We have recently isolated a novel C-type cyclin (called Cyclin T) which interacts directly with HIV-1 Tat through its activation domain. Cyclin T (CycT) is a predominant cyclin partner for CDK9 (PITALRE), the catalytic subunit of the positive-acting transcription elongation factor complex, P-TEFb. We determined that the interaction of Tat with CycT dramatically enhances its binding to TAR RNA, and confers a requirement for critical sequences in the loop of TAR which are not recognized by the free (uncomplexed) Tat protein. Structure-function analysis of the cyclin T:Tat interaction. Here we propose to characterize the interaction between Tat and CycT in molecular detail, and to identify other factors present in the Tat-associated kinase (TAK)/P-TEFb complex that may be required for the regulation of transcription elongation by Tat.
In Specific Aim 1 we will identify residues within CycT and in Tat that are necessary for their interaction and for binding to Tar RNA using site-directed mutagenesis and UV cross-linking techniques.
In Specific Aim 2, we will analyze the ability of wild-type and mutant CycT proteins to support basal and Tat-mediated transactivation in vivo and in cell-free transcription reactions that have been immunodepleted of hCycT, or blocked with trans-dominant Tat (1-48) protein. We will also determine whether additional P-TEFb components are needed for Tat activity. We have recently cloned the murine CycT protein (mCycT) and find that it is unable to support Tat transactivation through TAR.
In Specific Aim 3, we will characterize the defect in mCycT and identify the minimal changes needed to restore Tat transactivation in vivo. Finally, in Specific Aims 4 and 5 we propose to clone and characterize two novel proteins which we find to be tightly associated with TAK/P- TEFb in nuclear extracts, either alone or when bound to TAR RNA. Taken together, these studies will provide important new information on the mechanism of HIV Tat transactivation.
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