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A human papillomavirus type 1 (HPV-1)/pBR322 recombinant plasmid was constructed consisting of two complete HPV-1 genomes in a head-to-tail arrangement, inserted into the BamHI site of pBR322. To obtain established human keratinocytes keratinocytes carrying dimeric HPV-1, origin-minus simian virus 40 (SV40) DNA was used as a dominant transforming marker in co-transfection experiments performed with cultured human foetal keratinocytes. Using the Southern blotting technique, HPV-1 DNA was detected in one out of five SV40-transformed human keratinocyte cell lines which were obtained in this way. Further blotting experiments using SV40, pBR322 and HPV-1 DNA as probes revealed patterns of hybridization which were consistent with co-integration of SV40 DNA with between two and four copies of the HPV-1 genome. The HPV-1 sequences in this cell line were virtually non-methylated and were transcribed at a lower level than SV40 mRNAs in the same cultured cells. A low level of monomeric HPV form I DNA was detected by blotting of undigested total cellular DNA. No evidence for a stimulation of form I HPV DNA replication could be obtained by blotting analysis of total DNA extracted from keratinizing cysts, which were formed by subcutaneous inoculation of these cells into nude mice.

journal of general virology

Establishment of a Human Keratinocyte Cell Line Carrying Complete Human Papillomavirus Type 1 Genomes: Lack of Vegetative Viral DNA Synthesis upon Keratinization