Cyclin T and HIV1 Tat Transactivation

描述（由申请人提供）：本提案的目标是了解HIV-1 Tat蛋白如何调节HIV-1 LTR的转录，Tat直接与细胞P-TEFb（正转录延伸因子）复合体中CDK9的调节亚基Cyclin T1相互作用。Tat和cyct11与病毒TAR RNA协同结合，将P-TEFb招募到HIV-1启动子上，并促进cdk9介导的RNAPII大亚基c端结构域（CTD）的磷酸化。我们最近发现了刺激Tat活性的其他蛋白质：SKIP（滑雪相互作用蛋白）和gu - α RNA解旋酶II酶，后者在提取物中以RNA依赖的方式与Tat结合。这两种蛋白质在体外增强HIV-I Tat转录，并可能在RNAPII延伸期间发挥作用。为了将这些和其他伸长因子整合到Tat转录激活的整体机制中，我们开发了一种Tat诱导细胞系，该细胞系包含一个整合的HIV-1 LTR- egfp报告基因，可用于染色质免疫沉淀实验，以检查Tat和相关伸长因子在体内向HIV-1 LTR募集的精确动力学和模式。本文提出了五个具体目标：1)鉴定纯化的天然Tat:P-TEFb:TAR复合物的蛋白质组成和翻译后修饰。2)通过鉴定体内和体外转录所需的功能域，以及鉴定与SKIP相互作用以刺激延伸的细胞蛋白，来检验SKIP作为HIV-1转录的积极效应者的作用。3)研究gu - α等RNA解旋酶在体内和体外对Tat活性的影响，以及RNA解旋酶在转录过程中对Tat:P-TEFb:TAR复合体分解的影响。4)表征直接结合Tat和/或CycT1的其他因子和RNA加工酶。5)通过染色质免疫沉淀实验分析整合HIV- 1 LTR启动子中tat定向募集细胞延伸因子、RNA加工因子和染色质重塑活性。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand how the HIV-1 Tat protein regulates transcription from the HIV-1 LTR. Tat interacts directly with Cyclin T1, the regulatory subunit of CDK9 in the cellular P-TEFb (positive transcription elongation factor) complex. Tat and CycT1 1 bind co-operatively to the viral TAR RNA to recruit P-TEFb to the HIV-1 promoter and facilitate CDK9-mediated phosphorylation of the C-terminal domain (CTD) of the RNAPII large subunit. We recently identified additional proteins that stimulate Tat activity: SKIP (ski-interacting protein), and the GU-alpha RNA helicase II enzyme, which associates with Tat in an RNA-dependent manner in extracts Both proteins enhance HIV-I Tat transcription in vitro and may function during RNAPII elongation. To integrate these and other elongation factors into the overall mechanism of Tat transactivation, we developed a Tat-inducible cell line containing an integrated HIV-1 LTR-eGFP reporter gene that can be used for chromatin immunoprecipitation experiments to examine the precise kinetics and pattern of recruitment of Tat and associated elongation factors to the HIV-1 LTR in vivo. Five specific aims are proposed here: 1) Identify the protein composition and post-translational modifications of purified native Tat:P-TEFb:TAR complexes. 2) Examine the role of SKIP as a positive effector of HIV-1 transcription by identifying the functional domains required for transcription in vivo and in vitro and identifying cellular proteins that interact with SKIP to stimulate elongation. 3) Examine the role of GU-alpha and other RNA helicases on Tat activity in vivo and in vitro, and effects of RNA helicases on the disassembly of the Tat:P-TEFb:TAR complex during transcription. 4) Characterize additional factors and RNA processing enzymes that bind directly to Tat and/or CycT1. 5) Analyze the Tat-directed recruitment of cellular elongation factors, RNA processing factors, and chromatin remodeling activities at the integrated HIV- 1 LTR promoter by chromatin immunoprecipitation experiments.