Transactivation of the human T-cell lymphotropic virus type 1 tax₁- 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 Tax₁ gene product has been shown to transactivate transcription of viral and cellular promoters. To examine the biochemical mechanism of Tax₁ transactivation, we have developed an in vitro transactivation assay in which wild-type Tax₁ is able to specifically transactivate a polymerase II promoter through upstream Tax₁-responsive elements. The in vitro system utilizes the HTLV-1 21-bp repeats cloned upstream of the ovalbumin promoter and G-free cassette. Purified Tax₁ specifically transactivates this template 5- to 10-fold in a concentration- dependent manner. No transactivation of the ovalbumin promoter (pLovTATA) template control was observed. Tax₁ transactivation was inhibited by low concentrations of α-amanitin and was effectively neutralized by anti-Tax₁ but not control sera. Consistent with in vivo transactivating activity, Tax₁ NF-κB mutant M22, but not cyclic AMP-responsive element-binding protein mutant M47, transactivated the template containing the tandem 21-bp repeat. In a reconstituted in vitro transcription assay, Tax₁ transactivation was dependent upon basal transcription factors TFIIB, TFIIF, Pol II, TFIID, and TFIIA. TATA-binding protein did not functionally substitute for TFIID in the transactivation assay by Tax₁ but was sufficient for basal transcription. Finally, we have used anti-TFIIA antibody (p55) to ask if Tax₁ transactivation required TFIIA activity. Addition of TFIIA antibody to in vitro transcription reactions, as well as depletion of TFIIA by preclearing with antibody, showed that TFIIA was required for Tax₁ transactivation. Only a slight (twofold) drop of basal transcription was observed. Tax₁ transactivation was restored when purified HeLa TFIIA was added back into the reconstituted system. We propose that Tax₁ utilizes a transactivation pathway involving the activator regulated basal transcription factors TFIID and TFIIA.