Establishing HIV-1 chromatin in resting T cells: Vpr, latency, and H2A.Z

项目摘要 静息的CD4T细胞潜伏的HIV感染在寻求治疗净化方面是一个巨大的挑战 人体内的艾滋病病毒。延迟在很大程度上是通过修改 结合在病毒启动子区域周围的核小体。重新激活潜伏的艾滋病毒的药物必须影响 这些结构才能允许HIV转录和病毒表达。然而，由于未知的原因，并不是所有的 完整的前病毒对潜伏期反转剂有反应，因此给治愈带来了特别麻烦的障碍。 对HIV-1染色质的调节、核小体及其组蛋白的安装有更深入的了解 翻译后修饰(PTM)将极大地促进对HIV的治疗控制的发展。 1延迟。该项目寻求这样的理解，并建立在广泛的初步研究基础上，揭示 对这些过程的重要新见解。我们首次研究了HIV-1染色质的初始阶段 安装，发现它同时发生或在逆转录后不久发生，并且在 整合到细胞DNA中。我们发现，在没有病毒蛋白的情况下，抑制性染色质是存在的 VPR正在病毒粒子中传递。病毒粒子vpr显著增加转录活性的数量 静息CD4T细胞感染后前4d出现前病毒。当VPR存在时，核小体 控制HIV表达被以促进HIV复制和阻止安装抑制物的方式进行修改 染色质。例如，替代组蛋白H_2A.Z仅在病毒粒子VPR可用时安装，而H_2A.Z是 由暂停但反应迅速的推动者组成的中央组织者。如果没有VPR，潜伏期前病毒的染色质就会 在更受抑制的结构上，导致更多的前驱体对潜伏期反转剂不起作用。 新的rna-seq数据显示病毒粒子vpr重新编程染色质中心的基因表达途径。 组织和转录调控。这个项目将描述HIV染色化的两个初始步骤 以及导致可逆潜伏期和不可逆转压抑的长期过程。目标1将系统地 静息CD4T细胞早期前染色质的分析及VPR和TAT定向转录的影响 表观遗传景观。我们的中心假设是VPR指导转录启动前的安装 静息T细胞基础Pre-Tat转录的复合体。目标2将分析导致VPR依赖的通路 这些结构。RNA-SEQ数据将被扩展并用于研究新的VPR调控靶标，并已知 将调查VPR通路对早期事件的影响。目标3将检查长期感染和 从目标1和目标2收集的结构和通路的影响下的潜伏期。我们的假设是VPR 保护前病毒免受表观遗传抑制和不可逆转的潜伏期。拟议的研究将提供许多 所需的信息将有助于开发可将艾滋病毒从体内清除或 永久抑制病毒复制，而无需持续的抗病毒治疗。

Project Summary Latent HIV infection of resting CD4 T cells present a formidable challenge in the pursuit of treatments to purge HIV from the body. Latency is established and regulated in large part through the modifications of the nucleosomes that are bound around the viral promoter region. Drugs that reactivate latent HIV must influence these structures in order to allow HIV transcription and virus expression. However, for unknown reasons not all intact proviruses respond to latency reversing agents and thus present a particularly troublesome barrier to cure. A greater understanding of the regulation of HIV-1 chromatin, the installation of nucleosomes and their histone post translational modifications (PTM) will contribute greatly to the development of therapeutic control over HIV- 1 latency. This project seeks such an understanding and builds upon extensive preliminary studies revealing important new insights into these processes. For the first time we investigate the initial stages of HIV-1 chromatin installation, finding that it occurs either contemporaneously or soon after reverse transcription, and before integration into the cellular DNA. We find that repressive chromatin is installed in the absence of the viral protein Vpr being delivered in the virion. Virion Vpr dramatically increases the number of transcriptionally active proviruses in the first 4 days after infection of resting CD4 T cells. When Vpr is present, the nucleosomes that control HIV expression are modified in ways that facilitate HIV replication and block installation of repressive chromatin. For example, the alternate histone H2A.Z is installed only when virion Vpr is available, and H2A.Z is a central organizer of paused but responsive promoters. Without Vpr, the chromatin of latent proviruses takes on a more repressed structure, resulting in more proviruses that do not respond to latency reversing agents. Novel RNA-seq data demonstrate that virion Vpr reprograms gene expression pathways central to chromatin organization and transcriptional regulation. This project will describe both the initial steps in HIV chromatization and the long term processes that lead to reversible latency and irreversible repression. Aim 1 will systematically analyze early proviral chromatin in resting CD4 T cells and the influence of Vpr and Tat-directed transcription on the epigenetic landscape. Our central hypothesis is that Vpr directs installation of the transcriptional pre-initiation complex for basal pre-Tat transcription in resting T cells. Aim 2 will analyze Vpr-dependent pathways leading to these structures. RNA-seq data will be expanded and used to study novel Vpr targets of regulation, and known Vpr pathways will be investigated for their influence on early events. Aim 3 will examine long term infection and latency under the influence the structures and pathways gleaned from Aims 1 and 2. Our hypothesis is that Vpr protects the provirus from epigenetic repression and irreversible latency. The proposed studies will provide much needed information that will assist in the development of therapeutics that can purge HIV from the body or permanently repress virus replication without continual antiviral treatments.