TRANSACTIVATION OF THE HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-1 TAX(1)-RESPONSIVE 21-BASE-PAIR REPEATS REQUIRES HOLO-TFIID AND TFIIA

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia and tropical spastic paraparesis/HTLV -1-associated myelopathy. The HTLV-1 Tax(1) gene product has been show n. to transactivate transcription of viral and cellular promoters. To examine the biochemical mechanism of Tax(1) transactivation, we have d eveloped an in vitro transactivation assay in which wild-type Tax(1) i s able to specifically transactivate a polymerase II promoter through upstream Tax(1)-responsive elements. The in vitro system utilizes the HTLV-1 21-bp repeats cloned upstream of the ovalbumin promoter and G-f ree cassette. Purified Tax(1) specifically transactivates this templat e 5- to 10-fold in a concentration-dependent manner. No transactivatio n of the ovalbumin promoter (pLovTATA) template control was observed. Tax(1) transactivation was inhibited by low concentrations of alpha-am anitin and was effectively neutralized by anti-Tax(1) but not control sera. Consistent with in vivo transactivating activity, Tax(1) NF-kapp a B mutant M22, but not cyclic AMP-responsive element-binding protein mutant M47, transactivated the template containing the tandem 21-bp re peat. In a reconstituted in vitro transcription assay, Tax(1) transact ivation was dependent upon basal transcription factors TFIIB, TFIIF, P ol II, TFIID, and TFIIA. TATA-binding protein did not functionally sub stitute for TFIID in the transactivation assay by Tax(1) but was suffi cient for basal transcription. Finally, we have used anti-TFIIA antibo dy (p55) to ask if Tax(1) transactivation required TFIIA activity. Add ition of TFIIA antibody to in vitro transcription reactions, as well a s depletion of TFIIA by preclearing with antibody, showed that TFIIA w as required for Tax(1) transactivation. Only a slight (twofold) drop o f basal transcription was observed. Tax(1) transactivation was restore d when purified HeLa TFIIA was added back into the reconstituted syste m. We propose that Tax(1) utilizes a transactivation pathway involving the activator regulated basal transcription factors TFIID and TFIIA.