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

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 机制存在于静止记忆的CD4T细胞中，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CD4T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。

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.