Regulation of Transcriptional Elongation by HIV-1 Tat

HIV-1 Tat 对转录延伸的调节

• 批准号: 7925117
• 负责人: QIANG ZHOU
• 金额: $ 31.48万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7925117
• 关键词: Affinity Chromatography; Anti-HIV Therapy; Binding; Biochemical; Bromodomain; Calcineurin; Cells; Characteristics; Chromatin; Column Chromatography; Complex; Data; Elongation Factor; Fractionation; Funding; General Transcription Factors; Genes; Genetic Transcription; HIV; HIV-1; Human; In Vitro; Infection; Investigation; Lead; Lentivirus Vector; Libraries; Maintenance; Mediating; Minor; Nuclear; Nuclear Protein; Nuclear Proteins; Phosphorylation; Phosphotransferases; Positive Transcriptional Elongation Factor B; Process; Protein Dephosphorylation; Protein Isoforms; Proteins; RNA; RNA Interference; RNA Polymerase II; Recruitment Activity; Regulation; Signal Transduction; Small Interfering RNA; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Staging; Stimulus; Structure; TAF7 gene; Tars; Testing; Transcript; Transcription Elongation; Transcriptional Regulation; Viral; Virus; base; cofactor; cyclin T1; cyclin-dependent kinase-activating kinase; extracellular; genetic regulatory protein; in vivo; insight; negative elongation factor; novel; overexpression; public health relevance; research study; response; tat Protein; therapeutic gene; trafficking; transcription factor TFIIH; vector

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.

描述（由申请人提供）：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，位于所有病毒转录物5'末端的达特和TAR RNA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。此外，核P-TEFb的主要储库被隔离在无活性的7SK snRNP中。进一步的分析表明，响应于Ca 2+信号传导，P-TEFb在保守的Cdk 9 T环被PP 11和PP 2B去磷酸化后从7SK snRNP释放。去磷酸化的P-TEFb优先被Brd 4结合，Brd 4将其募集到转录前起始复合物中。由于Cdk 9 T环的磷酸化对于P-TEFb活性是必需的，因此预期T环在稍后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的完全活性。鉴于P-TEFb是生产性HIV-1感染所必需的，本提案的目的是研究P-TEFb调控的各种模式如何由其相关因子，一个假定的Cdk 9特异性CAK和HIV-1达特/TAR将影响HIV-1的转录和复制。提出的实验是研究是否可以操纵各种P-TEFb相关因子的表达和活性来控制HIV-1复制和潜伏期。结合有针对性的调查和全面的，公正的筛选将被用来确定Cdk 9特异性CAK和阐明其磷酸化P-TEFb的机制和功能意义。为了确定P-TEFb的达特/TAR活化的机制，将检查达特/TAR对HIV-1转录的不同阶段的Cdk 9 T环的磷酸化状态的影响、Tat-TAR-P-TEFb复合物内可能存在的新组分以及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，以确定有助于激活P-TEFb和HIV-1转录的新因素，并提供有关P-TEFb如何刺激HIV-1和细胞基因转录的新见解。更好地理解P-TEFb控制HIV-1复制和潜伏期的机制以及达特/TAR在调节该过程中的多功能性将为抗HIV治疗的新靶点的鉴定提供信息。公共卫生相关性：P-TEFb的Cdk 9-细胞周期蛋白T1异二聚体是HIV-1转录的达特激活的宿主细胞辅因子。所提出的实验将使我们能够研究P-TEFb依赖的HIV-1转录和复制如何受到以下因素的控制：（1）各种P-TEFb相关的细胞因子;（2）推定的Cdk 9活化激酶;以及（3）HIV-1达特蛋白和TAR RNA，它们不仅仅是简单地将P-TEFb募集到病毒LTR中。这些实验将导致识别有助于激活P-TEFb和HIV-1转录的新因子，并为P-TEFb如何控制HIV-1和细胞基因的转录延伸提供新的见解。更好地了解P-TEFb调节HIV-1复制和潜伏期的机制以及达特/TAR在调节该过程中的多功能性可能导致抗HIV治疗的新靶点的鉴定。