Assembly of HIV -1 and Cellular Transcription Elongation Complexes

转录对于HIV-1转录其基因组和产生新的病毒颗粒， 使感染永久化。HIV-1的转录程序依赖于达特，一种病毒编码的转录因子， 激活子，其通过与新生病毒上的转录延伸因子P-TEFb组装而起作用。 RNA激活起始和延伸之间的过渡。详细剖析达特的步骤 组装成转录复合物、P-TEFb募集和激活仍然是不完全的。本研究 计划将使用跨学科的方法，如病毒学，生物化学/结构生物学和全基因组 为揭示达特激活机制和艾滋病发病机制提供了新的思路。我 将表征HIV-1转录前起始复合物的组装，并定义 复合物处于其非活性、Tat活化和Tat抑制状态。该项目将在UCSF进行， 生物化学和生物物理系的丰富社区，拥有最先进的技术， 一个非常互动的气氛，合作者在病毒学以外的领域有着丰富的经验，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， HIV-1和特定细胞复合物的组装过程。在辅导阶段建议开展的工作 在很大程度上建立在初步数据和一套创新方法的基础上， 病毒转录复合物组装的模型，其中达特和无活性的P-TEFb在转录过程的早期被加载。 过程这些初步数据，沿着我们捕获转录复合物的方法，将是 探索如何定义达特组装和转录激活发生。在独立阶段，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， 细胞转录复合体的组装。我将定义1）达特调节的基因子集， 在其启动子处的组装，2）启动子调控元件和复合物组成，以及3）相关性 利用基于P-TEFb的达特表达和RNAi以及所选因子的基因表达谱。

Transcription is essential for HIV-1 to transcribe its genome and for ttie 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.