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

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Abstract
A human papillomavirus type 1 (HPV-1)/pBR322 recombinant plasmid was constructed consisting of 2 complete HPV-1 genomes in a head-to-tail arrangement, inserted into the BamHI site of PBR322. To obtain established human keratinocytes carrying dimeric HPV-1, origin-minus SV40 DNA was used as a dominant transforming marker in co-transfection experiments performed with cultured human fetal keratinocytes. Using the Southern blotting technique, HPV-1 DNA was detected in 1 of 5 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 2 and 4 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 mRNA 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 s.c. inoculation of these cells into nude mice.