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: 10591851
• 负责人: Susana T Valente
• 金额: $ 43.79万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591851
• 关键词: Identification; characterization; chromatin; regulators; HIV

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.

摘要 在HIV将前病毒DNA整合到宿主基因组中后，病毒可以保持潜伏或激活 抄写。病毒的Tat蛋白是开关，它可以增强HIV-1启动子的转录延长。 在这两个州之间。由于TAT位于同一启动子的控制之下，因此它提高了自己的能力 通过正反馈循环转录。我们发现二氢皮质酮A(DCA)是一种非常有效的 TAT的抑制剂(1，2)。在从无氧血症患者分离的人CD4+T细胞中，结合DCA和ART 加速HIV-1抑制并防止治疗中断期间病毒反弹，因为HIV-1启动子 仍然受到表观遗传的抑制。HIV-1转录抑制物具有减少颗粒的独特性质 从受感染的细胞中产生。DCA是一种概念证明，这类新的分子可以服从 阻断和锁定功能治疗方法，旨在减少ART和限制期间的残余病毒血症 病毒式反弹。因此，重要的是要了解不仅解释DCA抑制 重新激活，但也调节HIV-1潜伏期的机制，在一般的CD4+T记忆T细胞，以展开 “阻断并锁定”方法，并探索抑制逆转录病毒的替代方案。 人类大约有320种染色质调节剂，它们可以“写”、“擦除”或“读”染色质 修改或重塑核小体拓扑结构。基因表达的特异性来自于组合 染色质修饰的性质，以及相关染色质调节亚基的组装。这样做的理由是 建议是，建立HIV-1潜伏期的因素对病毒重新激活很重要，并且通过识别 抑制它们，就可以达到一种对重新激活具有极强抵抗力的“锁定”沉默状态。 我们建议结合核小体的全面高分辨率作图 HIV病毒启动子区染色质重塑复合体的组织和定位 潜伏期，通过强大的shRNAs池筛选方法询问所有染色质调节因子 在一次实验中并行进行。一次和二次筛选将在新开发的 捕获真正的HIV-1潜伏期并成功脱离CD4+T细胞的原代细胞系统 治疗过艾滋病毒感染的捐赠者。 在项目的R61阶段，我们将能够关联高分辨率核小体 架构数据与其绑定到所有染色质重塑机器家族，并开发全面的 潜伏期内驱动HIV-1基因组染色质活性的信号和因素图。此数据 将支持稳健的假设和目标，以便在R33阶段进行详细测试。我们从这些方面预料到了 候选人，我们可以推断HIV潜伏期是如何被控制的，并开发合理的治疗方法来 调节HIV潜伏期。