Multiple Free Viral DNA Copies in Polyoma Virus-Transformed Mouse Cells Surviving Productive Infection

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Abstract
Mouse [fibroblast] 3T6 cells were infected with polyoma virus at high multiplicity, and survivors were isolated. Clones from single cells were then established and were resistant to a 2nd infection. In some clones viral functions could at least be partially expressed during reinfection, as judged from a stimulation of nuclear tumor antigen expression. One such clone was studied in detail. These cells were transformed and produced low amounts of virus (< 1 PFU [plaque forming units]/cell per generation). The persistent infection did not seem to be a carrier-state phenomenon, since infectious-center assays showed that most cells produced virus. The resistance of the cells to reinfection can be explained by interference from viral DNA present in the cells, averaging about 1500 free copies/cell. This DNA had the normal physical characteristics of polyoma DNA. It had a slightly larger size than authentic polyoma DNA. Mapping with [HpaII, EcoRI, BamHI and HhaI] restriction endonucleases showed that the addition to the DNA was about 5% of the wild-type genome and was located close to the origin of DNA replication. This DNA was infectious, although it had a 10-fold lower infectivity than wild-type polyoma DNA. Both virus and DNA from the polyoma-resistant cells had a small-plaque morphology, as opposed to the large-plaque morphology of the virus used for the initial selection of cells.