Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA

Abstract

Oligonucleotide-directed mutagenesis was used to construct a nonsense mutation in open reading frame (ORF) E2 of bovine papillomavirus DNA. A single base substitution mutation was constructed which converted a TAC codon into a TAG amber stop codon at a position in the ORF that did not overlap with any other viral ORFs. Full-length viral DNA containing the mutation induced only approximately 2% of the transformed foci of mouse C127 cells that were induced by wild-type DNA. In a different transformation assay, approximately one-half of the C127 cells which had acquired the mutant DNA gave rise to colonies containing at least some cells with transformed morphology. The constructed mutation was maintained in cell lines derived from cells which had acquired the mutant viral DNA, but the viral DNA appeared to be integrated into the host cell genome. Genetic mapping experiments proved that the constructed amber mutation caused the decrease in focus-forming activity and the integration of the mutant viral DNA. These results suggest that ORF E2 encodes a protein which is involved either directly or indirectly in some aspects of oncogenic transformation by bovine papillomavirus and in maintaining the viral DNA as a plasmid in transformed cells.