INHIBITION OF TAT-MEDIATED TRANSACTIVATION AND HIV REPLICATION WITH TAT MUTANT AND REPRESSOR DOMAIN FUSION PROTEINS

Strategies to inhibit the spread of HIV infection consist of a number of specific molecular approaches. Since viral production is dependent upon Tat-mediated transactivation of the HIV promoter through the Tat activating region (TAR), tat antisense RNA, anti-tat ribozymes, TAR de coys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant prot eins, including a newly generated form Tat Delta 58, for the ability t o inhibit Tat-mediated transactivation and HIV production. In addition , we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (Tat Delta 53) and the Drosophila Engrailed (Eng) transcri ption repressor domain to test the hypothesis that transcriptional rep ression can be targeted to the HIV promoter. This fusion mutant was al so examined for its capacity to block both Tat-mediated transactivatio n and HIV replication. We show that three mutants Tat Delta 53, Tat De lta 58 and Tat Delta 53/Eng result in a transdominant phenotype inhibi ting wild-type Tat-mediated transactivation, and that the inhibiting p otential is increased by the presence of the entire basic domain or th e fusion of a repressor domain. However, only the transdominant mutant s Tat Delta 58 and Tat Delta 53/Eng significantly inhibit HIV-1 replic ation after infection of transfected T cell lines. These results demon strate the potent inhibiting activity of Tat mutants on HIV replicatio n, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.