HIV latency is reversed by ACSS2-driven histone crotonylation.
ABSTRACT: Eradication of HIV-1 (HIV) is hindered by stable viral reservoirs. Viral latency is epigenetically regulated. While the effects of histone acetylation and methylation at the HIV long-terminal repeat (LTR) have been described, our knowledge of the proviral epigenetic landscape is incomplete. We report that a previously unrecognized epigenetic modification of the HIV LTR, histone crotonylation, is a regulator of HIV latency. Reactivation of latent HIV was achieved following the induction of histone crotonylation through increased expression of the crotonyl-CoA-producing enzyme acyl-CoA synthetase short-chain family member 2 (ACSS2). This reprogrammed the local chromatin at the HIV LTR through increased histone acetylation and reduced histone methylation. Pharmacologic inhibition or siRNA knockdown of ACSS2 diminished histone crotonylation-induced HIV replication and reactivation. ACSS2 induction was highly synergistic in combination with either a protein kinase C agonist (PEP005) or a histone deacetylase inhibitor (vorinostat) in reactivating latent HIV. In the SIV-infected nonhuman primate model of AIDS, the expression of ACSS2 was significantly induced in intestinal mucosa in vivo, which correlated with altered fatty acid metabolism. Our study links the HIV/SIV infection-induced fatty acid enzyme ACSS2 to HIV latency and identifies histone lysine crotonylation as a novel epigenetic regulator for HIV transcription that can be targeted for HIV eradication.
Project description:DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5' LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5' LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients. However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG methylation might have important implications for strategies aimed at eradicating HIV-1 infection.
Project description:<h4>Background</h4>Current antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a "block and lock" approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs).<h4>Results</h4>J-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP<sup>-</sup> and GFP<sup>+</sup> cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators.<h4>Conclusion</h4>The active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our "block and lock" approach constitutes a novel way of enforcing HIV-1 "super latency" through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.
Project description:Various epigenetic marks at the HIV-1 5'LTR suppress proviral expression and promote latency. Cellular antisense transcripts known as long noncoding RNAs (lncRNAs) recruit the polycomb repressor complex 2 (PRC2) to gene promoters, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), thus promoting nucleosome assembly and suppressing gene expression. We found that an HIV-1 antisense transcript expressed from the 3'LTR and encoding the antisense protein ASP promotes proviral latency. Expression of ASP RNA reduced HIV-1 replication in Jurkat cells. Moreover, ASP RNA expression promoted the establishment and maintenance of HIV-1 latency in Jurkat E4 cells. We show that this transcript interacts with and recruits PRC2 to the HIV-1 5'LTR, increasing accumulation of the suppressive epigenetic mark H3K27me3, while reducing RNA Polymerase II and thus proviral transcription. Altogether, our results suggest that the HIV-1 ASP transcript promotes epigenetic silencing of the HIV-1 5'LTR and proviral latency through the PRC2 pathway.
Project description:Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-?B motifs within the viral 5'LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5'LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.
Project description:Cells latently infected with HIV represent a currently insurmountable barrier to viral eradication in infected patients. Using the J-Lat human T-cell model of HIV latency, we have investigated the role of host factor binding to the kappaB enhancer elements of the HIV long terminal repeat (LTR) in the maintenance of viral latency. We show that NF-kappaB p50-HDAC1 complexes constitutively bind the latent HIV LTR and induce histone deacetylation and repressive changes in chromatin structure of the HIV LTR, changes that impair recruitment of RNA polymerase II and transcriptional initiation. Knockdown of p50 expression with specific small hairpin RNAs reduces HDAC1 binding to the latent HIV LTR and induces RNA polymerase II recruitment. Similarly, inhibition of histone deacetylase (HDAC) activity with trichostatin A promotes binding of RNA polymerase II to the latent HIV LTR. This bound polymerase complex, however, remains non-processive, generating only short viral transcripts. Synthesis of full-length viral transcripts can be rescued under these conditions by expression of Tat. The combination of HDAC inhibitors and Tat merits consideration as a new strategy for purging latent HIV proviruses from their cellular reservoirs.
Project description:HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.
Project description:Eradication of HIV infection depends on the elimination of a small, but stable population of latently infected T cells. After the discontinuation of therapy, activation of latent virus can rekindle infection. To purge this reservoir, it is necessary to define cellular signaling pathways that lead to activation of latent HIV. We used the SCID-hu (Thy/Liv) mouse model of HIV latency to analyze a broad array of T cell-signaling pathways and show in primary, quiescent cells that viral induction depends on the activation of two primary intracellular signaling pathways, protein kinase C or nuclear factor of activated T cells (NF-AT). In contrast, inhibition or activation of other important T cell stimulatory pathways (such as mitogen-activated protein kinase, calcium flux, or histone deacetylation) do not significantly induce virus expression. We found that the activation of NF-kappaB is critical to viral reactivation; however, all pathways that stimulate NF-kappaBdonot reactivate latent virus. Our studies further show that inhibition of NF-kappaB does not prevent activation of HIV by NF-AT, indicating that these pathways can function independently to activate the HIV LTR. Thus, we define several molecular pathways that trigger HIV reactivation from latency and provide evidence that latent HIV infection is maintained by the functional lack of particular transcription factors in quiescent cells.
Project description:HIV-1 latency in resting CD4(+) T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). Eliminating the latent HIV-1 reservoir may require the reactivation of viral gene expression in latently infected cells. Most approaches for reactivating latent HIV-1 require nonspecific T cell activation, which has potential toxicity. To identify factors for reactivating latent HIV-1 without inducing global T cell activation, we performed a previously undescribed unbiased screen for genes that could activate transcription from the HIV-1 LTR in an NF-kappaB-independent manner, and isolated an alternatively spliced form of the transcription factor Ets-1, DeltaVII-Ets-1. DeltaVII-Ets-1 activated HIV-1 transcription through 2 conserved regions in the LTR, and reactivated latent HIV-1 in cells from patients on HAART without causing significant T cell activation. Our results highlight the therapeutic potential of cellular factors for the reactivation of latent HIV-1 and provide an efficient approach for their identification.
Project description:To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5' long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4<sup>+</sup> T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4<sup>+</sup> T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4<sup>+</sup> T cells, and crucially in CD4<sup>+</sup> T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency. <b>IMPORTANCE</b> A reservoir of latent HIV-1 infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency-maintaining host factors and functionally validated a subset of 10 of those in additional T-cell-based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1's promoter, the 5' LTR, while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor topiramate as a viable latency reversal agent with clinical potential.
Project description:The presence of latent HIV-1 in infected individuals represents a major barrier preventing viral eradication. For that reason, reactivation of latent viruses in the presence of antiretroviral regimens has been proposed as a therapeutic strategy to achieve remission. We screened for small molecules and identified several benzotriazole derivatives with the ability to reactivate latent HIV-1. In the presence of IL-2, benzotriazoles reactivated and reduced the latent reservoir in primary cells, and, remarkably, viral reactivation was achieved without inducing cell proliferation, T cell activation, or cytokine release. Mechanistic studies showed that benzotriazoles block SUMOylation of phosphorylated STAT5, increasing STAT5's activity and occupancy of the HIV-1 LTR. Our results identify benzotriazoles as latency reversing agents and STAT5 signaling and SUMOylation as targets for HIV-1 eradication strategies. These compounds represent a different direction in the search for "shock and kill" therapies.