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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：
10629307
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 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10629307
关键词：
Affinity Antibodies Behavior Binding Binding Sites Biochemical Biochemistry Biogenesis Biological Biological Assay CDK9 Protein Kinase Cell Line Cell model Cells ChIP-seq Circulation Complex DNA Data Set Development Dissociation Enzymes Epigenetic Process Equilibrium FRAP1 gene Fishes 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 Nucleoplasm Pathway interactions Patients Positive Transcriptional Elongation Factor B Post-Translational Protein Processing Productivity 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 Technology Tissue Sample Tissues Transactivation Transcription Elongation Transcriptional Elongation Factors Transcriptional Regulation Translations Viral Work antiretroviral therapy cofactor cyclin T1 experimental study fluorescence imaging in vivo inhibitor memory CD4 T lymphocyte peripheral blood pharmacologic phenotypic biomarker promoter protein complex reactivation from latency 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.

我们对艾滋病毒潜伏期和持久性的理解因数字较少而变得复杂循环中发现的潜伏感染细胞，难以获得全面的患者的组织样本，缺乏已知的表型标志物，可以潜伏地区分来自未感染细胞的感染细胞，以及关于组织储存库行为的有限信息在活体内。主要使用HIV潜伏期的细胞系模型进行的机械性研究已经研究表明，病毒的重新激活需要由表观遗传沉默的前病毒通过病毒Tat蛋白与宿主转录延伸共因子P-TEFb的复合体伸长复合体(SEC)。对于HIV潜伏期的研究至关重要的是，额外的P-TEFb控制静息记忆中的CD4+T细胞存在这种机制，其中CycT1蛋白水平急剧上升减少了。我们最近还在原代T细胞中显示CDK9处于非活性状态与Hsp90/CDC37结合。因此，需要依次激活特定的T细胞信号通路在原代细胞中组装具有功能的7SK SnRNP复合体。使用精致的高度可重复性的 HIV潜伏期的原代细胞模型(QUECEL模型)，我们将解决两个关键问题，但关于HIV潜伏期转录控制的基本问题：(1)T细胞信号如何调控P-TEFb、7SK-SnRNP和SEC在记忆CD4+T细胞中的组装？ (2)TAT依赖和独立的T细胞分子机制允许这种交换 P-TEFb从7SK SnRNP到SEC，最终到潜伏的HIV前病毒？我们的特定 AIMS将研究后7SK SnRNP的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前病毒的递送(目标2)和定义 7SK SnRNP在前病毒再激活过程中P-TEFb交换的生化(AIM) 3)。关键的技术突破，使这项工作有别于几乎所有以前的工作对HIV转录调控的研究是我们现在有了可靠的原代细胞模型用于HIV潜伏期和重新激活。使用原代细胞可能具有挑战性，因为相对可用的单元格数量有限。因此，我们强调使用成像实验在我们的大多数实验中，高灵敏的CHIP-Seq和RNA-Seq分析。定义 P-TEFb生物发生及其转移的分子和细胞生物学机制 HIV启动子应提供明确的药理靶标标识，即开发新型高效的潜伏期反转剂所需。