Cyclin T and HIV1 Tat Transactivation

描述（由申请方提供）：本提案的目标是了解HIV-1达特蛋白如何调节HIV-1 LTR的转录。达特直接与细胞周期蛋白T1相互作用，细胞周期蛋白T1是细胞P-TEFb（正转录延伸因子）复合物中CDK 9的调节亚基。达特和CycT 11协同结合病毒TAR RNA以将P-TEFb募集至HIV-1启动子并促进RNAPII大亚基的C-末端结构域（CTD）的CDK 9介导的磷酸化。我们最近发现了其他刺激达特活性的蛋白质：SKIP（SKI相互作用蛋白）和GU-α RNA解旋酶II酶，其在提取物中以RNA依赖的方式与达特结合。这两种蛋白质在体外增强HIV-I达特转录，并可能在RNAPII延伸过程中发挥作用。为了将这些和其他延伸因子整合到达特反式激活的整体机制中，我们开发了一种Tat诱导型细胞系，其含有整合的HIV-1 LTR-eGFP报告基因，该报告基因可用于染色质免疫沉淀实验，以检查达特和相关延伸因子在体内向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）鉴定纯化的天然达特：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结构域，并鉴定与SKIP相互作用以刺激延伸的细胞蛋白，检查SKIP作为HIV-1转录的阳性效应子的作用。3)检查GU-alpha和其他RNA解旋酶在体内和体外对达特活性的作用，以及RNA解旋酶在转录过程中对达特：P-TEFb：TAR复合物分解的影响。4)表征直接与达特和/或CycT 1结合的其他因子和RNA加工酶。5)通过染色质免疫沉淀实验分析Tat定向募集的细胞延伸因子、RNA加工因子和在整合的HIV- 1 LTR启动子处的染色质重塑活性。

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.