RESUMO
Understanding the mechanisms that drive HIV expression and latency is a key goal for achieving an HIV cure. Here we investigate the role of the SETD2 histone methyltransferase, which deposits H3K36 trimethylation (H3K36me3), in HIV infection. We show that prevention of H3K36me3 by a potent and selective inhibitor of SETD2 (EPZ-719) leads to reduced post-integration viral gene expression and accelerated emergence of latently infected cells. CRISPR/Cas9-mediated knockout of SETD2 in primary CD4 T cells confirmed the role of SETD2 in HIV expression. Transcriptomic profiling of EPZ-719-exposed HIV-infected cells identified numerous pathways impacted by EPZ-719. Notably, depletion of H3K36me3 prior to infection did not prevent HIV integration but resulted in a shift of integration sites from highly transcribed genes to quiescent chromatin regions and to polycomb repressed regions. We also observed that SETD2 inhibition did not apparently affect HIV RNA levels, indicating a post-transcriptional mechanism affecting HIV expression. Viral RNA splicing was modestly reduced in the presence of EPZ-719. Intriguingly, EPZ-719 exposure enhanced responsiveness of latent HIV to the HDAC inhibitor vorinostat, suggesting that H3K36me3 can contribute to a repressive chromatin state at the HIV locus. These results identify SETD2 and H3K36me3 as novel regulators of HIV integration, expression and latency.
Assuntos
Infecções por HIV , HIV-1 , Histona-Lisina N-Metiltransferase , Latência Viral , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Humanos , Latência Viral/fisiologia , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Infecções por HIV/genética , HIV-1/fisiologia , HIV-1/genética , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/metabolismo , Regulação Viral da Expressão GênicaRESUMO
Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.
Assuntos
Infecções por HIV , HIV-1 , Inibidores de Histona Desacetilases , Nanopartículas , Latência Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Humanos , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Antígenos HIV/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Latência Viral/efeitos dos fármacosRESUMO
Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Cromatina/metabolismo , Epigenômica/métodos , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Transcrição/metabolismo , Latência Viral , Sítios de Ligação , Linfócitos T CD4-Positivos/virologia , Cromatina/genética , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Células Jurkat , Fatores de Transcrição/genética , Ativação ViralRESUMO
Despite the success of antiretroviral therapy, human immunodeficiency virus (HIV) cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency, we employed an integrated single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) approach to simultaneously profile the transcriptomic and epigenomic characteristics of â¼ 125,000 latently infected primary CD4+ T cells after reactivation using three different latency reversing agents. Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identified cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrated that a machine learning model trained on these data was 75%-79% accurate at predicting viral reactivation. Finally, we validated the role of two candidate HIV-regulating factors, FOXP1 and GATA3, in viral transcription. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency.
Assuntos
Linfócitos T CD4-Positivos , Fator de Transcrição GATA3 , HIV-1 , Análise de Célula Única , Ativação Viral , Latência Viral , Latência Viral/genética , Humanos , Ativação Viral/genética , Análise de Célula Única/métodos , HIV-1/genética , HIV-1/fisiologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD4-Positivos/metabolismo , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Infecções por HIV/virologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Transcriptoma/genética , Regulação Viral da Expressão GênicaRESUMO
A long-lived latent reservoir of HIV-1-infected CD4 T cells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 T cells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory T cells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.
Assuntos
Infecções por HIV , Histona Desacetilases , Humanos , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Latência Viral/genética , Inibidores de Histona Desacetilases/farmacologia , Epigênese GenéticaRESUMO
Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.
Assuntos
Infecções por HIV , HIV-1 , Animais , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , RNA , Fatores de Transcrição/metabolismo , Ativação Viral , Latência ViralRESUMO
To acquire and maintain directed cell motility, Caenorhabditis elegans sperm must undergo extensive, regulated cellular remodeling, in the absence of new transcription or translation. To regulate sperm function, nematode sperm employ large numbers of protein kinases and phosphatases, including SPE-6, a member of C. elegans' highly expanded casein kinase 1 superfamily. SPE-6 functions during multiple steps of spermatogenesis, including functioning as a "brake" to prevent premature sperm activation in the absence of normal extracellular signals. Here, we describe the subcellular localization patterns of SPE-6 during wild-type C. elegans sperm development and in various sperm activation mutants. While other members of the sperm activation pathway associate with the plasma membrane or localize to the sperm's membranous organelles, SPE-6 surrounds the chromatin mass of unactivated sperm. During sperm activation by either of two semiautonomous signaling pathways, SPE-6 redistributes to the front, central region of the sperm's pseudopod. When disrupted by reduction-of-function alleles, SPE-6 protein is either diminished in a temperature-sensitive manner (hc187) or is mislocalized in a stage-specific manner (hc163). During the multistep process of sperm activation, SPE-6 is released from its perinuclear location after the spike stage in a process that does not require the fusion of membranous organelles with the plasma membrane. After activation, spermatozoa exhibit variable proportions of perinuclear and pseudopod-localized SPE-6, depending on their location within the female reproductive tract. These findings provide new insights regarding SPE-6's role in sperm activation and suggest that extracellular signals during sperm migration may further modulate SPE-6 localization and function.