? Project Research Objectives: 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. We hypothesize that CDK2 has a regulatory role in HIV transcription. We propose that the mechanism whereby 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.
In specific aim 1, we will investigate the physiological importance of Tat phosphorylation for HIV-1 transcription. Specifically, we will mutate Ser16 and Ser46 residues of tat in the pNL4-3 provirus and determine the kinetics of replication of mutant viruses and analyze Tat phosphorylation during viral replication. We will also analyze the effect of mutations of Tat Ser16 and Ser46 residues on Tat binding to P-TEFb and TAR RNA; to histone acetyltransferases and whether phosphorylation of Tat facilitates its ubiquitination or affects the stability of Tat protein.
In specific aim 2, we will analyze kinetics of Tat phosphorylation by CDK2 and interaction of phosphorylated Tat with transcriptional co-activators. We will study the kinetics of the phosphorylation of wild and mutant Tat by CDK2/cyclin E in vitro; interaction of phosphorylated Tat with transcriptional co-activators and interaction of Tat with CDK2/cyclin E using molecular modeling.
In specific aim 3, 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 or 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. ? ? Project Developmental Objectives: (1) develop into an independent investigator by establishing my own laboratory using SC1 funding and by presenting my research at SCORE-funded and international meetings; (2) prepare and apply for non-SCORE support at year 2 of the SC1 support and participate in a MORE-sponsored grant-writing workshop to facilitate grant-writing skills; and (3) initiate new research and educational programs at Howard University. The underlying theme of the proposed research is to reduce health disparities, to enhance excellence of research at Howard University and to permit my transition into an independent investigator. The proposed research may point to host CDK2 as a potential new target for anti-HIV-1 therapeutics. ? ? ?
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