Most of the currently available anti-HIV drugs inhibit two essential viral enzymes, reverse transcriptase and viral protease. However, due to onset of viral drug resistance resulting in infection persistence, alternative targets of HIV-1 are necessary. Transcription of HIV-1 viral genes is uniquely dependent on the interaction of the viral HIV-1 Tat protein with host cell factors. Our lab has previously shown that cell-cycle dependent kinase 2 (CDK2) phosphorylates HIV-1 Tat and that inhibition of this phosphorylation through the mutation of Tat or inactivation of CDK2 inhibits HIV -1 replication. Our preliminary results indicate that CDK2 might phosphorylate CDK9 and affect the kinase activity of CDK9/cyclin T1 complex in the cells that have a reduced CDk2 expression. We hypothesize that CDK2-mediated phosphorylation of CDK9 helps to maintain the activity of CDK9/cyclin T1. In the proposed studies, we will utilize a 293T cell line that stably expresses shRNA against CDK2 to analyze the effect of CDK2 inactivation on CDK9/cyclin T1 activity. In Specific Aim 1 we will analyze the activity of CDK9/cyclin T1 complex in this cell line. In Specific Aim 2, we will determine if CDK2 regulates the cellular activity of CDK9/cyclin T1 complex by analyzing the phosphorylation of short CDK9- derived peptides in a CDK2-knock-out cell line. We will also elucidate the site of phosphorylation on CDK9 by CDK2 by analyzing the sites of CDK9 phosphorylation, mutate those sites, and analyze the activity of CDK2. In Specific Aim 3 we will analyze the affect of CDK9 phosphorylation on HIV-1 transcription and replication by mutating CDK9. The proposed research is designed to help elucidate the mechanism of interaction between CDK2 and CDK9 and the effect of this interaction on HIV-1 transcription and replication. These findings will provide valuable information regarding the use of CDK2 as a possible target for HIV-1 drug therapy. PUBLIC HEALTH RELEVANCE: Most of the currently available anti-HIV drugs inhibit two essential viral enzymes, reverse transcriptase and viral protease. However, due to onset of viral drug resistance resulting in infection persistence, alternative targets of HIV-1 are necessary. Our findings from this research will provide valuable information regarding the use of CDK2 as a possible target for HIV-1 drug therapy.