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由于数量少而变得复杂 在循环中发现的潜伏感染细胞，获得全面的成套的 组织样本，缺乏已知的表型标记，可以区分潜在的 受感染的细胞与未受感染的细胞之间的差异，以及关于组织储库行为的有限信息 in vivo.主要使用HIV潜伏期的细胞系模型进行的机制研究， 表明病毒的再活化需要表观遗传学沉默的前病毒的反式激活， 病毒达特蛋白与宿主转录延伸辅因子P-TEFb和超转录因子P-TEFb复合 延伸复合物（SEC）。对于HIV潜伏期的研究至关重要的是，额外的P-TEFb对照 机制存在于静息记忆CD 4 + T细胞中，其中CycT 1蛋白水平急剧升高。 降低我们最近还在原代T细胞中发现，CDK 9以非活性状态存在， 与Hsp 90/Cdc 37结合。因此，需要激活特定的T细胞信号通路， 以在原代细胞中组装功能性7SK snRNP复合物。使用精制的高重现性 艾滋病毒潜伏期的主要细胞模型（QUECEL模型），我们将解决两个关键未解决的问题，但 HIV潜伏期的转录控制的基本问题：（1）T细胞信号如何 通路调节记忆性CD 4 + T细胞中P-TEFb、7SK snRNP和SEC的组装？ (2)什么样的Tat依赖性和非依赖性T细胞分子机制允许交换 P-TEFb从7SK snRNP到SEC并最终到潜伏的HIV前病毒？我们的具体 目的是研究7SK snRNP的生物合成和拆卸的调节， 翻译修饰和T细胞信号通路（目标1），应用荧光成像， P-TEFb向潜伏前病毒的时空分布和递送（目的2），并定义 在前病毒再活化过程中，7SK snRNP中P-TEFb交换的生物化学（目的 3）。关键的技术突破，使这项工作与以往几乎所有 研究HIV转录调控的一个重要原因是我们现在有了可靠的原代细胞模型 艾滋病毒潜伏期和再激活使用原代细胞可能具有挑战性，因为相对而言， 可用的单元数量有限。因此，我们强调使用成像实验 和高灵敏度的ChIP-Seq和RNA-Seq检测。限定 导致P-TEFb生物合成的分子和细胞生物学机制及其向 HIV启动子应该提供药理学靶点的确定性鉴定， 开发新的和有效的延迟逆转剂类别所需的。

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.