Papillomaviruses induce benign tumors in a variety of vertebrate species including man and, in some cases, these viral-induced lesions can progress to carcinomas. The intent of our laboratory's investigations is to define the mechanisms by which the papillomaviruses """"""""transform"""""""" both immortalized or primary cells in vitro and to determine how they contribute to tumorigenesis in vivo. Specifically, we are studying the transforming activities of bovine and human papillomavirus DNA as determined by focus information and immortalization assays of cultured murine and human epithelial cells. These studies also involve genetic definition of the viral genes responsible for in vitro transformation. We are also committed to identifying the protein products of the papillomavirus transforming genes and to characterizing their mode of action. To date, we have been able to demonstrate that the bovine papillomavirus (BPV) E5 ORF directs the synthesis of a small, hydrophobic transforming protein which is responsible for the major in vitro transforming activity of BPV. Cell fractionation studies have shown that most of the E5 protein is present in cell membranes but that some remains associated with the nucleus (presumeably with nuclear membranes). We have also shown that the E5 protein forms dimers via cysteines which are located near the COOH terminus of the molecule. By mutational analysis of the E5 ORF, we also have defined the initiation codon for E5 protein translation and demonstrated that NH2 terminal deletions, insertions, or substitutions do not interfere with the ability of the E5 protein to associate with cell membranes or to form dimers. Analysis of BPV-transformed hamster cells indicates that there is a direct correspondence between viral protein expression and the tumorigenic phenotype and that there is a threshold level of viral protein expression which is required for cellular transformation. Finally, we have shown that BPV-transformed hamster cells can resist allograft rejection, unlike hamster cells transformed by adenovirus type 2 or 12.
