Assembly of HIV -1 and Cellular Transcription Elongation Complexes

描述(由Candiate提供)：转录对于HIV-1转录其基因组和产生新的病毒颗粒以维持感染是必不可少的。HIV-1的转录程序依赖于TAT，TAT是一种病毒编码的转录激活因子，它通过与新生病毒RNA上的转录延伸因子P-TEFb组装来激活起始和延伸之间的转换。将TAT组装成转录复合体的详细步骤，即P-TEFb的招募和激活，仍未完成。这项研究计划将使用跨学科方法，如病毒学、生物化学/结构生物学和全基因组方法，以揭示TAT激活机制和艾滋病发病机制的新见解。我将描述HIV-1转录预启动复合体的组装，并定义复合体在非活性、TAT激活和TAT抑制状态下的组成。该项目将在加州大学旧金山分校生物化学和生物物理系的丰富社区进行，该学院拥有最先进的技术和非常互动的氛围，合作者在病毒学以外的领域拥有丰富的经验，如体外分析、蛋白质组学、相互作用网络和结构生物学。我的职业发展计划包括接受蛋白质组学和结构生物学方面的培训和指导，以完成HIV-1组装和选定的细胞复合体的特征。在指导阶段提出的工作在很大程度上建立在初步数据和一系列创新方法的基础上，这些方法导致提出了一个修订的病毒转录复合体组装模型，其中TAT和非活性P-TEFb在该过程的早期被加载。这些初步数据，以及我们捕获转录复合体的方法，将被探索以定义TAT组装和转录激活是如何发生的。对于独立的阶段，我将利用在体外分析、蛋白质组学和全基因组方法中获得的经验来检查细胞转录复合体的组装。我将定义1)TAT通过在其启动子上组装而调节的基因亚集，2)启动子调控元件和复合体组成，以及3)基于P-TEFb的TAT表达和RNAi以及选定的因子与基因表达谱的相关性。 相关性：该项目将使用创新的体外分析和全基因组范围的方法来表征HIV-1 Tat和延伸因子组装成病毒和细胞转录复合体的特征。我们的目标是揭示转录复合体组装的基础，以控制细胞的动态平衡，了解TAT在艾滋病发病机制中的作用，并帮助设计替代策略来对抗病毒复制。

DESCRIPTION (provided by candidate): Transcription is essential for HIV-1 to transcribe its genome and for the production of new viral particles to perpetuate the infection. The transcriptional program of HIV-1 relies on Tat, a viral encoded transcriptional activator, which functions by assembling with the transcription elongation factor P-TEFb on nascent viral RNAs to activate the transition between initiation and elongation. Detailed dissection of the steps of Tat assembly into transcription complexes, P-TEFb recruitment and activation, is still incomplete. This Research Plan will use interdisciplinary approaches, such virology, biochemistry/structural biology and genome-wide approaches to uncover new insights into the mechanism of Tat activation and the pathogenesis of AIDS. I will characterize the assembly of HIV-1 transcription preinitiation complexes and define the composition of complexes in their inactive, Tat-activated and Tat-inhibited states. The project will be conducted at UCSF, in the rich community of the Dept of Biochemistry and Biophysics, which has state-of-the-art technologies and a very interactive atmosphere of collaborators with vast experience in fields other than virology, such as in vitro assays, proteomics, network of interactions and structural biology. My career development plan includes receiving training and mentorship in proteomics and structural biology to complete characterization of the assembly of HIV-1 and selected cellular complexes. The work proposed during the mentored phase builds heavily on preliminary data and a set of innovative approaches that led to the proposal of a revised model for the assembly of viral transcription complexes, where Tat and inactive P-TEFb are loaded early in the process. This preliminary data, along with our approach to capture transcription complexes, will be explored to define how Tat assembly and transcription activation occurs. For the independent phase, I will make use of the experience gained in in vitro assays, proteomics and genome-wide approaches to examine the assembly of cellular transcription complexes. I will define 1) the subset of genes that Tat regulates by assembly at their promoters, 2) promoter regulatory elements and complexes composition, and 3) correlation with gene expression profiling based on Tat expression and RNAi of P-TEFb and selected factors. RELEVANCE: This project will use innovative in vitro assays and genomic-wide scale approaches to characterize the assembly of HIV-1 Tat and elongation factors into viral and cellular transcription complexes. We aim at uncovering the basis of transcription complex assembly for cell homeostasis control, learning the role of Tat in the pathogenesis of AIDS and helping in the design of alternative strategies to combat viral replication.