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 structural features of a typical RNA polymerase II (pol II) template. The promoter contains a TATA box 30 bp upstream of the transcription initiation site and binding sites for several pol II transcription factors, and long poly(A)+ RNA is synthesized from the integrated HTLV-I proviral DNA in vivo. Consistent with these characteristics, HTLV-I transcription 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 requires RNA pol II. In HeLa whole cell extracts, however, the HTLV-I long terminal repeat also contains an overlapping transcription unit (OTU). HTLV-I OTU transcription is initiated at the same nucleotide site as the RNA isolated from the HTLV-I-infected cell line MT-2 but was not inhibited by the presence of alpha-amanitin at concentrations which inhibited the adenovirus major late pol II promoter (6 micrograms/ml). HTLV-I transcription was inhibited when higher concentrations of alpha-amanitin (60 micrograms/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 contains overlapping promoters, a typical pol II promoter and a unique pol III promoter which requires a distinct set of transcription factors.
Piras
G
G
Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20892.
Kashanchi
F
F
Radonovich
M F
MF
Duvall
J F
JF
Brady
J N
JN
D000546:Amanitins / Q000633:toxicity*; D015153:Blotting, Western; D004352:Drug Resistance, Microbial; D006367:HeLa Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011061:Poly A / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016385:TATA Box; D035561:Transcription Factor TFIIA; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D000546:Amanitins; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D035561:Transcription Factor TFIIA; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D011061:Poly A; D012313:RNA; D012319:RNA Polymerase II; C056604:transcription factor TFIIF