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。该研究旨在描述宿主细胞周期依赖性激酶2 （CDK2）在HIV-1 Tat蛋白介导的HIV-1转录调控中的作用。HIV-1 Tat通过结合CDKg/cyclin T1和诱导RNA聚合酶II (RNAPII) c端结构域（CTD）的过度磷酸化来刺激病毒转录的延伸。我们的初步数据表明，HIV-1 Tat也可能利用CDK2/cyclin E增加CTD磷酸化，并在细胞周期的G1/S期刺激HIV-1转录。我们假设，与CDKg一起，CDK2是转录延伸复合体的一部分，这是tat依赖性转录所必需的。我们提出(1)CDK2/cyclin E结合Tat的活化结构域并使Tat磷酸化，(2)Tat与CDK2/cyclin E的这种动态关联以及Tat与CTD的相互作用刺激了CTD heptapetide repeat的位置2丝氨酸（Ser-2）的磷酸化，以及(3)CDK2对Ser-2的磷酸化参与了HIV-1转录伸长的调节。我们还提出，CDK2诱导的Ser-2磷酸化与CDK9诱导的Ser-5磷酸化互补，从而刺激HIV-1转录本的延伸。在Aim 1中，我们将确定CDK2是否与培养细胞中的Tat相互作用，以及这种相互作用是否对HIV-1转录过程中RNAPII七元体Ser-2的磷酸化很重要。在Aim 2中，我们将确定CDK2是否会使培养细胞中的Tat磷酸化，以及Tat的磷酸化对HIV-1转录是否重要。在Aim 3中，我们将确定Tat是否与周期蛋白E相互作用，以及这种相互作用是否调节HIV-1转录。拟议的研究将确定CDK2/cyclin E和CDK9/cyclin T1在体内是否是tat转激活的共激活因子。该研究的新颖之处在于建立了Tat在HIV-1转录中作为酶促因子的先前未被认识的功能，它同时结合酶CDK2和底物CTD，以促进CTD Ser-2的磷酸化。这项研究很重要，因为它可能描述宿主CDK2调控HIV-1激活转录的机制，并可能指出宿主CDK2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.