This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. After HIV infects a cell and a reverse transcribed copy of its RNA genome has integrated into the host genome, generation of viral RNAs requires the transactivation activity of a viral protein, Tat, in conjunction with the host's P-TEFb. Tat and the specific form of P-TEFb that contains Cdk9 and cyclin T1 bind to a stem and loop structure (TAR) in the 5?R end of the nascent HIV transcript. Recruitment of P-TEFb in this way promotes the transition of engaged polymerases at the HIV LTR into productive elongation. This recruitment mechanism initially seemed bizarre, and TAR still represents perhaps the only example of a eukaryotic """"""""RNA enhancer"""""""". However, the recent discovery of the P-TEFb regulatory machinery provides evidence that HIV took advantage not only of P-TEFb, but the whole P-TEFb control process. There are significant similarities between the Tat-TAR-P-TEFb complex and the HEXIM-7Sk-P-TEFb complex. Indeed, the RNA binding domain of Tat will functionally substitute for the RNA binding domain of HEXIM1 in a chimeric HEXIM1-Tat protein. The main difference between the two P-TEFb complexes is that HEXIM1 and 7SK inhibit P-TEFb, while Tat and TAR do not. Understanding the details of the Tat-TAR-P-TEFb complex afforded by the proposed structural studies may provide rational targets for small molecules to block the complex formation and, thereby, inhibit HIV replication. All general P-TEFb inhibitors, including Flavopiridol, block HIV replication, but at slightly higher concentrations also exhibit cellular toxicity. Molecules that block the formation of the Tat-TAR-P-TEFb complex would likely be more specific for HIV transcription than general P-TEFb inhibitors.
The aim of this proposal is to solve the structures of Tat-P-TEFb,Tat-TAR-P-TEFb and HEXIM-7Sk-P-TEFb complexes.
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