Regulation of HIV-1 Transcription by CDK2

描述(由申请人提供) 项目研究目的：耐药HIV-1毒株的出现对新药的设计提出了挑战。靶向参与HIV-1复制调控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CDK2在HIV转录中具有调节作用。我们认为，CDK2调节HIV-1转录的机制可能包括：(1)CDK2使TAT磷酸化，从而增强P-TAT与细胞周期蛋白T1或组蛋白乙酰转移酶的相互作用，并在HIV-1转录复合体中募集共激活子，或使P-TAT泛素化，从而增强其转录活性或稳定性；(2)CDK2/Cyclin E介导的RNAPII CTD七肽重复序列的磷酸化。在特定的目标1中，我们将研究TAT磷酸化对HIV-1转录的生理意义。具体来说，我们将突变pNL4-3前病毒中Tat的Ser16和Ser46残基，并确定突变病毒的复制动力学，并分析病毒复制过程中Tat的磷酸化。我们还将分析TAT Ser16和Ser46残基突变对TAT与P-TEFb和TAR RNA结合、与组蛋白乙酰转移酶结合的影响，以及TAT的磷酸化是否促进其泛素化或影响TAT蛋白的稳定性。在特定的目标2中，我们将分析CDK2对TAT的磷酸化动力学以及磷酸化的TAT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TAT被CDK2/Cyclin E体外磷酸化的动力学，磷酸化TAT与转录共激活子的相互作用，以及TAT与CDK2/Cyclin E的相互作用。在具体目标3中，我们将确定CDK2影响HIV-1转录的机制。我们将研究抑制CDK2是否会影响人工靶向Cyclin T1或VP16诱导的转录，以及抑制CDK2是否会影响CDK9的细胞活性。我们将确定在体外和培养细胞中，CDK2对TAT的磷酸化是否促进了CDK2与RNAPII转录复合体和CTD的磷酸化之间的联系。 项目发展目标：(1)通过使用SC1资助建立自己的实验室，并通过在SC1资助和国际会议上展示我的研究，发展成为一名独立的调查者；(2)准备并申请SC1资助的第二年的非评分支持，并参加更多赞助的赠款撰写研讨会，以促进赠款撰写技能；以及(3)在霍华德大学启动新的研究和教育项目。拟议研究的基本主题是减少健康差距，提高霍华德大学研究的卓越水平，并允许我转变为一名独立调查员。这项拟议的研究可能指出，宿主CDK2可能成为抗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.