The long-term goals of this project are to understand the biochemical mechanism of Tat (transactivator) action and to utilize basic knowledge of Tat function to design antagonists of HIV replication. First, to understand Tat function, the investigator proposes to focus on a new human cellular protein of 36 kDa that he has shown to (1) bind tightly to Tat, (2) form a complex with Tat in vitro, and (3) potentiate Tat transactivation when introduced into rodent cells. The author will further characterize this 36 kDa protein, delineate the Tat recognition sequences for binding it, determine cell species and cell type expression, clone its cDNA and study its role in Tat transactivation. Second, in studies to understand Tat uptake, the author will further characterize a 90 kDa cell surface protein that he has found to bind to Tat in vivo and in vitro. He proposes to analyze the role of this 90 kDa protein in Tat cellular uptake by cross-linking studies and Scatchard analysis, to delineate the Tat recognition sequences for binding 90 kDa, and to determine by amino acid sequence analysis whether the 90 kDa is a new protein. If warranted, he will isolate a cDNA clone encoding 90 kDa for further studies. Third, in studies to develop further Tat peptide antagonists of HIV replication, the investigator will utilize the results of mapping studies of Tat recognition sequences for (1) cell uptake, (2) TAR binding, and (3) 36 kDa binding in attempts to design small Tat peptides that will be taken up by cells and block HIV replication. As a long-range goal, analogues of promising Tat peptides will be synthesized by peptide mimetic technology to yield stable and more efficient Tat antagonists. Fourth, to explore the use of Tat peptide sequences to transport foreign polypeptides into cells, the author proposes to analyze the cellular uptake, nuclear transport, and biological function of fusion polypeptides containing Tat """"""""delivery"""""""" sequences. He proposes to first examine the 116 amino acid HIV Rev (regulator of virion protein expression [formerly art/trs]) protein fused to Tat.
