Identification and genetic definition of a bovine papillomavirus type 1 E7 protein and absence of a low-copy-number phenotype exhibited by E5, E6, or E7 viral mutants

The bovine papillomavirus type 1 (BPV-1) genome replicates as a multiple-copy plasmid in murine C127 cells transformed to neoplasia by virus infection or by transfection with BPV-1 DNA. It was reported previously that BPV-1 genomes harboring frameshift mutations in the E6 or E7 open reading frame (ORF) replicated in C127 cells transformed by these mutants at a low copy number. Furthermore, the characterization of a BPV-1 mRNA in which the E6 and E7 ORFs were spliced together in frame has led to the assumption that an E6/7 fusion protein is expressed in virus-transformed C127 cells. To define the number and nature of the E6 and E7 gene products expressed in BPV-1-transformed cells, we performed immunoprecipitation experiments with antisera raised to bacterially expressed BPV-1 E6 and E7 fusion proteins. By employing cell culture conditions which induce BPV-1 E2 transactivator expression and viral early region transcription in virus-transformed C127 cell lines, we detected a single immunoprecipitated E6 protein species with an apparent molecular mass of 17 kDa and a single E7 protein species with an apparent molecular mass of 15 kDa. To characterize further these E6 and E7 proteins, C127 cells were transformed by transfection with BPV-1 genomes containing mutations predicted to prevent expression of specific E6 or E7 gene products, and the transformed cells were subjected to immunoprecipitation analysis with the E6 or E7 antiserum. The results of these experiments confirmed that the E6 and E7 ORFs encode distinct proteins and failed to establish the existence of an E6/7 fusion protein. We did not find a significant difference in the viral genome copy number between clonal C127 cell lines transformed by wild-type BPV-1 or by mutant viral genomes unable to express the E6 or the E7 protein. Furthermore, in contrast to two previous reports suggesting that expression of the BPV-1 E5 gene was required for the establishment or maintenance of a high viral plasmid copy number, we observed a two- to fourfold increase over wild-type BPV-1 plasmid copy number in C127 cells transfected with a BPV-1 E5-minus mutant and subsequently selected by neoplastic focus formation.