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

项目摘要 潜伏的HIV感染静止的CD 4 T细胞在寻求治疗以清除HIV感染方面提出了一个艰巨的挑战。 艾滋病毒从身体。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，以使艾滋病毒的转录和病毒的表达。然而，由于未知的原因， 完整的前病毒对潜伏期逆转剂有反应，因此对治愈提出了特别棘手的障碍。 更好地了解HIV-1染色质的调节，核小体及其组蛋白的安装 翻译后修饰（PTM）将大大有助于开发对HIV的治疗控制， 1延迟。该项目寻求这样一种理解，并建立在广泛的初步研究基础上， 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，发现它同时发生或逆转录后不久，和之前， 整合到细胞DNA中。我们发现在没有病毒蛋白的情况下， Vpr在病毒体中传递。病毒体Vpr显著增加了转录活性的 在感染静息的CD 4 T细胞后的前4天内，当Vpr存在时， 控制HIV表达的基因以促进HIV复制和阻止抑制性转录因子的安装的方式被修饰。 染色质例如，只有当病毒体Vpr可用时，才安装替代组蛋白H2A.Z，并且H2A.Z是 一个暂停但反应灵敏的发起人的中心组织者。没有Vpr，潜伏原病毒的染色质 在一个更压抑的结构，导致更多的前病毒不响应潜伏逆转剂。 新的RNA-seq数据表明，病毒体Vpr重新编程对染色质至关重要的基因表达途径 组织和转录调控。这个项目将描述艾滋病毒染色的最初步骤 以及导致可逆潜伏和不可逆抑制的长期过程。目标1将系统地 分析静息CD 4 T细胞中的早期前病毒染色质以及Vpr和Tat指导的转录对CD 4 T细胞的影响。 表观遗传景观我们的中心假设是Vpr指导转录前起始的安装， 在静息T细胞中基础前Tat转录的复合物。目标2将分析导致以下结果的Vpr依赖性途径： 这些结构。RNA-seq数据将被扩展并用于研究新的Vpr调控靶点， 将研究Vpr通路对早期事件的影响。目标3将检查长期感染， 在目标1和2中收集的结构和途径的影响下的潜伏期。我们假设Vpr 保护前病毒免受表观遗传抑制和不可逆潜伏。拟议的研究将提供许多 所需的信息，这将有助于开发治疗，可以清除艾滋病毒从身体或 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.