Identification of three transforming proteins encoded by cottontail rabbit papillomavirus

Cottontail rabbit papillomavirus (CRPV) provides an animal model for human papillomaviruses associated with a high risk of cancer development. So far, nothing is known about the transforming functions of CRPV genes because of the lack of an assay system. We have recently developed two systems to assay for CRPV transforming functions. One is based on the finding that transformation of NIH 3T3 cells by CRPV is considerably increased by deleting sequences in open reading frame L2. The second one is based on the use of a cottontail rabbit skin epithelial cell line, sf1Ep (C. Meyers and F. O. Wettstein, Virology 181:637-646, 1991). Mutations were introduced which abolished expression of the full-length E6 protein (LE6), the short E6 protein (SE6) initiated at the second ATG of E6, the E7 protein, or the E5 protein. Mutations affecting LE6 or E7, but not SE6, reduced transformation of NIH 3T3 and sf1Ep cells. Transformed NIH 3T3 cell lines with mutations in LE6 and E7 did not grow in soft agar, while those with mutations in SE6 and E5 grew with a reduced efficiency. The cell lines with mutations in LE6, SE6, or E7 still did induce tumors in nude mice. These mutations, however, abolished the ability to induce papillomas in rabbits. When expressed individually with a retroviral vector, LE6, SE6, or E7, but not E5, conferred anchorage-independent growth. The level of viral protein expression in these cell lines was generally low, and a comparison of the abundance of virus-specific mRNA showed that cell lines contained 20 to 50 times less mRNA than a cottontail rabbit papilloma. These data demonstrate that CRPV encodes at least three transforming proteins.