HIV-1 encodes a transactivating protein, tat, that activates viral gene expression, is essential for HIV replication, and thus is a potential target for development of AIDS therapy. Our major goals are to understand tat structure-function relationships, to study tat transport and posttranslation modifications, to identify dominant negative mutant tat peptides as potential tat antagonists, and to develop methods to deliver and test the efficacy of tat peptide antagonists as inhibitors of HIV expression. We have developed a mammalian cell microinjection assay for tat transactivation and have chemically synthesized the biologically active full length 86 amino acid tat protein and several deletion peptides encoding domains possessing high transactivating activity. We propose to precisely delineate these domains to facilitate structure-function studies and to develop peptide antagonists of tat function. Single amino acid substitutions will be made in minimal tat domain peptides. This will allow the identification of essential amino acid resides and can lead to antagonists of tat function. Several tat mutant peptides have already been identified in this manner which inhibit transactivation by wild type tat. We will further develop tat mutant antagonists by additional amino acid substitutions and test their ability to inhibit HIV replication in T lymphocytes, as an indication of their potential use for therapy of AIDS. Structure-function requirements for uptake and nuclear transport of tat peptides into T lymphocytes will be studied. Synthetic genes encoding tat antagonist peptides will be cloned into adenovirus vectors for infection of HIV infected T4 lymphocytes as a test of the efficacy of tat peptide antagonists. These vectors may also have clinical use for AIDS therapy. As a long range goal, we will develop expression vector systems based on tat peptide transactivation of HIV-LTR regulated genes.
