EVIDENCE FOR FUNCTIONAL INTERACTION BETWEEN THE HIV-1 TAT TRANSACTIVATOR AND THE TATA BOX-BINDING PROTEIN IN-VIVO

Tat strongly activates transcription of the HIV-1 provirus by stimulat ing both initiation and elongation. This transactivator binds to the T AR RNA element, but can also associate with cellular transcription fac tors, interacting with upstream promoter sequences. To achieve a bette r understanding of the role of Tat in the assembly of the transcriptio nal initiation complex in the living cell, we have examined how the ac tivity of this protein is modified when the general transcription fact or involved in the first step of this process, TBP, is overexpressed. The activity of Tat, either wild-type or fused to the DNA binding doma in of GAL4 (GBTat), was tested using reporter constructs containing GA L4 binding sites upstream of a minimal promoter corresponding to the H IV-1 TATA box, with or without the TAR element. We found that overexpr ession of TBP led to a dramatic increase in the activity of the GBTat protein. In order to activate GBTat, TBP must be able to interact with the TATA box. Analysis of several Tat mutants indicated that both the cysteine-rich and the core domains of this transactivator are necessa ry and sufficient to activate transcription when TBP is overexpressed. In vitro experiments showed that Tat binds specifically to TBP. There was a correlation between the ability of different Tat mutants to bin d TBP and their capacity to activate transcription in vivo. With the n atural HIV-1 promoter, overexpression of TBP first stimulated and then suppressed the Tat-induced activity. This inhibition was abrogated by an increase in the intracellular levels of Tat. These experimental da ta indicate that Tat stimulates initiation of transcription by interac ting with TBP in vivo.