EXPRESSION AND DNA-BINDING OF BUDGERIGAR FLEDGLING DISEASE VIRUS LARGE T-ANTIGEN

Budgerigar fledgling disease virus (BFDV) represents the first non-mam malian member of the polyomavirus genus and possesses uncommon structu ral and biological properties. Recombinant baculoviruses were construc ted to express BFDV small t antigen, large T antigens, as well as a la rge T deletion mutant T-d and beta-galactosidase-T-d fusion proteins t o 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 l arge T antigen derivatives. Recombinant T as well as authentic T antig en proteins from infected chicken embryo fibroblasts were purified usi ng both immunoaffinity and DNA affinity column chromatography. We pres ent evidence that the large T antigen interacts specifically with DNA sequences present in the non-coding region of BFDV; by indirect DNA im munoprecipitation mapping and DNase I footprinting, four regions inclu ding 12 DNA-binding sites have been determined that cover most of the BFDV non-coding region. The T antigen binding pattern observed suggest s a protein-DNA interaction system considerably different from those o f simian virus 40 and other polyomaviruses.