Efficient replication of the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) requires the viral transactivator protein known as Tat. Tat stimulates transcriptional elongation and the switch from latency to active replication in infected individuals has been suggested to occur at this level. Therefore Tat represents an attractive target for anti-viral agents. To understand the mechanism of action of Tat, it is essential to identify and characterize the cellular factor(s) that interacts with Tat. Recently, a cellular protein kinase, TAK (Tat-associated kinase), that binds specifically to the activation domain of the Tat proteins, was identified using an in vitro kinase assay. TAK fulfills the stringent genetic criteria established for a Tat cofactor: TAK binds in vitro to the activation domains of the Tat proteins of HIV-1, HIV-2, and the distantly related lentivirus equine infectious anemia virus, but not to Tat mutants that are nonfunctional for transactivation in vivo. Furthermore, a biochemical characterization of TAK suggests that its activity is consistent with proposed roles of a putative Tat cofactor. First, TAK is sensitive to DRB, a nucleoside analog that inhibits a limited number of kinases and is known to inhibit Tat transactivation in vivo and in vitro. Second, TAK hyperphosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II. Phosphorylation of the CTD has been proposed to trigger the transition from initiation to active elongation and also to influence later stages during elongation. TAK also phosphorylates Tat-2, but not Tat- 1, in vitro. Together, these results implicate TAK as a very promising candidate for a cellular factor that mediates Tat transactivation. This grant application proposes to test the hypothesis that TAK is a key cellular factor directly involved in the Tat function.
The specific aims of this proposal are: 1). to purify TAK for use in biochemical experiments and in cloning of its cDNA(s) and generating reagents with which to study TAK function; 2). to investigate the role of TAK in Tat transactivation using purified TAK and reagents generated by the cDNA cloning; 3). to examine the biological relevance of CTD phosphorylation by TAK in vivo and 4). to map the in vivo and in vitro phosphorylation sites of the HIV-2 Tat protein and determine the significance of the phosphorylation on the protein's function. The role of TAK in Tat transactivation will be examined using biochemical assays including tat-dependent in vitro transcription systems, add- back experiments, depletion experiments, and a TAR RNA binding assay. Additional experiments are proposed to investigate the biological significance of TAK phosphorylation in vivo. Elucidation of the mechanism of action of Tat may lead to the rational design of novel inhibitors of Tat activity.
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