Comparisons of two early gene functions essential for transformation in polyoma virus and SV-40

Abstract

We have compared ts-a mutants of polyoma virus with ts-A mutants of SV-40, and hr-t mutants of polyoma virus with the viable deletion mutants of SV-40 mapping between 0.54 and 0.59 map unit (referred to as dl). All four groups of mutants are either totally or partially defective in inducing stable transformation as assayed by anchorage-independent growth. In each virus system, two classes of mutants—hr-t and ts-a of polyoma virus and dl and tsA of SV-40—complement to induce stable transformation. Two distinct functions essential for transformation are therefore encoded within the early regions of these papova viruses. Two approaches have been taken in attempts to define the roles of these early viral genes in cell transformation. In the first approach, a clonal analysis was made of cells transformed at the permissive temperature by ts-a/A mutants. Selections were carried out either for anchorage-independent growth or for focus formation. Although the variation in expression of the selected parameter of transformation among multiple clones derived with the same virus and cell line is often high, the majority of clones show no temperature dependence of either selected or unselected properties when compared to wild-type virus-transformed clones. In some instances, temperature-sensitive clones are observed. No correlation is seen between the appearance of a temperature-sensitive phenotype in individual clones and the expression of T-antigen species at permissive and nonpermissive temperatures determined by immunofluorescence or immunoprecipitation of [ 35Slmethio-nine-labeled proteins. In the second approach, mutants of all four groups were tested for their ability to induce abortive transformation measured as the transient loss of anchorage-dependent growth. This assay circumvents the problem of clonal variation and gives a clearcut result. ts-a/A mutants retain the ability to induce abortive transformation, behaving like wild-type virus at the nonpermissive temperature. hr-t mutants are virtually negative, while the dl mutants show a reduced ability to induce abortive transformation. The simplest explanation which is adequate for the majority of the results is that the ts-a/A function is required only transiently to carry out an initiation event which stabilizes transformation, while the hr-t/dl function acts to induce parameters of the transformed phenotype in the manner of a maintenance function. Additional interpretations are put forward to explain the results with temperature-sensitive clones transformed by ts-a/A mutants.