TRANSCRIPTION OF THE HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE-I PROMOTER BY AN ALPHA-AMANITIN-RESISTANT POLYMERASE

The human T-lymphotropic virus type I (HTLV-I) promoter contains the s tructural features of a typical RNA polymerase II (pol II) template. T he promoter contains a TATA box 30 bp upstream of the transcription in itiation site and binding sites for several pol II transcription facto rs, and long poly(A)(+) RNA is synthesized from the integrated HTLV-I proviral DNA in vivo. Consistent with these characteristics, HTLV-I tr anscription activity was reconstituted in vitro by using TATA-binding protein, TFIIA, recombinant TFIIB, TFIIE, and TFIIF, TFIIH, and pol II . Transcription of the HTLV-I promoter in the reconstituted system req uires RNA pol II. In HeLa whole cell extracts, however, the HTLV-I lon g terminal repeat also contains an overlapping transcription unit (OTU ). HTLV-I OTU transcription is initiated at the same nucleotide site a s the RNA isolated from the HTLV-I-infected cell line MT-2 but was not inhibited by the presence of cu-amanitin at concentrations which inhi bited the adenovirus major late pol II promoter (6 mu g/ml). HTLV-I tr anscription was inhibited when higher concentrations of cx-amanitin (6 0 mu g/ml) were used, in the range of a typical pol III promoter (VA-I ). Neutralization and depletion experiments with three distinct pol II antibodies demonstrate that RNA pol II is not required for HTLV-I OTU transcription. Antibodies to basal transcription factors TATA-binding protein and TFIIB, but not TFIIIC, inhibited HTLV-I OTU transcription . These observations suggest that the HTLV-I long terminal repeat cont ains overlapping promoters, a typical pol II promoter and a unique pol III promoter which requires a distinct set of transcription factors.