 phosphorylation after 2hr treatment of PEP005 in panel A. Relative band intensities from three independent experiments in J-Lat A1 cells (Left panel) or PBMCs (Right panel) as determined using ImageJ (NIH) are shown in the bar graph. * p<0.05, ** p<0.01.</p

PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART.

<p>Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 μM Ionomycin, or DMSO for 6 hours. Induction of HIV transcription was measured using RT-qPCR for the 5’ LTR region or Poly A region of the virus. **, p<0.01.</p

Expression of T cell activation markers in PEP005-treated primary CD4+ T cells.

<p><b>(A)</b> CD4+ T cells were isolated from uninfected control subjects and treated with 12 nM PEP005 for 24hrs. Total RNA was extracted, and gene expression of CD38, CD69, CD25, or HLA-DR was analyzed by RT-qPCR. (<b>B-F</b>) PBMCs isolated from peripheral blood of healthy HIV-negative donors were treated for 24 or 72 hours with 12 nM of PEP005, and the expression of CD38 <b>(B)</b>, CD69 <b>(C, D)</b>, and HLA-DR <b>(E, F)</b> was evaluated using flow cytometry after co-staining with the CD3 T cell marker.</p