Regulation of HIV Transcription Elongation

描述（由申请人提供）：对建立和维持潜伏期的分子事件的研究因潜伏感染细胞的稀有性和不可接近性而受到阻碍。 HIV原病毒在转录水平受到调节，涉及转录起始、染色质组织和延伸的变化。特别是，转录延伸已被证明是 HIV 表达的限制步骤，克服这一阻碍是病毒因子 Tat 的主要活性。我们假设调节转录延伸和过早转录终止的细胞因素对 HIV 转录有直接影响，包括消除 HIV 转录和建立潜伏期。初步数据表明，在没有 Tat 的情况下，Pol II 持续性是 HIV 转录的检查点，并且 NELF 和 Pcf11 这两个因素负向调节 HIV 转录延伸。使用多种生化方法，包括染色质免疫沉淀、Pol II 足印以及体内和体外转录系统，我们建议确定生化机制并表征负调节 HIV 转录延伸的细胞因子。我们期望这些研究将为艾滋病毒潜伏期的建立、维持和逆转提供新的见解。了解 HIV 转录延伸的调节将为控制和清除不同细胞库中的 HIV 提供新的细胞靶点。公共卫生相关性：成功根除艾滋病毒感染需要了解病毒表达不良的细胞储存库是如何建立和维持的，以及确定是否可以清除这些人群中的艾滋病毒。该提案重点关注抑制 HIV 转录的生化机制，以便深入了解调节 HIV 潜伏期的因素，长期目标是确定控制不同细胞中 HIV 表达的新靶点。

DESCRIPTION (provided by applicant): Studies of the molecular events that establish and maintain latency have been hampered by the rarity and inaccessibility of latently infected cells. HIV provirus is regulated at the level of transcription and involves changes in transcription initiation, chromatin organization and elongation. In particular, transcription elongation has been demonstrated to be a limiting step for HIV expression and overcoming this block is the primary activity of the viral factor Tat. We hypothesize that cellular factors that regulate transcription elongation and premature transcription termination have a direct impact on HIV transcription, including extinguishing HIV transcription and establishing latency. Preliminary data show that Pol II processivity is a check point for HIV transcription in the absence of Tat and that two factors, NELF and Pcf11, negatively regulate HIV transcription elongation. Using a variety of biochemical approaches, including chromatin immunoprecipitation, Pol II foot printing and in vivo and in vitro transcription systems, we propose to determine the biochemical mechanisms and characterize the cellular factors that negatively regulate HIV transcription elongation. We expect that these studies will provide new insights into the establishment, maintenance and reversal of HIV latency. Understanding the regulation of HIV transcription elongation will provide novel cellular targets for controlling and purging HIV in different cellular reservoirs. PUBLIC HEALTH RELEVANCE: Successfully eradicating HIV infection will require understanding how cellular reservoirs that poorly express virus are established and maintained as well as determining whether these populations can be purged of HIV. This proposal focuses on the biochemical mechanisms that repress HIV transcription, in order to gain insights into factors that regulate HIV latency with long term goals of identifying novel targets for controlling HIV expression in different cells.