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

基本信息

批准号：
10158438
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
项目类别：
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/10158438
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 Structure 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/antagonist memory CD4 T lymphocyte peripheral blood phenotypic biomarker promoter protein complex response snRNP Biogenesis spatiotemporal stem tat Protein transcriptome sequencing virtual

项目摘要

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.

由于数量较少，我们对艾滋病毒潜伏期和持久性的理解变得复杂循环中发现的潜伏感染细胞的数量，获得全面的数据集的难度来自患者的组织样本，缺乏可以潜在区分的已知表型标记来自未感染细胞的感染细胞，以及有关组织储存库行为的有限信息体内。主要使用 HIV 潜伏期细胞系模型进行的机制研究已研究表明，病毒重新激活需要表观遗传沉默的原病毒的反式激活病毒 Tat 蛋白与宿主转录延伸辅助因子 P-TEFb 和超级蛋白复合物伸长复合体（SEC）。对于 HIV 潜伏期的研究至关重要的是，额外的 P-TEFb 控制机制存在于静息记忆 CD4+ T 细胞中，其中 CycT1 蛋白水平急剧下降减少。我们最近还在原代 T 细胞中发现 CDK9 以非活性状态存在结合 Hsp90/Cdc37。因此，需要激活特定的T细胞信号传导途径在原代细胞中组装功能性 7SK snRNP 复合物。使用精制的高度再现性 HIV潜伏期的原代细胞模型（QUECEL模型），我们将解决两个未解决的关键问题，但是关于 HIV 潜伏期转录控制的基本问题：（1）T 细胞信号如何传导通路调节记忆 CD4+ T 细胞中 P-TEFb、7SK snRNP 和 SEC 的组装？（2）Tat依赖和独立的T细胞分子机制允许什么交换 P-TEFb从7SK snRNP到SEC并最终到潜伏的HIV原病毒？我们的具体目标将研究后处理对 7SK snRNP 生物发生和分解的调节翻译修饰和 T 细胞信号通路（目标 1），应用荧光成像 P-TEFb 的时空分布和向潜伏原病毒的传递（目标 2）并定义原病毒再激活过程中 P-TEFb 从 7SK snRNP 交换的生物化学（目的 3）。关键技术突破，使这项工作与几乎所有以前的工作区分开来 HIV转录调控的研究表明我们现在拥有可靠的原代细胞模型 HIV潜伏期和重新激活。使用原代细胞可能具有挑战性，因为相对可用的细胞数量有限。因此我们强调使用成像实验我们的大部分实验都采用高度灵敏的 ChIP-Seq 和 RNA-Seq 检测。定义导致 P-TEFb 生物发生及其转移的分子和细胞生物学机制 HIV 启动子应提供药理靶点的明确鉴定，即开发新型高效的延迟逆转代理所需。