REGULATION OF HIV-1 TRANSCRIPTION BY CDK2

描述（由申请人提供）：耐药HIV-1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。本研究旨在阐明宿主细胞周期依赖性激酶2（CDK 2）在HIV-1达特蛋白介导的HIV-1转录调控中的作用。HIV-1达特通过与CDKg/细胞周期蛋白T1结合并诱导RNA聚合酶II（RNAPII）的C末端结构域（CTD）的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-1达特也可能利用CDK 2/细胞周期蛋白E来增加CTD磷酸化，并刺激HIV-1在细胞周期的G1/S期的转录。我们假设，沿着CDKg，CDK 2是Tat依赖性转录所需的转录延伸复合物的一部分。我们提出（1）CDK 2/细胞周期蛋白E与达特的活化结构域结合并磷酸化达特，（2）达特与CDK 2/细胞周期蛋白E的这种动态结合沿着达特与CTD的相互作用刺激CTD七肽重复序列的2位丝氨酸（Ser-2）的磷酸化，（3）CDK 2对Ser-2的磷酸化参与HIV-1转录延长的调节。我们还提出，Tat诱导的丝氨酸-2磷酸化的CDK 2补充Tat诱导的丝氨酸-5磷酸化的CDK 9刺激延长的HIV-1转录。在目的1中，我们将确定CDK 2是否与培养细胞中的达特相互作用，以及这种相互作用是否对HIV-1转录期间RNAPII七肽的Ser-2磷酸化重要。在目的2中，我们将确定CDK 2是否使培养细胞中的达特磷酸化，以及达特的磷酸化是否对HIV-1转录重要。在目标3中，我们将确定达特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转录。这项研究将确定CDK 2/细胞周期蛋白E沿着CDK 9/细胞周期蛋白T1是否是体内Tat反式激活的共激活因子。该研究的新奇在于建立了以前未被认识到的达特作为HIV-1转录中的酶辅因子的功能，同时结合CDK 2（酶）和CTD（底物），以促进CTD Ser-2的磷酸化。这项研究很重要，因为它可以描述宿主CDK 2调节HIV-1激活转录的机制，并可能指出宿主CDK 2作为抗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.