Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat.
We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2- to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (U5RE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: 110-, 80- and 70-kDa proteins. The 110-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70- and 80-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV-I basal transcriptional repression.
Okumura
K
K
Shionogi Institute for Medical Science, Osaka, Japan.
Takagi
S
S
Sakaguchi
G
G
Naito
K
K
Minoura-Tada
N
N
Kobayashi
H
H
Mimori
T
T
Hinuma
Y
Y
Igarashi
H
H
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004265:DNA Helicases; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D016356:Gene Products, tax / Q000378:metabolism; D006367:HeLa Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D013601:T-Lymphocytes; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D001324:Autoantigens; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D016356:Gene Products, tax; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D015500:Chloramphenicol O-Acetyltransferase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen