Analysis of stable and unstable viral forms in SV40-infected human keratinocytes.
We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.
Naimski
P
P
Steinberg
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ML
D002460:Cell Line; D002472:Cell Transformation, Viral; D002999:Clone Cells; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D000078404:Epidermal Cells; D005814:Genes, Viral; D006801:Humans; D007633:Keratins / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D013539:Simian virus 40 / Q000235:genetics*
D004279:DNA, Viral; D007633:Keratins; D004262:DNA Restriction Enzymes