Introduction of cloned human papillomavirus genomes into mouse cells and expression at the RNA level.
The entire DNA genomes of five different human papillomaviruses (HPVs) were cloned into the BamHI site of pBR322 (HPV-1a, HPV-3, HPV-4, and HPV-9) or the EcoRI site of pBR325 (HPV-2), using as starting materials virus preparations isolated from papillomas of individual patients. Under stringent hybridization conditions (Tm-28 degrees), the five cloned HPVs exhibited less than 10% homology with one another. To establish model cell systems that may be useful for the identification of HPV genes and HPV gene products, mouse thymidine kinase negative (tk-) cells were cotransformed to the tk+ phenotype with the herpesvirus thymidine kinase gene and each of the five HPV cloned DNAs (either as intact recombinants or excised HPV DNA without removal of pBR). In most tk+ cell clones, a complex pattern of multiple high molecular weight inserts of HPV DNA were present in high copy number. Most of the HPV DNA sequences in the cotransformed cells were not present as unit-length episomal viral DNA. Analyses of the integration pattern (DNA blot) and RNA expression (RNA blot) of several HPV-1a and HPV-3 transformed cell lines suggest that some copies of the viral genome are integrated in a similar manner in different cell lines leading to the expression of identical viral RNA-containing species. Two of the cell lines transformed by the intact HPV-1a/pBR322 recombinant synthesized substantial amounts of four discrete viral polyadenylated cytoplasmic RNA species of 1.9, 3.2, 3.8, and 4.5 kb. Two cell lines transformed by the intact HPV-3/pBR322 recombinant synthesized 4-5 polyadenylated cytoplasmic viral RNA species ranging from 0.8 to 4.6 kb. The analysis shows that each viral RNA species appears to be a hybrid RNA molecule containing both HPV and pBR322 sequences. Based on these findings and the molecular organization of the HPV-1a genome (O. Danos, M. Katinka, and M. Yaniv (1982). EMBO J. 1, 231-237), it is possible that transcription of each of the HPV-1a RNA species is initiated using the HPV early promoter and terminated in pBR322.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005814:Genes, Viral; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D011061:Poly A / Q000032:analysis / Q000096:biosynthesis; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D012333:RNA, Messenger; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger; D012367:RNA, Viral; D011061:Poly A; D012313:RNA