TRANSCRIPTIONAL ACTIVATION IN-VITRO BY THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT PROTEIN - EVIDENCE FOR SPECIFIC INTERACTION WITH A COACTIVATOR(S)

The Tat protein encoded by human immuno deficiency virus type 1 is a s trong transcriptional activator of gene expression from the viral long terminal repeat and is essential for virus replication. We have inves tigated the molecular mechanism of Tat trans activation by using a cel l-free transcription system. We find that the trans-activation domain of Tat, amino acid residues 1-48 [Tat-(1-48)], can inhibit specificall y-i.e., ''squelch,'' transcriptional activation by full-length Tat [Ta t-(1-86)]. Squelching depends upon the functional integrity of the Tat trans activation domain because the mutant [Ala(41)]Tat-(1-48), which is defective in Tat trans-activation in vivo and in vitro, does not s quelch in vitro Tat trans-activation. Inhibition is selective because Tat-activated transcription, but not Tat-independent transcription, is squelched. Preincubation experiments with Tat or Tat-(1-48) and nucle ar extracts show that the trans-activation region of Tat can interact with cellular coactivator(s) required for Tat trans-activation and tha t this interaction can occur in the absence of the human immunodeficie ncy virus long terminal repeat promoter. Furthermore, the putative coa ctivator(s) mediating trans-activation by Tat differ from those mediat ing trans-activation by the acidic activator VP16, as shown by recipro cal squelching experiments in vitro. Our results suggest that specific cellular coactivator(s) are required for mediating activated transcri ption by human immunodeficiency virus type 1 Tat.