The Tat gene of human immunodeficiency virus (HIV) plays a central role in the activation and life cycle of HIV. Tat exerts its effects at the level of transcriptional initiation and elongation. Here we report that Tat binds directly to the basal transcription factor TFIID. The transcriptional activity of HeLa extracts was depleted after chromatography on a Tat affinity column, which specifically retained the polymerase II-specific transcription factor TFIID. Direct interaction of Tat with holo-TFIID composed of TATA-binding protein (TBP) and associated factors (TAFs) was observed. Tat, through amino acids 36-50, binds directly to the TBP subunit of TFIID. Our results suggest that Tat may transduce upstream or downstream regulatory signals by direct interaction with the basal transcription factor TFIID. Second-site revertants from replication-incompetent molecular clones of HIV contain base substitutions adjacent to the TATA motif. The altered TATA box motifs were analyzed for their effect(s) on virus infectivity, long terminal repeat (LTR)-directed expression in transient transfection assays, in vitro RNA synthesis, and assembly of the TFIID/TFIIA preinitiation complex. The revertant TATA boxes accelerated the kinetics of HIV replication when present in the context of an LTR containing a Sp1 mutation. In CAT assays and in vitro transcription systems, the altered TATA box motifs led to elevated basal levels of RNA synthesis, but did not increase responsiveness to Tat transactivation. The revertant TATA boxes accelerated the binding of TFIID and TFIIA to the LTR and stabilized their association with the promoter. These results suggest that in the context of an impaired enhancer/promoter (viz. three mutated Sp1 elements), a series of HIV revertants emerge which contain LTR alterations that significantly augment basal RNA synthesis. The TATA motif revertants are capable of rescuing the enhancer/promoter defect and sustain virus infectivity.
