Regulation of Transcriptional Elongation by HIV-1 Tat

描述(申请人提供)：通用转录因子P-TEFb，由CDK9和细胞周期蛋白T组成，强烈刺激RNA聚合酶II的延伸。它也是激活HIV-1转录的宿主细胞辅因子。越来越多的证据表明，TAT和位于所有病毒转录本5‘端的TAR RNA不仅可以将P-TEFb募集到HIV-1 LTR中，而且还可以引起CDK9激酶的激活。对于细胞基因的一般转录，在当前资助期间获得的数据表明，P-TEFb被溴结构域蛋白Brd4招募到染色质模板中。此外，核P-TEFb的一个主要储存库被隔离在不活跃的7SK SnRNP中。进一步的分析表明，当PP11和PP2B对保守的CDK9 T环进行去磷酸化时，P-TEFb从7SK SnRNP中释放出来，作为对钙信号的响应。去磷酸化的P-TEFb优先与Brd4结合，Brd4将其招募到转录前起始复合体中。由于CDK9T-loop的磷酸化对P-TEFb的活性至关重要，T-loop有望在稍后阶段被一种未知的CDK激活激酶(CAK)重新磷酸化，以恢复P-TEFb的全部活性。鉴于P-TEFb对于HIV-1的生产性感染是必不可少的，本提案的目的是研究其相关因子、假定的CDK9特异性CAK和HIV-1TAT/TAR所实施的P-TEFb调控的各种模式将如何影响HIV-1的转录和复制。建议进行实验，以调查各种P-TEFb相关因子的表达和活性是否可以被操纵来控制HIV-1的复制和潜伏期。我们将采用针对性研究和全面、无偏见的筛选相结合的方法来鉴定CDK9特异性的CAK，并阐明其磷酸化P-TEFb的机制和功能意义。为了确定P-TEFb的TAT/TAR激活机制，我们将检测TAT/TAR在HIV-1转录的不同阶段对CDK9 T环磷酸化状态的影响，TAT-TAR-P-TEFb复合体中可能存在新的成分，以及TFIIH和TAF7抑制P-TEFb激活的能力。这些实验将提供一个令人兴奋的机会，以确定有助于激活P-TEFb和HIV-1转录的新因素，并为P-TEFb如何刺激HIV-1和细胞基因的转录提供新的见解。更好地理解P-TEFb控制HIV-1复制和潜伏期的机制以及TAT/TAR在调节这一过程中的多功能性将有助于确定抗HIV治疗的新靶点。公共卫生相关性：P-TEFb的CDK9-Cyclin T1异源二聚体是激活HIV-1转录的TAT的宿主细胞辅助因子。拟议的实验将使我们能够研究依赖P-TEFb的HIV-1转录和复制如何受到以下因素的控制：(1)各种与P-TEFb相关的细胞因子；(2)可能的CDK9激活蛋白；(3)HIV-1Tat蛋白和TAR RNA，其功能不仅仅是将P-TEFb招募到病毒LTR。这些实验将有助于识别有助于激活P-TEFb和HIV-1转录的新因素，并为P-TEFb如何控制HIV-1和细胞基因的转录延长提供新的见解。更好地了解P-TEFb调节HIV-1复制和潜伏期的机制以及TAT/TAR在调节这一过程中的多功能性可能会导致确定抗HIV治疗的新靶点。

DESCRIPTION (provided by applicant): The general transcription factor P-TEFb, consisting of Cdk9 and cyclin T, strongly stimulates RNA polymerase II elongation. It is also a host cell cofactor for Tat activation of HIV-1 transcription. Accumulating evidence suggests that Tat and the TAR RNA, located at the 5' end of all viral transcripts, not only recruit P-TEFb to the HIV-1 LTR but also cause the activation of Cdk9 kinase. For general transcription of cellular genes, data obtained during the current funding period indicate that P-TEFb is recruited to chromatin templates by the bromodomain protein Brd4. In addition, a major reservoir of nuclear P-TEFb is sequestered in the inactive 7SK snRNP. Further analyses indicate that in response to Ca2+signaling, P-TEFb is released from 7SK snRNP upon the dephosphorylation of the conserved Cdk9 T-loop by PP11 and PP2B. The dephosphorylated P- TEFb is preferentially bound by Brd4, which recruits it to the transcription pre-initiation complex. As the phosphorylation of Cdk9 T-loop is essential for P-TEFb activity, the T-loop is expected to undergo rephosphorylation by an as yet unidentified Cdk activating kinase (CAK) at a later stage in order to restore full activity to P-TEFb. Given that P-TEFb is essential for productive HIV-1 infection, the objective of this proposal is to examine how the various modes of P-TEFb regulation exerted by its associated factors, a putative Cdk9-specific CAK and the HIV-1 Tat/TAR will impact HIV-1 transcription and replication. Proposed are experiments to investigate whether the expression and activity of various P-TEFb-associated factors can be manipulated to control HIV-1 replication and latency. A combination of targeted investigations and comprehensive, unbiased screens will be employed to identify the Cdk9-specific CAK and elucidate the mechanism and functional significance of its phosphorylation of P-TEFb. To determine the mechanism of Tat/TAR activation of P-TEFb, the impact of Tat/TAR on phosphorylation status of the Cdk9 T-loop at different stages of HIV-1 transcription, the possible existence of novel components within the Tat-TAR-P-TEFb complex, and the ability of TFIIH and TAF7 to inhibit P-TEFb activation will be examined. These experiments will offer an exciting opportunity to identify novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb stimulates transcription of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb controls HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process will be informative toward the identification of new targets for anti-HIV therapy. PUBLIC HEALTH RELEVANCE: The Cdk9-cyclin T1 heterodimer of P-TEFb is a host cell cofactor for Tat activation of HIV-1 transcription. The proposed experiments will allow us to investigate how the P-TEFb-dependent HIV-1 transcription and replication can be controlled by: (1) the various P-TEFb-associated cellular factors; (2) a putative Cdk9 activating kinase; and (3) the HIV-1 Tat protein and TAR RNA that can do more than simply recruiting P-TEFb to the viral LTR. These experiments will lead to the identification of novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb controls transcriptional elongation of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb regulates HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process may lead to the identification of new targets for anti-HIV therapy.