Molecular and biological characterization of cottontail rabbit papillomavirus variant DNA sequences integrated in the VX7 carcinoma

Abstract

The transplantable VX7 carcinoma was derived from a tumor induced by a recoverable strain of cottontail rabbit papillomavirus (CRPV) able to replicate in domestic rabbits. Low levels of late viral gene expression have been retained through serial propagation in rabbits. We have cloned and characterized the three major types of CRPV sequences integrated in this tumor, a genome-length 8-kb DNA molecule and two rearranged 9- and 3.8-kb molecules. The VX7 8-kb DNA displays only a few differences in its restriction map, when compared to the wild-type (wt) CRPV DNA. The VX7 9- and 3.8-kb DNAs derive from the VX7 8-kb DNA since they share the same restriction site polymorphism. The VX7 9-kb DNA contains a duplication of the E6 open reading frame. The VX7 3.8-kb DNA results from the deletion of most of the E region and the insertion, between the borders of the deletion, of a 174-nucleotide-long segment of the long control region potentially driving the expression of a truncated L2 protein. Both VX7 9- and 3.8-kb species potentially allow the expression of abnormal E6 fusion proteins. Nineteen point mutations were detected in the 3.8-kb DNA, compared to the wt CRPV DNA. None of these molecules were able to induce warts in domestic rabbits, in contrast to wt CRPV DNA. Furthermore, when cloned VX7 DNAs were inoculated together with wt CRPV DNA, none of the VX7 CRPV sequences, as identifiable by their specific restriction enzyme cleavage patterns, could be detected in the resulting warts. This suggests that CRPV sequences integrated in the VX7 carcinoma are no longer able to replicate as episomes, which might be a prerequisite for the production of warts.