TRANSCRIPTIONAL TRANS ACTIVATION BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAT REQUIRES SPECIFIC COACTIVATORS THAT ARE NOT BASAL FACTORS

Expression of human immunodeficiency virus type 1 (HIV-1) genes is reg ulated by the trans activator Tat. Tat exerts its effects by increasin g the rate of transcription, but the mechanism by which it does so is still unknown. To study the cellular factors required for Tat trans ac tivation, we have expressed functional Gst-Tat fusion protein and used it to construct affinity columns. Our findings are as follows, (i) A Gst-Tat affinity matrix depleted HeLa nuclear extracts of a factor(s) required for Tat function. A Tat mutant bearing the missense mutation lysine to alanine at position 41 was incapable of this depletion. (ii) Tat trans activation was recovered by addition of unfractionated nucl ear extract, the 0.5 M KCl elution fraction from the Tat affinity colu mn, or sedimentation gradient fractions of HeLa extracts. The activity from the gradients sedimented with an apparent molecular mass of 200 kDa. (iii) Tat trans activation could not be recovered by use of recom binant human TATA-binding protein or partially purified TFIID. (iv) tr ans activation by Tat was blocked by heating of the nuclear extract un der conditions in which basal transcription was not decreased. Our dat a demonstrate for the first time the existence of unique Tat coactivat ors distinct from factors required for general basal transcription.