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: 10458119
• 负责人: Susana T Valente
• 金额: $ 92.18万
• 依托单位: UNIVERSITY OF 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458119
• 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; 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 在将病毒DNA整合到宿主基因组中后，病毒可以保持潜在或激活 转录。病毒TAT蛋白增强了来自HIV-1启动子的转录本的伸长率是开关 在这两个状态之间。由于Tat驻留在同一启动子的控制下，因此可以增强自己的 通过正反馈循环转录。我们确定了二乙基 - 皮质蛋白A（DCA）是非常有效的 TAT的抑制剂（1，2）。在人类CD4 +T细胞中，从阿维病个体分离出来，将DCA与ART结合 加速HIV-1抑制并防止治疗中的病毒反弹，因为HIV-1启动子 仍然表观遗传反射。 HIV-1转录抑制剂具有还原粒子的独特特性 受感染细胞的生产。 DCA是概念的证据，表明这种新颖的分子可以适合 阻止锁定功能治疗方法，旨在减少艺术期间残留的病毒血症和限制 病毒反弹。因此，重要的是要了解不仅解释DCA抑制的机制 重新激活，以及通常在CD4+T记忆T细胞中调查HIV-1潜伏期的机制，以扩展 “阻止锁”方法，并探索逆转录病毒抑制的替代选择。 大约有320个人类染色质调节剂，它们“写入”，“擦除”或“读”染色质 修饰或重塑核小体拓扑。基因表达的特异性来自组合 染色质修饰的性质和相关染色质调节剂亚基的组装。理由 建议是建立HIV-1潜伏期的因素对于病毒重新激活至关重要，并且通过识别 并抑制它们，可以实现一种“锁定”的沉默状态，对重新激活具有极大的抵抗力。 我们建议结合核小体的全面高分辨率映射 HIV期间，染色质重塑复合物的组织和定位 潜伏期，采用强大的合并shrnas筛选方法来询问所有染色质调节因素 在单个实验中并联。初级和次要屏幕将在新开发的 捕获真正的HIV-1潜伏期的主细胞系统，并从成功的CD4+T细胞中偏离 治疗的艾滋病毒感染者。 在项目的R61阶段，我们将能够将高分辨率核小体相关联 建筑数据及其与所有染色质重塑机家的结合，并开发全面的 在潜伏期期间，在HIV-1基因组上驱动染色质活性的信号和因素的图片。这个数据 将支持强大的假设和目标在R33阶段进行详细测试。我们预计从这些 候选人，我们可以推断如何控制艾滋病毒的历史，并开发理性疗法的方法 调节艾滋病毒潜伏期。