Regulation of Transcriptional Elongation by HIV-1 Tat

项目摘要/摘要 由CDK9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伸长。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TAR RNA位于所有病毒的5‘端 转录本不仅能将P-TEFb募集到HIV-1LTR中，而且还能激活CDK9激酶。 对于细胞基因的一般转录，在当前资助期内获得的数据表明 P-TEFb被溴结构域蛋白Brd4招募到染色质模板中。此外，a 核P-TEFb的主要储存库被隔离在非活动的7SK SnRNP中。进一步分析 表明响应于钙信号，P-TEFb从7SK的SnRNP中释放出来 PP1和PP2B对保守的CDK9 T环的去磷酸化作用。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。AS CDK9 T-环的磷酸化是P-TEFb活性所必需的，T-环有望 在较晚的阶段经历一种尚不确定的CDK激活激酶(CAK)的重新磷酸化 以恢复P-TEFb的全部活性。鉴于P-TEFb对于生产性HIV-1感染是必不可少的， 这项提案的目标是研究P-TEFb监管的各种模式是如何由 其相关因素，假定的CDK9特异性CAK和HIV-1Tat/TAR将影响HIV-1 转录和复制。建议进行实验，以调查表达式和 各种P-TEFb相关因子的活性可以被操纵来控制HIV-1的复制和 延迟。有针对性的调查和全面、公正的筛查相结合将是 用于鉴定CDK9特异性的CAK并阐明其机制和功能 其磷酸化P-TEFb的意义。确定TAT/TAR活化的机理 P-TEFb，TAT/TAR对不同阶段CDK9 T环磷酸化状态的影响 HIV-1转录，TAT-TAR-P-TEFb中可能存在新的成分 TFIIH和TAF7抑制P-TEFb激活的能力将被检测。这些 实验将提供一个令人兴奋的机会来确定有助于激活的新因素 并为P-TEFb如何刺激HIV-1转录提供了新的见解 HIV-1和细胞基因的转录。更好地理解这一机制 P-TEFb控制HIV-1复制和潜伏期以及TAT/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艾滋病毒治疗的新靶点。

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.