Introduction of Cloned Human Papillomavirus 1a DNA into Rat Fibroblasts: Integration, de novo Methylation and Absence of Cellular Morphological Transformation

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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-Ia). Molecularly cloned HPV-Ia genomes were introduced into cultured Rat-2 cells in cotransfection experiments using a cloned herpes simplex virus thymidine kinase gene as a selectable phenotypic marker. In each of 13 HPV-Ia-positive cell clones examined the papillomavirus DNA sequences were associated with the high MW fraction of the cellular DNA; 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 resitance of the viral DNA to complete cleavage with the HpaII endonuclease. Two of 3 cell lines harboring multiple copies of the HPV-1a genome contained detectable levels of HPV-1a transcripts; no transcripts were deteced in the 3rd such cell line in which the viral HpaII sites were methylated virtually to completion. Thus, 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. Methylation of the HPV-1a sequences may be responsibl for the low levels of expression of the viral genome.