Regulation of Transcriptional Elongation by HIV-1 Tat

项目总结/摘要 由Cdk 9和细胞周期蛋白T组成的通用转录因子P-TEFb强烈刺激 RNA聚合酶II延伸。也是达特激活HIV-1转录的宿主细胞辅因子。 越来越多的证据表明，达特和TAR RNA，位于所有病毒的5'端， 转录物，不仅将P-TEFb募集到HIV-1 LTR，而且还引起Cdk 9激酶的活化。 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Brd 4募集到染色质模板。另外还有按 核P-TEFb的主要储库被隔离在无活性的7SK snRNP中。进一步分析 表明响应于Ca 2 +-信号传导，P-TEFb从7SK snRNP释放， 通过PP 1和PP 2B使保守的Cdk 9 T环去磷酸化。去磷酸化的P- TEFb优先与Brd 4结合，Brd 4将其募集到转录前起始复合物中。作为 Cdk 9 T环的磷酸化对于P-TEFb活性是必需的，预期T环 在晚期阶段通过尚未鉴定的Cdk活化激酶（CAK）进行再磷酸化， 以恢复P-TEFb的全部活性。考虑到P-TEFb对于产生性HIV-1感染是必不可少的， 本建议的目的是研究如何通过各种方式的P-TEFb调节 其相关因子，一个假定的Cdk 9特异性CAK和HIV-1达特/TAR将影响HIV-1 转录和复制。提出的实验，以调查是否表达和 可以操纵各种P-TEFb相关因子的活性来控制HIV-1复制， 延迟。将有针对性的调查和全面、公正的筛选相结合， 本研究旨在鉴定Cdk 9特异性CAK，并阐明其作用机制和功能。 其磷酸化P-TEFb的意义。为了确定达特/TAR激活的机制， P-TEFb，达特/TAR对Cdk 9 T环在不同阶段的磷酸化状态的影响。 HIV-1转录，Tat-TAR-P-TEFb内可能存在新组分 将检查TFIIH和TAF 7抑制P-TEFb活化的能力。这些 实验将提供一个令人兴奋的机会，以确定新的因素，有助于激活 的P-TEFb和HIV-1转录，并提供新的见解P-TEFb如何刺激 HIV-1和细胞基因的转录。更好地理解 P-TEFb控制HIV-1复制和潜伏期以及达特/TAR在调节这一过程中的多功能性 这一过程将有助于确定抗HIV治疗的新靶点。

Project Summary/Abstract 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 PP1¿ 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.