Expression and DNA binding of budgerigar fledgling disease virus large T antigen.
Budgerigar fledgling disease virus (BFDV) represents the first non-mammalian member of the polyomavirus genus and possesses uncommon structural and biological properties. Recombinant baculoviruses were constructed to express BFDV small t antigen, large T antigens, as well as a large T deletion mutant Td and beta-galactosidase-Td fusion proteins to high levels in infected insect cells. A recombinant virus containing a genomic copy of the BFDV early region was used for small t antigen expression, and corresponding intron-deleted cDNAs for production of large T antigen derivatives. Recombinant T as well as authentic T antigen proteins from infected chicken embryo fibroblasts were purified using both immunoaffinity and DNA affinity column chromatography. We present evidence that the large T antigen interacts specifically with DNA sequences present in the non-coding region of BFDV; by indirect DNA immunoprecipitation mapping and DNase I footprinting, four regions including 12 DNA-binding sites have been determined that cover most of the BFDV non-coding region. The T antigen binding pattern observed suggests a protein-DNA interaction system considerably different from those of simian virus 40 and other polyomaviruses.
Luo
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D
Department of Medicine, University of Alberta, Edmonton, Canada.
Müller
H
H
Tang
X B
XB
Hobom
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G
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D001717:Birds / Q000382:microbiology; D002451:Cell Compartmentation; D004268:DNA-Binding Proteins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011120:Polyomavirus / Q000235:genetics*; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins