title: Introduction of cloned human papillomavirus 1a DNA into rat fibroblasts: integration, de novo methylation and absence of cellular morphological transformation.
abstract: The morphologically normal rat fibroblast cell line Rat-2 was used as a target cell type to test the transforming ability of a human papillomavirus (HPV-1a). To this end, molecularly cloned HPV-1a genomes were introduced into cultured Rat-2 cells in cotransfection experiments using a cloned herpes simplex virus thymidine kinase gene as a selectable phenotypic maker. In each of 13 HPV-1a-positive cell clones examined the papillomavirus DNA sequences were associated with the high molecular weight fraction of the cellular DNA, and restriction endonuclease plus Southern blotting analyses revealed patterns of hybridization which were consistent with integration of the viral genomes. Even Rat-2 clones containing multiple copies of the entire HPV-1a genome retained the normal, i.e. flat, cell morphology and were unable to grow in soft agar. De novo methylation of the HPV-1a sequences in many Rat-2 cell clones was evidenced by resistance of the viral DNA to complete cleavage with the HpaII endonuclease. Two out of three cell lines harbouring multiple copies of the HPV-1a genome contained detectable levels of HPV-1a transcripts, whereas no transcripts were detected in the third such cell line in which the viral HpaII sites were methylated virtually to completion. These results are consistent with the notion that HPV-1a genes are expressed inefficiently in Rat-2 cells; consequently integration of the viral DNA occurs, and there is no effect of the virus on the growth properties of this cell type. It is possible that methylation of the HPV-1a sequences is responsible for the low levels of expression of the viral genome.
authors.forename: T S
authors.initials: TS
authors.lastname: Burnett
authors.forename: P H
authors.initials: PH
authors.lastname: Gallimore
mesh_terms: D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts; D005814:Genes, Viral; D006801:Humans; D008745:Methylation; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids; D051381:Rats; D011995:Recombination, Genetic; D014162:Transfection
chemical_list: D004279:DNA, Viral