Expression and DNA binding of budgerigar fledgling disease virus large T antigen

Abstract

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.