REGULATION OF HIV-1 TRANSCRIPTION BY CDK2

描述(申请人提供)：抗药性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。本研究旨在阐明宿主细胞周期依赖蛋白2(CDK2)在HIV-1Tat蛋白介导的HIV-1转录调控中的作用。HIV-1 Tat通过与CDKg/Cyclin T1结合并诱导RNA聚合酶II(RNAPII)C末端结构域(CTD)过度磷酸化来刺激病毒转录的延长。我们的初步数据表明，HIV-1Tat还可能利用CDK2/Cyclin E来增加CTD的磷酸化，并在细胞周期的G1/S阶段刺激HIV-1的转录。我们假设，与CDKg一起，CDK2是TAT依赖的转录所需的转录延伸复合体的一部分。我们认为(1)CDK2/Cyclin E结合到TAT的激活区并磷酸化TAT，(2)TAT与CDk2/Cyclin E的这种动态结合以及TAT与CTD的相互作用刺激CTD七肽重复序列的第二位丝氨酸(Ser-2)的磷酸化，以及(3)CDK2对Ser-2的这种磷酸化参与了HIV-1转录延伸的调节。我们还提出了TAT诱导CDK2对Ser-2的磷酸化和CDK9对TAT诱导的Ser-5的磷酸化来刺激HIV-1转录本的延长。在目标1中，我们将确定在培养细胞中CDK2是否与TAT相互作用，以及这种相互作用是否对HIV-1转录过程中RNAPII七联体的Ser-2的磷酸化起重要作用。在目标2中，我们将确定CDK2是否使培养细胞中的TAT磷酸化，以及TAT的磷酸化是否对HIV-1转录起重要作用。在目标3中，我们将确定TAT是否与细胞周期蛋白E相互作用，以及这种相互作用是否调节HIV-1的转录。这项拟议的研究将确定CDK2/Cyclin E和CDK9/Cyclin T1是否是体内TAT反式激活的共同激活剂。这项研究的创新之处在于建立了TAT在HIV-1转录中作为酶辅助因子的先前未知的功能，同时结合酶CDK2和底物CTD，以促进CTD Ser-2的磷酸化。这项研究具有重要意义，因为它可能揭示宿主CDK2调控HIV-1激活转录的机制，并可能指出宿主CDK2可能成为抗HIV-1治疗的潜在新靶点。

DESCRIPTION (provided by applicant): 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. The proposed research is designed to delineate the role of host cell-cycle dependent kinase 2 (CDK2) in the regulation of HIV-1 transcription mediated by HIV-1 Tat protein. HIV-1 Tat stimulates elongation of viral transcription by binding to CDKg/cyclin T1 and inducing hyperphosphorylation of the C-terminal domain (CTD) of RNA Polymerase II (RNAPII). Our preliminary data indicate that HIV-1 Tat may also utilizes CDK2/cyclin E to increase CTD phosphorylation and to stimulate HIV-1 transcription at the G1/S phase of the cell cycle. We hypothesize that, along with CDKg, CDK2 is part of the transcription elongation complex that is required for Tat-dependent transcription. We propose (1) that CDK2/cyclin E binds to the activation domain of Tat and phosphorylates Tat, (2) that this dynamic association of Tat with CDK2/cyclin E along with the interaction of Tat with CTD stimulates phosphorylation of position 2 serines (Ser-2) of the CTD heptapetide repeats, and (3) that this phosphorylation of Ser-2 by CDK2 participates in the regulation of HIV-1 transcription elongation. We also propose that Tat-induced phosphorylation of Ser-2 by CDK2 complements Tat-induced phosphorylation of Ser-5 by CDK9 to stimulate elongation of the HIV-1 transcripts. In the Aim 1 we will determine if CDK2 interacts with Tat in cultured cells and whether this interaction is important for the phosphorylation of Ser-2 of RNAPII heptads during HIV-1 transcription. In the Aim 2 we will determine if CDK2 phosphorylates Tat in cultured cells and whether the phosporylation of Tat is important for HIV-1 transcription. In the Aim 3 we will determine if Tat interacts with cyclin E and whether this interaction regulates HIV-1 transcription. The proposed research will determine whether CDK2/cyclin E along with CDK9/cyclin T1 is a co-activator of Tat-transactivation in vivo. The novelty of the research lies in establishing a previously unrecognized function of Tat as an enzymatic cofactor in HIV-1 transcription, binding simultaneously CDK2, the enzyme, and CTD, the substrate, to facilitate the phosphorylation of CTD Ser-2. The proposed research is important because it may delineate the mechanism of host CDK2 regulation of HIV-1 activated transcription, and may point to host CDK2 as a potential new target for anti-HIV-1 therapeutics.