Studies of HIV and Host Transcriptional Control by Tat

描述（由申请人提供）：病毒已经进化出复杂的机制来劫持细胞资源进行复制和传播。转录调控对HIV至关重要 来转录它的基因组并持续感染。HIV的转录程序依赖于达特，其通过与新生病毒RNA（TAR）上的细胞转录延伸因子P-TEFb组装来激活转录起始和延伸之间的转换而发挥作用。达特从启动子组装的7SK snRNP复合物中捕获P-TEFb以激活P-TEFb激酶。然而，P-TEFb从7SK snRNP释放和P-TEFb激酶在与TAR结合时的活化的步骤的详细剖析仍然是不完整的。除了控制病毒转录外，达特似乎还调节宿主转录以促进感染的建立并影响免疫系统的生物学。然而，达特的基因靶点和所使用的分子机制在很大程度上仍不清楚。本研究计划将使用跨学科的方法来阐明达特从非活性储库中捕获P-TEFb以激活病毒转录的机制，并定义达特如何利用细胞复合物来施加建立感染所需的广泛基因表达变化。该项目将在UT西南医学中心进行，该中心拥有最先进的技术和非常丰富的合作者氛围，而不是病毒学领域，如免疫学和计算生物学。所提出的工作主要建立在初步数据的基础上，这是一组生化、遗传、病毒学测定，这些测定导致鉴定出由达特招募到7SK snRNP以释放P-TEFb并激活病毒转录的n酶。此外，一组基因组方法导致直接达特靶基因的鉴定，并定义了达特如何改变达特辅因子的占用率和全基因组范围内的染色质状态图。这些初步数据，沿着计算方法，揭示了由共同机制调节的基因类别，并揭示了细胞蛋白质复合物和染色质修饰酶作为重编程过程的调节剂。该研究项目将：（1）进一步确定P-TEFb通过达特从7SK snRNP释放的机制，（2）描述达特如何靶向细胞复合物以重编程宿主转录，和（3）确定选定的达特靶基因在AIDS发病机制中的作用。总的来说，这些发现将更好地阐明达特激活的机制，以及揭示达特如何操纵生物学相关的细胞基因来改变T细胞过程。

DESCRIPTION (provided by applicant): Viruses have evolved intricate mechanisms for hijacking cellular resources to replicate and spread. Transcription regulation is essential for HIV to transcribe its genome and for persistent infection. The transcriptional program of HIV relies on Tat, which functions by assembling with the cellular transcription elongation factor P-TEFb on nascent viral RNAs (TAR) to activate the transition between transcription initiation and elongation. Tat captures P-TEFb from the promoter-assembled 7SK snRNP complex to activate the P-TEFb kinase. However, detailed dissection of the steps of P-TEFb release from 7SK snRNP and activation of the P-TEFb kinase when bound to TAR, is still incomplete. In addition to controlling viral transcription, Tat appears to modulate host transcription to favor the establishment of infection and affect the biology of immune system. However, the gene targets of Tat and the molecular mechanisms used remain largely unclear. This Research Plan will use interdisciplinary approaches to elucidate the mechanism by which Tat captures P-TEFb from the inactive reservoir to activate viral transcription as well as define how Tat exploits cellular complexes to impose widespread gene expression changes required for the establishment of infection. The project will be conducted at UT Southwestern Medical Center, which has state-of-the-art technologies and a very rich atmosphere of collaborators in fields other than virology such as immunology and computational biology. The work proposed builds heavily on preliminary data, a set of biochemical, genetic, virology assays that led to the identification of n enzyme recruited by Tat to 7SK snRNP to release P-TEFb and activate viral transcription. In addition, a set of genomic approaches led to the identification of direct Tat target genes, and defined how Tat alters the occupancy of Tat cofactors and the chromatin-state maps in a genome-wide scale. This preliminary data, along with a computational approach, revealed gene classes regulated by common mechanisms and revealed cellular protein complexes and chromatin-modifying enzymes as modulators of the reprogramming process. This research project will: (1) further define the mechanism of P-TEFb release from 7SK snRNP by Tat, (2) delineate how Tat targets cellular complexes to reprogram host transcription, and (3) define the role of selected Tat target genes in the pathogenesis of AIDS. Collectively, these discoveries will better elucidate the mechanism of Tat activation as well as reveal how Tat manipulates biologically relevant cellular genes to alter T cell processes.