Regulation of HIV-1 Transcription by CDK2
Prinicipal Investigator: Sergei A. Nekhai, PhD
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 pf HIV-1 to anti-viral agents. HIV-1 Tat stimulates elongation of viral transcription by inducing phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II (RNAPII). Tat recruits a cell cycle dependent kinase 9 (CDK9) to RNAPII through a transactivation response (TAR) RNA element and stimulates phosphorylation of CTD. Our previous studies indicate that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro; and that HIV-1 transcription and viral replication is inhibited by RNA interference directed to CDK2 or by pharmacological inhibitors of CDK2. We hypothesize that 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.
- We will investigate the physiological importance of Tat phosphorylation for HIV-1 transcription. We will mutate Ser16 and Ser46 residues of Tat binding to P-TEFb and TAR RNA or to histone acetyltransferases in vitro and in cultured cells. We will also analyze whether phosphorylation of tat facilitates its ubiquitination of affects stability of Tat protein.
- We will analyze the kinetics of Tat phosphorylation by CDK2 and interaction of Tat by CDK2/cyclin E using molecular modeling.
- 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 of 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. The proposed research is designed to determine whether CDK2 phosphorylates HIV-1 Tat during HIV-1 transcription and whether this phosphorylation is physiologically important for viral replication.