Kilham polyomavirus: Activation of gene expression and DNA replication in mouse fibroblast cells by an enhancer substitution

The Kilham strain of polyomavirus (KV) infects vascular endothelial cells i n vivo (J. E. Greenlee, Infect. Immun. 26:705-713, 1979), but no permissive cell type for growth of the virus in vitro has been identified. The failur e of KV DNA to replicate in mouse fibroblast cells after transfection sugge sted that viral gene expression had narrow cell specificity. A KV substitut ion mutant having a part of the regulatory region of KV DNA replaced with a segment of the polyomavirus transcriptional enhancer was constructed. The substitution mutant was able to replicate in transfected 3T3 cells, and the newly replicated viral DNA associated with protein to form particles with the density of virions in CsCl equilibrium gradients. However, these partic les were noninfectious when tested on 3T3 cells, suggesting that absorption or uptake of virus particles was defective for these cells. Analysis of ea rly and late promoter activities by luciferase reporter gene expression sho wed that the enhancer substitution had a moderate positive effect on early gene expression and a large effect on the expression of the late genes. KV large T antigen inhibited the activities of both the wild-type and the subs titution mutant early promoter, whereas only the mutant late promoter was a ctivated under the same conditions. A comparison of the KV and polyomavirus large T antigens showed that they were not interchangeable in the initiati on of KV and polyomavirus DNA synthesis. Furthermore, the wild-type KV orig in of DNA replication was less active than the mutant structure in the pres ence of saturating amounts of KV large T antigen. Together, our data demons trate several differences between the two types of large T antigen in their interactions with cellular proteins.