Biochemical and functional interaction of the human immunodeficiency virus type 1 Tat transactivator with the general...

Tat strongly stimulates transcription of the human immunodeficiency type 1 (HIV-1) provirus by interacting with various cellular transcription factors, including TFIID. The results presented in this report indicate that the effect exerted by Tat also involves an interaction with TFIIB. A direct protein-protein interaction between Tat and TFIIB was observed in vitro. Detailed analysis of this interaction showed that the cysteine-rich and core domains of Tat bind to the N-terminal moiety of the general transcription factor. The role of the interaction between Tat and TFIIB in the activation of the entire HIV-1 promoter was analysed. Transfection experiments performed using a reporter construct containing the HIV-1 long terminal repeat fused to a reporter gene showed that overexpression of TFIIB progressively suppressed Tat-induced transcription. This effect was weakened by an increase in the intracellular concentration of Tat. A similar consequence of TFIIB overexpression was observed in a HeLa cell line stably transformed with a construct corresponding to the lacZ gene under the control of the HIV-1 promoter. Mutants of TFIIB which differed in their ability to interact with Tat and to function in basal transcription were analysed. The ability of TFIIB mutants defective for basal transcription to inhibit Tat-induced activity of the HIV-1 promoter depended on their capacity to interact with Tat. Mutants of TFIIB functional for basal transcription, but defective for the interaction with Tat, exhibited a dominant negative effect. From these data we propose a model in which interaction between Tat and both general transcription factors TBP and TFIIB maintains the transcriptional initiation complex in an active configuration.