Studies on the Molecular Biology of the Mouse Pneumotropic Polyomavirus

Sammanfattning: The Murine Pneumotropic Virus (MPtV), in contrast to the other MurinePolyomavirus (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in Escherichia coli DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.