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

在静息 T 细胞中建立 HIV-1 染色质：Vpr、潜伏期和 H2A.Z

• 批准号: 10329921
• 负责人: DAVID N LEVY
• 金额: $ 39.63万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10329921
• 关键词: Apoptosis; Binding; Biological Assay; CD4 Positive T Lymphocytes; Cell Cycle Arrest; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Chronic; Complex; DNA; DNA Damage; Data; Disputes; Epigenetic Process; Event; Evolution; Gene Expression; Genes; Genetic Transcription; Glean; HIV; HIV Infections; HIV-1; Histone Deacetylation; Histone H2A; Histones; Immediate-Early Proteins; Infection; Lead; Lentivirus; Mediating; Modification; Molecular; Nucleosomes; Pathway interactions; Pharmaceutical Preparations; Positive Transcriptional Elongation Factor B; Post-Translational Protein Processing; Process; Promoter Regions; Protein Isoforms; Proteins; Proviruses; RNA; Regulation; Reporter; Repression; Rest; Reverse Transcription; Role; Signal Transduction; Stimulus; Structure; T-Lymphocyte; Testing; Time; Transactivation; Transcriptional Regulation; Transformed Cell Line; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Replication; Work; design; epigenetic regulation; histone methylation; insight; latent infection; mutant; novel; preservation; promoter; purge; recruit; response; small molecule inhibitor; therapeutic development; transcriptome sequencing; vpr Gene Products

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.

项目概要 静息 CD4 T 细胞的潜伏 HIV 感染对寻求清除治疗提出了巨大的挑战 艾滋病病毒从体内排出。延迟的建立和调节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。重新激活潜伏艾滋病毒的药物必须影响 这些结构是为了让 HIV 转录和病毒表达。然而，由于未知的原因，并非所有 完整的原病毒对潜伏期逆转剂有反应，因此为治疗带来了特别麻烦的障碍。 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）将极大地促进艾滋病毒治疗控制的发展 1 延迟。该项目寻求这样的理解，并建立在广泛的初步研究的基础上，揭示了 对这些过程的重要新见解。我们首次研究 HIV-1 染色质的初始阶段 安装，发现它发生在逆转录的同时或之后不久，并且之前 整合到细胞 DNA 中。我们发现在没有病毒蛋白的情况下安装了抑制性染色质 Vpr 在病毒粒子中传递。 Virion Vpr 显着增加转录活性的数量 感染静息 CD4 T 细胞后的前 4 天。当 Vpr 存在时，核小体 控制 HIV 表达的方式被修改，以促进 HIV 复制并阻止抑制性的安装 染色质。例如，仅当病毒粒子 Vpr 可用时才安装替代组蛋白 H2A.Z，并且 H2A.Z 是 暂停但反应迅速的发起人的中央组织者。如果没有 Vpr，潜伏原病毒的染色质就会发生变化。 在更受抑制的结构上，导致更多的原病毒对潜伏期逆转剂没有反应。 新的 RNA-seq 数据表明病毒粒子 Vpr 重新编程染色质核心基因表达途径 组织和转录调控。该项目将描述 HIV 染色的初始步骤 以及导致可逆潜伏期和不可逆抑制的长期过程。目标1将系统地 分析静息 CD4 T 细胞中的早期原病毒染色质以及 Vpr 和 Tat 定向转录对 表观遗传景观。我们的中心假设是 Vpr 指导转录前启动的安装 静息 T 细胞中基础前 Tat 转录的复合物。目标 2 将分析 Vpr 依赖性途径，导致 这些结构。 RNA-seq 数据将被扩展并用于研究新的 Vpr 调控目标，以及已知的 将研究 Vpr 途径对早期事件的影响。目标 3 将检查长期感染和 潜伏期受到目标 1 和目标 2 收集的结构和通路的影响。我们的假设是 Vpr 保护原病毒免受表观遗传抑制和不可逆潜伏期的影响。拟议的研究将提供很多 需要的信息将有助于开发能够清除体内艾滋病毒的疗法，或 无需持续抗病毒治疗即可永久抑制病毒复制。