Regulation of HIV-1 Transcription by CDK2

描述（由申请人提供） 项目研究目的：HIV-1耐药毒株的出现对新药的设计提出了挑战。靶向参与调节HIV-1复制的宿主细胞因子可能是克服HIV-1对抗病毒药物耐药性的一种方法。我们假设CDK 2在HIV转录中具有调节作用。我们认为CDK 2调节HIV-1转录的机制可能包括：（1）CDK 2磷酸化达特，增强P-达特与细胞周期蛋白T1或组蛋白乙酰转移酶的相互作用，募集共激活因子到HIV-1转录复合物中，或泛素化P-达特，增强转录活性或稳定性;和（2）RNAPII CTD七肽重复的CDK 2/细胞周期蛋白E介导的磷酸化。在具体目标1中，我们将研究达特磷酸化对HIV-1转录的生理重要性。具体而言，我们将突变pNL 4 -3前病毒中tat的Ser 16和Ser 46残基，并确定突变病毒的复制动力学，并分析病毒复制期间的达特磷酸化。我们还将分析达特Ser 16和Ser 46残基突变对达特与P-TEFb和TAR RNA结合的影响;组蛋白乙酰转移酶以及达特的磷酸化是否促进其泛素化或影响达特蛋白的稳定性。在具体目标2中，我们将分析CDK 2对达特磷酸化的动力学以及磷酸化的达特与转录共激活因子的相互作用。我们将研究野生型和突变型达特在体外被CDK 2/细胞周期蛋白E磷酸化的动力学;磷酸化的达特与转录辅激活因子的相互作用以及达特与CDK 2/细胞周期蛋白E的相互作用。在具体目标3中，我们将确定CDK 2影响HIV-1转录的机制。我们将研究抑制CDK 2是否影响人工靶向细胞周期蛋白T1或VP 16诱导的转录，以及抑制CDK 2是否影响CDK 9的细胞活性。我们将确定在体外和培养细胞中HIV-1转录期间，CDK 2对达特的磷酸化是否促进CDK 2与RNAPII转录复合物的结合以及CTD的磷酸化。 项目发展目标：（1）通过使用SC 1资金建立自己的实验室并在SCORE资助的国际会议上展示我的研究，发展成为独立的研究者;（2）在SC 1支持的第二年准备并申请非SCORE支持，并参加MORE赞助的赠款写作研讨会，以促进赠款写作技能;（3）在霍华德大学启动新的研究和教育计划。拟议研究的基本主题是减少健康差距，提高霍华德大学的研究卓越性，并允许我过渡到一个独立的调查员。这项研究可能将宿主CDK 2作为抗HIV-1治疗的潜在新靶点。

DESCRIPTION (provided by applicant) Project Research Objectives: The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to overcome the resistance of HIV-1 to anti-viral agents. We hypothesize that CDK2 has a regulatory role in HIV transcription. We propose that the mechanism whereby CDK2 regulates HIV-1 transcription may include (1) phosphorylation of Tat by CDK2 that may enhance interaction of P-Tat with cyclin T1 or histone acetyltransferases and recruitment of co-activators to HIV-1 transcription complex, or ubiquitination of P-Tat that may enhance its transcriptional activity or its stability; and (2) CDK2/cyclin E-mediated phosphorylation of RNAPII CTD heptapeptide repeats. In specific aim 1, we will investigate the physiological importance of Tat phosphorylation for HIV-1 transcription. Specifically, we will mutate Ser16 and Ser46 residues of tat in the pNL4-3 provirus and determine the kinetics of replication of mutant viruses and analyze Tat phosphorylation during viral replication. We will also analyze the effect of mutations of Tat Ser16 and Ser46 residues on Tat binding to P-TEFb and TAR RNA; to histone acetyltransferases and whether phosphorylation of Tat facilitates its ubiquitination or affects the stability of Tat protein. In specific aim 2, we will analyze kinetics of Tat phosphorylation by CDK2 and interaction of phosphorylated Tat with transcriptional co-activators. We will study the kinetics of the phosphorylation of wild and mutant Tat by CDK2/cyclin E in vitro; interaction of phosphorylated Tat with transcriptional co-activators and interaction of Tat with CDK2/cyclin E using molecular modeling. In specific aim 3, we will determine the mechanism whereby CDK2 influences HIV-1 transcription. We will investigate if inhibition of CDK2 affects transcription induced by artificially targeted cyclin T1 or VP16 and whether inhibition of CDK2 affects cellular activity of CDK9. We will determine if phosphorylation of Tat by CDK2 promotes the association of CDK2 with RNAPII transcription complex and phosphorylation of CTD during HIV-1 transcription in vitro and in cultured cells. Project Developmental Objectives: (1) develop into an independent investigator by establishing my own laboratory using SC1 funding and by presenting my research at SCORE-funded and international meetings; (2) prepare and apply for non-SCORE support at year 2 of the SC1 support and participate in a MORE-sponsored grant-writing workshop to facilitate grant-writing skills; and (3) initiate new research and educational programs at Howard University. The underlying theme of the proposed research is to reduce health disparities, to enhance excellence of research at Howard University and to permit my transition into an independent investigator. The proposed research may point to host CDK2 as a potential new target for anti-HIV-1 therapeutics.