BIOCHEMICAL AND FUNCTIONAL INTERACTION OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT TRANSACTIVATOR WITH THE GENERAL TRANSCRIPTION FACTOR TFIIB

Tat strongly stimulates transcription of the human immunodeficiency ty pe 1 (HIV-1) provirus by interacting with various cellular transcripti on factors, including TFIID. The results presented in this report indi cate 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 t he cysteine-rich and core domains of Tat bind to the N-terminal moiety of the general transcription factor. The role of the interaction betw een Tat and TFIIB in the activation of the entire HIV-1 promoter was a nalysed. Transfection experiments performed using a reporter construct containing the HIV-1 long terminal repeat fused to a reporter gene sh owed that overexpression of TFIIB progressively suppressed Tat-induced transcription. This effect was weakened by an increase in the intrace llular concentration of Tat. A similar consequence of TFIIB overexpres sion was observed in a HeLa cell line stably transformed with a constr uct corresponding to the lacZ gene under the control of the HIV-1 prom oter. Mutants of TFIIB which differed in their ability to interact wit h Tat and to function in basal transcription were analysed. The abilit y of TFIIB mutants defective for basal transcription to inhibit Tat-in duced activity of the HIV-1 promoter depended on their capacity to int eract with Tat. Mutants of TFIIB functional for basal transcription, b ut defective for the interaction with Tat, exhibited a dominant negati ve effect. From these data we propose a model in which interaction bet ween Tat and both general transcription factors TBP and TFIIB maintain s the transcriptional initiation complex in an active configuration.