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 stimulating both initiation and elongation. This transactivator binds to the TAR RNA element, but can also associate with cellular transcription factors, interacting with upstream promoter sequences. To achieve a better understanding of the role of Tat in the assembly of the transcriptional initiation complex in the living cell, we have examined how the activity of this protein is modified when the general transcription factor involved in the first step of this process, TBP, is overexpressed. The activity of Tat, either wild-type or fused to the DNA binding domain of GAL4 (GBTat), was tested using reporter constructs containing GAL4 binding sites upstream of a minimal promoter corresponding to the HIV-1 TATA box, with or without the TAR element. We found that overexpression 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 necessary 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 bind TBP and their capacity to activate transcription in vivo. With the natural 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 data indicate that Tat stimulates initiation of transcription by interacting with TBP in vivo.