Involvement of TFIID and USA Components in Transcriptional Activation of the Human Immunodeficiency Virus Promoter by NF-κB and Sp1

ABSTRACT

The purified Rel/NF-κB (p50/p65) complex and Sp1 markedly activate transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a highly purified HeLa reconstituted transcription system. Transcriptional activation by NF-κB and Sp1 requires both TFIID and the USA fraction. The USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator activity, both by repressing the basal level of transcription and by potentiating activator function to yield large increases in the levels of transcription induction. Under limiting concentrations, PC2 and PC4 also show synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65 subunit of NF-κB is a potent activator when assayed as a Gal fusion in the reconstituted transcription system and interacts both with TATA-binding protein (TBP) and with several human TBP-associated factors (TAFs) that include TAF_II250. The p65 activation domain mediates transcription activation in the presence of partially reconstituted TFIID species that include a minimal complex containing only TBP and TAF_II250. These studies also show that, like USA components, TAFs can serve both to repress TBP-mediated transcription and, following activator interactions, to reverse the repression and effect a net increase in activity. Taken together, these data underscore the importance of both TAFs and specific USA-derived coactivators for optimal activation of the HIV-1 promoter, as well as certain parallels in their overall mechanisms of action.

ACKNOWLEDGMENTS

We thank C.-M. Chiang for the FLAG-Gal4 expression plasmid, D. K. Lee for the E1b construct, and T. Oelgeschläger for critical comments on the manuscript.

This work was supported by a grant from the Tebil Foundation to The Rockefeller University and by NIH grants AI32737 and CA 42567 to R.G.R.