Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors

Abstract

The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance. To define the viral genes which are necessary for this process, a series of specific mutations was constructed within the viral genome and the resulting mutant were assayed for their ability to replicate extrachromosomally in mouse C127 cells. The bovine papillomavirus type 1 trans-acting replication factors were encoded by at least 2 distinct viral genes since the mutants fell into 2 complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. Evidently, this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; the copy number of the mutant genomes were reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of 1 to 2, indicating that in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. Morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.