Control of P-TEFb biogenesis and HIV transcription in primary T-cells

原代 T 细胞中 P-TEFb 生物发生和 HIV 转录的控制

• 批准号: 10403547
• 负责人: JONATHAN KARN
• 金额: $ 40.25万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10403547
• 关键词: Address; Affinity; Antibodies; Behavior; Binding Sites; Biochemical; Biochemistry; Biogenesis; Biological; Biological Assay; Blood Circulation; CDK9 Protein Kinase; Cell Line; Cell model; Cells; ChIP-seq; Complex; DNA; Data Set; Development; Dissociation; Enzymes; Equilibrium; FRAP1 gene; Genes; Genetic Transcription; HIV; HIV Infections; Image; Immunofluorescence Immunologic; Immunoprecipitation; In Situ; In Vitro; Kinetics; Ligation; Location; Lymphoid Tissue; Mediating; Methods; Modeling; Modification; Molecular; Monitor; Mutation; Pathway interactions; Patients; Pharmacology; Positive Transcriptional Elongation Factor B; Post-Translational Protein Processing; Properdin; Protein Kinase C; Proteins; Proteomics; Proviruses; RNA; Receptor Signaling; Regulation; Reproducibility; Rest; Role; Signal Pathway; Signal Transduction; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; System; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TCR Activation; Time; Tissue Sample; Tissues; Transactivation; Transcription Elongation; Transcriptional Elongation Factors; Transcriptional Regulation; Translations; Viral; Work; antiretroviral therapy; base; cyclin T1; epigenetic silencing; experimental study; factor EF-P; fluorescence imaging; in vivo; inhibitor; memory CD4 T lymphocyte; peripheral blood; phenotypic biomarker; promoter; protein complex; response; snRNP Biogenesis; spatiotemporal; stem; tat Protein; transcriptome sequencing; virtual

Our understanding of HIV latency and persistence has been complicated by the small numbers of latently infected cells found in the circulation, the difficulty of obtaining comprehensive sets of tissue samples from patients, the lack of known phenotypic markers that can distinguish latently infected cells from uninfected ones, and limited information about the behavior of tissue reservoirs in vivo. Mechanistic studies, conducted primarily using cell line models of HIV latency, have shown that viral reactivation requires transactivation of epigenetically silenced proviruses by the viral Tat protein in complex with the host transcription elongation co-factors P-TEFb and the super elongation complex (SEC). Crucially for the study of HIV latency, additional P-TEFb control mechanisms exist in resting memory CD4+ T cells, where CycT1 protein levels are drastically reduced. We have also recently shown in primary T cells that CDK9 is present in an inactive state bound to Hsp90/Cdc37. Therefore, specific T-cell signaling pathways need to be activated in order to assemble a functional 7SK snRNP complex in primary cells. Using a refined highly reproducible primary cell model of HIV latency (the QUECEL model), we will address two key unsolved, but fundamental, questions on the transcriptional control of HIV latency: (1) How do T-cell signaling pathways regulate the assembly of P-TEFb, 7SK snRNP and the SEC in memory CD4+ T cells? (2) What Tat-dependent and independent T-cell molecular mechanisms allow for the exchange of P-TEFb from 7SK snRNP to the SEC and eventually to the latent HIV provirus? Our specific aims will investigate the regulation of the biogenesis and disassembly of 7SK snRNP by post- translational modifications and T-cell signaling pathways (Aim 1), apply fluorescence imaging of the spatiotemporal distribution and delivery of P-TEFb to the latent provirus (Aim 2) and define the biochemistry of the exchange of P-TEFb from 7SK snRNP during proviral reactivation (Aim 3). The key technological breakthrough, which distinguishes this work from virtually all previous studies of HIV transcription regulation is that we now have available reliable primary cell models for HIV latency and reactivation. Working with primary cells can be challenging since relatively limited numbers of cells are available. We therefore emphasize the use of imaging experiments and highly sensitive ChIP-Seq and RNA-Seq assays in the majority of our experiments. Defining the molecular and cell biological mechanisms leading to P-TEFb biogenesis and its transfer to the HIV promoter should provide the definitive identification of the pharmacological targets that is needed for the development of new and efficient classes of latency reversing agents.

我们对艾滋病毒潜伏期和持久性的理解使少数数字变得复杂 循环中发现的潜在感染细胞的难度 来自患者的组织样本，缺乏可以分辨的已知表型标记 未感染的细胞感染细胞，以及有关组织储层行为的有限信息 体内。机械研究主要使用HIV潜伏期的细胞系模型进行 表明病毒重新激活需要对表观遗传遗传沉默的病毒进行反式激活 与宿主转录伸长的副因素P-TEFB和SUPER相关的病毒TAT蛋白 伸长复合物（SEC）。至关重要的是研究HIV潜伏期，额外的P-TEFB控制 机制存在于静息记忆CD4+ T细胞中，其中Cyct1蛋白水平急剧 减少。我们最近在原代T细胞中还显示了CDK9存在于无活性状态 与HSP90/CDC37绑定。因此，需要按顺序激活特定的T细胞信号通路 在原代细胞中组装功能性的7SK SNRNP复合物。使用高度可重现的 HIV潜伏期的主要细胞模型（Quecel模型），我们将解决两个未解决的关键，但 基本，有关HIV潜伏期转录控制的问题：（1）T细胞信号如何 路径调节记忆CD4+ T细胞中P-TEFB，7SK SNRNP和SEC的组装？ （2）哪种tat依赖性和独立的T细胞分子机制可以交换 从7SK SNRNP到SEC的P-TEFB，最终到潜在的HIV病毒？我们的具体 AIMS将通过后调查7SK SNRNP的生物发生和拆卸的调节 翻译修改和T细胞信号通路（AIM 1），应用荧光成像 P-TEFB的时空分布和向潜在病毒的传递（AIM 2）并定义 病毒重新激活期间，从7SK SNRNP交换P-TEFB的生物化学（AIM 3）。关键的技术突破，将这项工作与几乎所有以前的工作区分开 HIV转录调节的研究是我们现在有可靠的可靠主要细胞模型 用于HIV潜伏期和重新激活。使用原始单元可能会具有挑战性，因为相对 可用数量的细胞数量有限。因此，我们强调使用成像实验 在我们大多数实验中，以及高度敏感的CHIP-SEQ和RNA-SEQ分析。定义 导致P-TEFB生物发生及其转移到的分子和细胞生物学机制 艾滋病毒启动子应提供对药理学靶标的明确识别 开发新的有效类别的潜伏期逆转代理所需的所需。