Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins

Abstract

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 decoys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant proteins, including a newly generated form TatΔ58, for the ability to inhibit Tat-mediated transactivation and HIV production. In addition, we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (TatΔ53) and the Drosophila Engrailed (Eng) transcription repressor domain to test the hypothesis that transcriptional repression can be targeted to the HIV pro- moter. This fusion mutant was also examined for its capacity to block both Tat-mediated transactivation and HIV replication. We show that three mutants TatΔ53, TatΔ58 and TatΔ53/Eng result in a transdominant phenotype inhibiting wild-type Tat-mediated transactivation, and that the inhibiting potential is increased by the presence of the entire basic domain or the fusion of a repressor domain. However, only the transdominant mutants TatΔ58 and TatΔ53/Eng significantly inhibit HIV-1 replication after infection of transfected T cell lines. These results demonstrate the potent inhibiting activity of Tat mutants on HIV replication, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.