LOSS OF INTEGRATED VIRAL-DNA SEQUENCES IN POLYOMA-TRANSFORMED CELLS IS ASSOCIATED WITH AN ACTIVE VIRAL-A FUNCTION

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated from through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the curing of lysogenic bacteria, revertants arising in the transformed rat populations were selected and it was determined whether these cells lost integrated viral genomes. It was further investigated whether the viral A function was necessary for curing by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluoresence for polyoma T [tumor] antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones were retransformed by superinfection and appeared to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo curing with about the same frequency at 33 or 39.degree. C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 than at 39.degree. C. In polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.