Identification and characterization of chromatin regulators of HIV-1 latency

HIV-1 潜伏期染色质调节因子的鉴定和表征

• 批准号: 9975693
• 负责人: Susana T Valente
• 金额: $ 93.1万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9975693
• 关键词: ATP phosphohydrolase; Affinity Chromatography; Anti-Retroviral Agents; Architecture; BLT mice; Bacterial Artificial Chromosomes; Binding; Biochemical; Biological; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell model; Cells; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data; Epigenetic Process; Family; Feedback; Gene Expression; Genes; Genetic Transcription; Genome; Goals; HIV; HIV Genome; HIV-1; Human; ISWI; Individual; Interruption; Investigation; Libraries; Link; Mediating; Modeling; Molecular; Mus; Nature; NuRD complex; Nucleosomes; Patients; Pharmacology; Phase; Phenotype; Positioning Attribute; Production; Property; RNA interference screen; Reactive Inhibition; Reproducibility; Residual state; Resistance; Resolution; Retroviral Vector; Sampling; Signal Transduction; Specificity; System; T memory cell; Techniques; Testing; Therapeutic; Tissues; Transcript; Transcription Initiation Site; Viral; Viremia; Virus; Virus Diseases; Writing; base; chromatin modification; chromatin remodeling; combinatorial; cortistatin; experimental study; follow-up; inhibitor/antagonist; insight; integration site; knock-down; mouse model; novel; particle; prevent; promoter; recruit; screening; small hairpin RNA; tat Protein; transcriptome sequencing; viral RNA; viral rebound

Abstract After HIV integration of the proviral DNA into the host genome, the virus can remain latent or activate transcription. The viral Tat protein, which enhances transcript elongation from the HIV-1 promoter, is the switch between these two states. Since Tat resides under the control of the same promoter, it enhances its own transcription via a positive feedback loop. We identified didehydro-Cortistatin A (dCA) as a very potent inhibitor of Tat (1, 2). In human CD4 +T cells isolated from aviremic individuals, combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound during treatment interruption, as the HIV-1 promoter remains epigenetically repressed. HIV-1 transcriptional inhibitors have the unique property of reducing particle production from infected cells. dCA is the proof-of-concept that this novel class of molecules is amenable to block-and-lock functional cure approaches, which aim at reducing residual viremia during ART and limit viral rebound. It is thus important to understand the mechanisms that explain not only dCA's inhibition of reactivation, but also mechanisms regulating HIV-1 latency in CD4+T memory T cells in general, to expand on “block-and-lock” approaches, and explore alternative options for retroviral suppression. There are approximately 320 human chromatin regulators, which “write”, “erase”, or “read” chromatin modifications, or remodel nucleosome topology. Specificity in gene expression derives from the combinatorial nature of chromatin modifications, and assembly of related chromatin regulator subunits. The rationale for this proposal is that factors that establish HIV-1 latency are important for viral reactivation, and that by identifying and inhibiting them, a “locked” state of silencing that is exceedingly resistant to reactivation can be achieved. We propose to combine a comprehensive high-resolution mapping of the nucleosome organization and positioning of chromatin remodeling complexes at the HIV promoter during HIV latency, with a robust pooled shRNAs screening approach to interrogate all chromatin regulatory factors in parallel during a single experiment. Primary and secondary screens will be performed in a newly developed primary cell system that captures bona fide HIV-1 latency, and departs from CD4+T cells from successfully treated HIV infected donors. During the R61 phase of the project we will be able to correlate high-resolution nucleosome architecture data with their binding to all chromatin remodeling machine families and develop a comprehensive picture of the signals and factors that drive chromatin activity at the HIV-1 genome during latency. This data will support robust hypotheses and targets to test in detail during the R33 phase. We anticipate that from these candidates, we can infer how HIV-latency is controlled and develop rational therapeutic approaches to modulate HIV latency.

抽象的 HIV 将原病毒 DNA 整合到宿主基因组中后，病毒可以保持潜伏或激活 转录。病毒 Tat 蛋白是开关，它可以增强 HIV-1 启动子的转录延伸 这两种状态之间。由于 Tat 处于同一发起人的控制之下，因此它增强了自己的能力 通过正反馈环进行转录。我们确定二脱氢皮质抑素 A (dCA) 是一种非常有效的 Tat (1, 2) 抑制剂。在从无病毒血症个体分离的人 CD4 + T 细胞中，将 dCA 与 ART 相结合 作为 HIV-1 启动子，加速 HIV-1 抑制并防止治疗中断期间病毒反弹 仍然受到表观遗传抑制。 HIV-1转录抑制剂具有独特的减少颗粒的性质 受感染细胞的生产。 dCA 是这一新型分子适合的概念验证 阻断和锁定功能性治疗方法，旨在减少 ART 期间的残留病毒血症并限制 病毒反弹。因此，了解 dCA 抑制的机制非常重要。 重新激活，以及调节 CD4+T 记忆 T 细胞中 HIV-1 潜伏期的机制，以扩展 “封锁和锁定”方法，并探索逆转录病毒抑制的替代方案。 人类约有 320 个染色质调节因子，负责“写入”、“擦除”或“读取”染色质 修饰，或重塑核小体拓扑。基因表达的特异性源自组合 染色质修饰的性质以及相关染色质调节亚基的组装。这样做的理由 该提议认为，确定 HIV-1 潜伏期的因素对于病毒重新激活非常重要，并且通过识别 并抑制它们，可以实现一种极难重新激活的“锁定”沉默状态。 我们建议结合核小体的全面高分辨率映射 HIV期间HIV启动子处染色质重塑复合物的组织和定位 潜伏期，采用强大的混合 shRNA 筛选方法来询问所有染色质调控因子 在单个实验中并行进行。主屏幕和辅助屏幕将在新开发的 原代细胞系统捕获真正的 HIV-1 潜伏期，并成功地从 CD4+T 细胞中分离出来 治疗感染艾滋病毒的捐赠者。 在该项目的 R61 阶段，我们将能够关联高分辨率核小体 架构数据及其与所有染色质重塑机器家族的结合，并开发一个全面的 潜伏期驱动 HIV-1 基因组染色质活性的信号和因素的图片。这个数据 将支持在 R33 阶段详细测试的稳健假设和目标。我们预计从这些 候选人，我们可以推断艾滋病毒潜伏期是如何控制的，并开发合理的治疗方法 调节 HIV 潜伏期。