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.
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