Abstract Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

Author Summary Stable latent viral reservoirs in HIV infected individuals are rapidly reactivated following the interruption of anti-retroviral therapy (ART). Despite an early initiation of ART, viral reservoirs are established and persist as demonstrated in the case of the Mississippi baby and from recent studies of the SIV model of AIDS. Therefore, new strategies are needed for the eradication of the latent HIV reservoirs. We found that ingenol-3-angelate (PEP005), a member of the new class of anti-cancer ingenol compounds, effectively reactivated HIV from latency in primary CD4+ T cells from HIV infected individuals receiving ART. Importantly, a combination of PEP005 and JQ1, a p-TEFb agonist, reactivated HIV from latency at level on average 7.5-fold higher compared to PEP005 alone. The potency of synergistic effects of PEP005 and JQ1 provide novel opportunities for advancing HIV eradication strategies in the future. In summary, ingenols represent a new group of lead compounds for combating HIV latency.

Citation: Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, et al. (2015) Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation. PLoS Pathog 11(7): e1005066. https://doi.org/10.1371/journal.ppat.1005066 Editor: Guido Silvestri, Emory University, UNITED STATES Received: January 3, 2015; Accepted: July 6, 2015; Published: July 30, 2015 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study is supported by NIH grants DK61297, AI43274, and UC Davis Research Investments in Science and Engineering (RISE) grant to SD and a post-doctoral fellowship from CAPES/Brazil (BEX 2951/12-6) to EAM. PK is supported by the Swiss National Science Foundation (PBZHP3_147260). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Anti-retroviral therapy (ART) is effective in suppressing HIV replication but it fails to eliminate latent viral reservoirs in HIV infected resting CD4+ T cells which, in blood, consist mainly of central and transitional memory CD4+ T cells [1–4]. Current ART options do not eradicate HIV from infected cells. In addition, these cells are invisible to the virus-specific immune responses in the setting of viral latency [5,6]. The viral reservoir is rapidly seeded and HIV latency might be established immediately after virus infection [7,8]. Despite initiation of ART in infants within hours of birth to HIV infected mothers, stable viral reservoirs were established and viral rebound occurred when therapy was interrupted [9]. In the simian immunodeficiency virus (SIV) model of AIDS, stable viral reservoirs are established within 2.5 days of infection [10]. The viral reactivation was detected in rhesus macaques following therapy interruption despite the initiation of ART at 3 days post SIV infection [10,11]. Collectively, these studies demonstrate that a very early initiation of ART may not be sufficient to prevent nor eliminate latent virus reservoirs [9,11,12]. It has been observed that the morbidity of HIV persistence in HIV-positive individuals on long-term ART includes drug toxicities and a higher risk of developing complications including dyslipidemia, cardiovascular disease and insulin resistance [13–15]. Therefore, a therapeutic cure of HIV is urgently needed that leads to viral eradication and experimental strategies for directly targeting HIV latent reservoirs are warranted. Recent studies have explored an experimental strategy for viral eradication of HIV infected CD4+ T cells by activating HIV transcription and viral antigen expression from the latent viral reservoirs in the presence of ART [6]. This would lead to the detection and clearance of infected cells by the virus-specific host immune responses while the ART prevents new rounds of infection. Cytopathic effects of the viral reactivation would further increase the clearance of the latent viral reservoir. This “shock and kill” strategy was applied in a pilot clinical trial using the histone deacetylase (HDAC) inhibitor, vorinostat, in patients receiving suppressive ART [16–18]. The findings from these studies showed some promise but failed to result in significant clearance of residual HIV reservoirs. Potential mechanisms of this failure include the modest induction of HIV by this earlier generation of latency reversing agents (LRAs) used singly and due to immune defects in clearance of infected cells in spite of the reactivation of viral expression [19,20]. These studies demonstrate an urgent need for the development of new strategies both for disrupting HIV latency and facilitating elimination of infected cells after HIV expression is reactivated. Several cell signaling pathways are critical for the establishment and maintenance of HIV latency [6,21,22]. Disruption of one or more of these pathways could lead to effective reactivation of HIV from latency. Various compounds have been tested for the disruption of HIV latency, and those inducing HIV reactivation from the viral long terminal repeat (LTR) through the stimulation of the protein kinase C (PKC)-NF-κB pathway showed high potency. These include phorbol esters (PMA and prostratin) and non-phorbol ester diterpenes (bryostatin and gnidimacrin) that induce NF-κB nuclear translocation and activation through the PKC pathway [22,23]. Some of these compounds effectively induce latent HIV reactivation in vitro at picomolar levels [24,25]. The LRAs, functioning through the PKC-NF-κB signaling, are able to reactivate latent HIV across a broad range of HIV latency models [20]. A recent study showed that LRAs stimulating PKC-NF-κB signaling may be most effective in inducing complete transcription of HIV from resting CD4+T cells of HIV infected individuals on suppressive ART [26]. Moreover, these compounds cause down-modulation of the expression of cell surface receptors, CD4, CXCR4 or CCR5, and protect cells against HIV infection [22]. Therefore, LRAs that activate PKC-NF-κB signaling are potential candidates for HIV cure studies. We previously reported that an ingenol ester, ingenol-3-hexanoate or IngB, is an excellent candidate for the reactivation of HIV from latency [24]. The modified ingenol-3-hexanoate was originally isolated from an Amazonian plant, Euphorbia tirucalli. It exerts low toxicity in CD4+ T cells and does not induce global T-cell activation. It caused reactivation of latent HIV at nanomolar levels [24]. However, since IngB induces expression and activation of both NF-κB and CyclinT1/CDK9, and stimulate IFNγ expression in primary CD4+ T cells, further search for new ingenol compounds with better HIV reactivation potential and lower cellular toxicity is needed [24,27,28]. Among previously identified ingenol compounds, ingenol-3-angelate (PEP005) is currently approved for clinical use. A recently FDA approved drug, PICATO, for the topical treatment of precancerous actinic keratosis contains ingenol-3-angelate as an active component [29]. A prior study suggested that ingenol-3-angelate could induce HIV expression from the U1 monocyte cell line harboring HIV genome [30]. In the current study, we report that PEP005 can effectively reactivate latent HIV through the activation of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB pathway in an HIV latency model in vitro but does not induce or increase NF-κB protein production by itself. It also reactivated full-length HIV transcription based on an assay targeting the poly A tail region of HIV transcripts in cells from ART-suppressed HIV-positive individuals, while exerting minimal toxicity and effects on T cell activation ex vivo [26]. Importantly, the effect of PEP005 was synergistic with JQ1, a p-TEFb activator, and the combination was highly potent in reactivating latent HIV expression both in vitro and ex vivo. Our findings identify this anti-cancer drug, PEP005, as having a distinct mechanism of molecular signaling and as a potential candidate for advancing to future HIV eradication studies.

Materials and Methods Cell culture J-Lat A1 cells (harboring a single copy of latent HIV LTR and one copy of green fluorescent protein gene under the HIV LTR control) or U1 cells (harboring two latent HIV genomes with defective Tat gene) were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS) and 1% Pen/strep in a 37°C incubator containing 5% CO 2 [24,31]. Both of the cell lines were obtained from NIH AIDS Reagent Program. For reactivation of HIV LTR, cells were treated with PMA (Sigma), JQ1 (Biovision), Prostatin (Sigma), SAHA (Santa Cruz), TNF-α (BD), GSK343 (Sigma), or PEP005 (Tocris Bioscience) for 24h. HIV reactivation was quantified by GFP expression using flow cytometry and the data were analyzed using FlowJo Software for J-Lat A1 cells, or by quantitative RT-PCR (RT-qPCR) for J-Lat A1 and U1 cells. Cell viability was evaluated using Live/Dead dye (Life Technologies) by flow cytometry. So far, there is not a single in vitro cell culture model available that captures all the features of HIV latency. However, PKC agonists seem to reactivate latent HIV in all the cell culture models of viral latency. Similar to other J-Lat cell lines, the J-Lat A1 clone has been widely used by researchers for HIV latency studies. The T-cell-derived J-Lat A1 cells harbor one copy of a construct containing a Tat and a green fluorescent protein (GFP) gene framed by the 5’ and 3’ HIV LTR [32]. The J-Lat A1 cells also contain the TAR loop located within the R region of HIV LTR (nt +1 to +60), which provides an opportunity for Tat-TAR interactions for the modulation of transcription and transcriptional elongation. Consequently, the J-Lat A1 cell model is a suitable cell culture model to investigate latency reversal by PKC agonists. Primary CD4+ T cell isolation and detection of T cell activation markers Peripheral blood samples were collected from 13 HIV-infected individuals receiving suppressive ART for >3 years except for one patient. All subjects had suppression of plasma viremia for more than 6 months (Average 5.84 years). At the time of the study enrollment, CD4+ T cell counts in peripheral blood samples ranged from 264 to 1100 cells/mm3 (Average 639 cell/mm3) and plasma viral loads were <20 copies per ml as measured by qPCR (Table 1). Isolation of the peripheral blood mononuclear cells (PBMC) and purification of CD4+ T cells using the EasySep kit (STEMCELL Technologies Inc. Vancouver, BC, Canada) were performed as previously described [24]. The purified CD4+ T cells were plated at a density of 1x106 cells/ml and treated with 200 ng/ml PMA plus 2 μM Ionomycin, 6–12 nM PEP005, 2 μM of JQ1, or 12 nM PEP005 plus 2 μM JQ1 for 6 hrs or 48 hrs and the cells were collected for RNA purification. To measure changes in the cell activation status of CD4+ and CD8+ T cell subsets, PBMCs were isolated from uninfected controls and 2x106 cells were incubated with DMSO, 200 ng/ml PMA plus 2 μM Ionomycin, 6–12 nM PEP005 for 24 hrs or 72 hrs, and immunostained with anti-CD3, anti-CD38, anti-CD69, or anti-HLA-DR antibodies (Biolegend) for 20 min at 4°C. Cells were fixed in 1% PFA and analyzed by flow cytometry (FlowJo software from TreeStar). In addition, PBMCs from HIV-negative uninfected controls were similarly treated for 24 or 72 hrs and cells were collected for cytokine analysis using RT-qPCR. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Characteristics of HIV-positive individuals receiving HAART. https://doi.org/10.1371/journal.ppat.1005066.t001 Cell viability and proliferation measurements Cells were placed in 96-well plates and incubated for 24 or 72h hrs with compounds. Cell viability was measured using MTT assay (Roche Laboratories), and cell proliferation/S-phase progression was determined by using BrdU ELISA kit (Cell signaling, #1863). Immunoblot analysis One million J-Lat A1 cells or PBMCs from HIV-negative uninfected controls were incubated with 12 nM PEP005 for 6 hrs. Whole cell protein extracts were prepared with RIPA buffer containing proteinase inhibitors and phosphatase inhibitors (Sigma). Expression of the isoforms of PKC protein or NF-κB/p65 was evaluated using the PKC Isoform Sampler Antibody Kit (Cell Signaling, 9960S) or anti-NF-κB/p65 (Abcam). The level of phosphorylation of PKC was determined using anti-Phospho-Ser664-PKCδ, anti-Phospho-Ser643/676 PKCδ or anti-Phospho-T538-PKCθ (Millipore), and the level of phosphorylation of IκB was determined using p-IκBα (Ser32), p-IκBβ (Thr19/Ser23) or p-IκBε (Ser18/22) antibodies (Millipore). HIV RNA quantification in patient samples Total RNA was extracted using the Qiagen RNeasy Kit that included a DNA digestion step. Quantitative RT-PCR was performed using Taqman Fast Virus 1-Step Master Mix (Applied Biosystems) in a ViiA7 real-time PCR system (Applied Biosystems). Transcripts containing the U5 region of the 5’ LTR were amplified using well-conserved primers (HXB2 559–543, 626–643) and fluorescent probe (HXB2 559–584) [24,33]. Transcripts containing the U3 to R region of the 3’ LTR including a Poly A tail were amplified in a one-step RT-PCR procedure related to a recently published assay with primers and probe binding sites as follows: gccctcagatgctrcatataa (HXB2 9496–9516), ttttttttttttttttttttttttttgaag (9632–9636 + poly T) and FAM-tgcctgtactgggtctctctggttag-MGB (HXB2 9529–9554) [26,34]. External HIV RNA standards were prepared from in vitro transcripts quantified by spectrophotometry. HIV RNA copy numbers were normalized to RNA input. Quantitative analysis of synergy of latency reversing agent combinations We adapted the Bliss independence model as implemented by Laird et al to test for synergy when PEP005 was combined with other latency reversing agents [35,36]. For drugs x and y, we used the equation fa xyP = fa x +fa y −(fa x )(fa y ), where fa xyP represents the predicted fraction affected by the combination of drug x and drug y given the observed effects of drug x (fa x ) and drug y (fa y ) used individually and fa xy,O = the observed effect when x and y were tested together. Calculation of fa x for U1 cells and patient derived T cells followed the approach of Laird for intracellular HIV RNA: fa x = (HIV RNA copies with drug x–background copies with DMSO)/ (HIV RNA copies with PMA–background copies with DMSO). For these analyses, we included data for which every parameter for the synergy analysis was available and excluded individual cases where some of the parameters were not available. In cases where one or more experimental drug conditions resulted in RNA expression exceeding the PMA condition, we imputed the highest HIV RNA value in that experiment +1 to represent the denominator for calculation of fa x . The calculation of fa x for J-Lat A1 cells used the % GFP positive cells in place of intracellular HIV RNA. With this model, Δ fa xy = fa xyO (the observed fraction affected by the drug combination)—fa xyP (the predicted fraction affected by the drug combination) provides an indication of synergy (Δ fa xy > 0), additive effect (Bliss independence) (Δ fa xy = 0) or antagonism (Δ fa xy < 0). Calculations analyzing synergy were done using Python. Statistical significance was determined using a one tailed ratio t-test executed in the statistical package “R”. Chromatin immunoprecipitation (ChIP) ChIP assay was performed as previously described [24,37]. Briefly, 1X106 J-Lat A1 cells were incubated with PEP005, or PKCδ/θ inhibitor (PKCδi, Millipore/Calbiochem), fixed in 1% formaldehyde then resuspended in lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1 (ChIP Assay Kit, Millipore) and protease inhibitor cocktail (Sigma-Aldrich). Lysates were sonicated to obtain DNA fragments of 200–1500 bp. The immune complex was retrieved by incubating for 45 min with 50 μl of protein A/G-agarose beads saturated with BSA/salmon sperm DNA. Following the washes, the chromatin was eluted and reverse cross-linked overnight. DNA was extracted (Qiagen PCR purification kit) and quantitative real-time PCR was performed using Agilent Brilliant Ultra-Fast SYBR Green QPCR reagent using the 7500 real-time PCR System. The upstream primer sequence was 5’-AGCTTGCTACAAGGGACTTTCC-3’, and the downstream primer sequence was 5’-ACCCAGTACAGGCAAAAAGCAG-3’. Real-time PCR analysis of GFP or HIV gene expression Total RNA was isolated from J-Lat A1 cells or U1 cells using the RNeasy Kit (Qiagen) followed by digestion with DNase I (Invitrogen). First strand cDNA was synthesized using Superscript II (Invitrogen). Real-time PCR (TaqMan) was performed on a ViiA 7 detector using the following primer/probe set: for J-Lat A1 cells, primer 1: 5’-GGAGCGACCATCTTCTTCA-3’, primer 2: 5’-AGGGTGTCGCCCTCGAA-3’, probe 5’-FAM CTACAAGACCC GCGCCGAGGTG TAMRA-3’, for U1 cells HIV 5’ LTR primers/probe were used (see above) [24]. The GAPDH primer/probe set was purchased from Applied Biosystems. Pre-treatment of primary CD4+ T cells with PEP005 and HIV-1 infection of primary CD4+ T cells Primary CD4+ T cells were isolated from peripheral blood samples of healthy HIV-negative donors and were pre-treated with PEP005 overnight. The CD4+ T cells were infected with HIV-1 (HIV-1 IIIB expanded in Jurkat T cells, 100ng p24-gag) through spinoculation as described previously [38]. Following an exposure of 24 hrs to HIV, the cells were washed. Cell supernatants were collected and HIV p24 levels were measured by ELISA. The cells were also collected for RNA extraction and measurement of viral transcripts by RT-qPCR. Ethics statement Studies were performed under informed written consent and a protocol approved by the University of California-Davis Institutional Review Board (IRB 219139–7). Statistical analysis Means and standard errors (SE) were calculated for all data points from at least 3 independent experiments in triplicates. Statistical significance was determined using the two-way Student t test, where p value<0.05 considered significant.

Discussion Since the discovery of stable, latent viral reservoirs in HIV-infected individuals in spite of long-term suppressive ART, several studies have been performed to better understand the mechanisms of the establishment of HIV latency as well as to identify crucial steps for the maintenance of the viral latency [5,6,21]. The identification of memory CD4+ T cells, mostly central memory CD4+ T cells, as the major viral reservoir has provided clues for designing novel strategies for HIV eradication [5,57]. Accordingly, a “shock and kill” strategy was proposed to eradicate latent HIV reservoirs [6]. Diterpene compounds are among the potential candidates for disrupting HIV latency. They are more potent than several other LRAs in inducing latent HIV expression in multiple HIV latency models including in vitro J-Lat cell lines, primary CD4+ T cell based cell cultures, and resting CD4+ T cells from the peripheral blood of HIV infected individuals [20,24]. Diterpenes can down-modulate the expression of cell surface receptors that are known to mediate HIV attachment and entry and can potentially prevent the spread of viral infection to bystander CD4+ T cells [27,52–54]. Therefore, it will be useful to identify diterpene compounds suitable for HIV cure studies that could reactivate HIV from the latent reservoirs with low to minimal cytotoxicity while preventing new viral infections of CD4+ T cells. The data from this study show, for the first time, that PEP005, a diterpene compound, can effectively reactivate latent HIV expression in vitro and ex vivo. We and others have previously reported that an ingenol ester, IngB, is also able to reactivate HIV from latency [24,27]. Both of these compounds showed efficacy in reactivation of HIV latency at low nanomolar levels without exerting apparent cytotoxicity. IngB could induce increased levels of protein expression of NF-κB, P-TEFb, and IFNγ [24,27] while PEP005 does not increase NF-κB protein expression levels. Activation of NF-κB and P-TEFb is essential for HIV reactivation from latency. However, excessive induction of these proteins may potentially be problematic because NF-κB is a master regulator for multiple signaling pathways, and P-TEFb is a general activator of transcriptional elongation, that also contributes to multiple signaling pathways [58,59]. Further studies are warranted to investigate whether IngB or PEP005 alone can induce protein expression of NF-κB or P-TEFb in resting CD4+ T cells, and not just in proliferating CD4+ T cells. Thirdly, IngB induced IFNγ protein expression [24,27] while PEP005 barely caused any increase in this or other inflammatory cytokine expression. Although PEP005 shares a similar core structure with IngB, its C3 side chain structure is different and quite distinct from IngB (Fig 1). Substitution of the C3-ester by different aliphatic and aromatic side chains confer markedly different biological properties [60,61], so it is likely that differences in molecular mechanisms of action and different T cell activation properties between PEP005 and Ingenol B are conferred by the structural differences of the C3-R. Consequently, PEP005 may offer several advantages over IngB. High levels of ingenols, up to 20 μM, induced NF-κB-independent cell death in Jurkat cells [62]. This concentration is about 1000-fold higher than the PEP005 concentrations used in this study, suggesting that low concentrations of PEP005 capable of HIV reactivation may trigger signaling pathways differently and result in its low-to-minimal cytotoxicity. Therefore, the use of PEP005 at low concentrations for reactivation of latent HIV will be important to minimize cytotoxic effects. In the recently FDA-approved drug PICATO, PEP005 is the only active component with PKC agonist activity. Pharmacokinetic studies of PEP005 have been performed in small animals in vivo. Similar to the safety of the topical application of PICATO, the systemic (intravenous) use of PEP005 in small animals (mini pig and rat model) was reported to be relatively safe, with the maximum nonlethal dose >73 μg/kg (See Assessment report of PICATO to European Medicines Agency, Sept 20, 2012). While additional safety data with systemic administration in non-human primates is needed, these existing data support further investigation of PEP005 as a potential candidate in HIV cure studies. Since multiple molecular signaling pathways are involved in establishment and maintenance of HIV latency, a single LRA may not be adequate to achieve disruption of the multi-pronged regulatory mechanisms promoting HIV latency. SAHA, an HDAC inhibitor, shows some ability to reactivate HIV. However, the efficacy of SAHA varies among HIV infected individuals indicating that SAHA alone may not be sufficient for effective reactivation of latent HIV for all HIV infected individuals [16,17,63]. Therefore, a combination of compounds should be explored to induce viral expression from latently infected cells [43]. Our data show that PEP005 alone potently reactivates latent HIV expression in vitro. However, a combination of PEP005 and JQ1 was substantially more effective in inducing latent HIV in J-Lat A1 cells or U1 cells and in ex vivo primary CD4+ T cells from infected patients receiving suppressive ART. This could be attributed to potentiating effects of the PKC-NF-kB pathway on p-TEFb mechanism for reactivating. Future investigations are warranted to determine the range of drug concentrations that will provide the best drug synergy by using PEP005 [64]. Synergistic reactivation of latent HIV also are achieved by combining JQ1 with other ingenol compounds as we previously observed that JQ1 boosted the reversal of HIV latency by IngB in J-Lat A1 cells [24]. However, it is not known whether this combined treatment leads to a synergistic effect on HIV expression in patient cells under ART ex vivo. Recently, Sillicano and colleagues showed that PKC agonists, such as Prostratin or Bryostatin 1 had a synergic effect with JQ1 on the reactivation latent HIV ex vivo [35]. It was intriguing to note that GSK343, an EZH2 inhibitor, had no capacity to reactivate latent HIV by itself, but displayed increased potency in combination with PEP005 in J-Lat A1 cells. These observations are complemented by a recently published study showing that GSK343 boosts SAHA or JQ1 reactivation of latent HIV in a primary resting T-cell model of HIV latency [65]. These findings suggest that pre-disruption of methylation of histone tails may facilitate efficient reactivation of latent HIV in a combination with a second LRA. PEP005 displayed potent activity to reactivate HIV from latency, producing polyadenylated viral transcripts while exerting minimal to low cytotoxicity and in the absence of major global T cell activation. Until now, only Bryostatin and PMA have been shown to stimulate comparable transcription of polyadenylated HIV RNA in primary CD4+ T cells isolated from HIV infected individuals on suppressive ART [26]. Considering that PEP005 had a similar capacity as PMA to reactivate latent HIV both in vitro and ex vivo (Figs 1D and 8), and that PEP005 could induce GFP protein expression in J-Lat A1 cells (Fig 1), it seems reasonable to consider that PEP005 as a potential candidate for inducing HIV virus expression in vivo. Further ex vivo investigations to confirm this using virus outgrowth assays are warranted. There was an indication that PEP005 up-regulated TNFα to some extent but these effects were not significantly different compared to controls. Similar to what is found with other PKC agonists, PEP005-induced NF-kB activation leads to increased expression of CD69 since its promoter contains multiple NF-κB binding sites [44]. However, it was interesting to note that cell proliferation was not significantly altered in J-Lat A1, U1 cells or primary CD4+ T cells, and inflammatory cytokine expression was not significantly enhanced by PEP005. These findings suggest that modulation of some of the T-cell activation markers may not necessarily lead to the global T-cell activation. It has also been proposed that a low level of T cell activation may be required for efficient reactivation of HIV latency [26]. The ability of PEP005 to reduce cell surface expression of CD4, CCR5, and CXCR4 on T cells directly impacts susceptibility of primary CD4+ T cells to in vitro HIV infection. This supports the concept that reactivation of latent HIV by PEP005 may block viral spread to uninfected bystander CD4+ T cells, making it an attractive potential candidate for advancing to clinical HIV cure studies. Our study in combination with previous reports suggests that ingenols, including PEP005, represent a new group of lead compounds for combating HIV latency for viral eradication that deserve further study.

Author Contributions Conceived and designed the experiments: GJ SD. Performed the experiments: GJ EAM PK YT IC. Analyzed the data: GJ EAM PK DPW JKW SD. Contributed reagents/materials/analysis tools: YT JEKH. Wrote the paper: GJ JKW PK SD. Coordinated patient samples: AF GPM GRT.