uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Kilham polyomavirus: activation of gene expression and DNA replication in mouse fibroblast cells by an enhancer substitution
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2001 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 75, no 21, 10015-10023 p.Article in journal (Refereed) Published
Abstract [en]

The Kilham strain of polyomavirus (KV) infects vascular endothelial cells in 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 failure of KV DNA to replicate in mouse fibroblast cells after transfection suggested that viral gene expression had narrow cell specificity. A KV substitution 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 particles were noninfectious when tested on 3T3 cells, suggesting that absorption or uptake of virus particles was defective for these cells. Analysis of early and late promoter activities by luciferase reporter gene expression showed 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 substitution mutant early promoter, whereas only the mutant late promoter was activated under the same conditions. A comparison of the KV and polyomavirus large T antigens showed that they were not interchangeable in the initiation of KV and polyomavirus DNA synthesis. Furthermore, the wild-type KV origin of DNA replication was less active than the mutant structure in the presence of saturating amounts of KV large T antigen. Together, our data demonstrate several differences between the two types of large T antigen in their interactions with cellular proteins.

Place, publisher, year, edition, pages
2001. Vol. 75, no 21, 10015-10023 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-90156DOI: 10.1128/JVI.75.21.10015-10023.2001PubMedID: 11581370OAI: oai:DiVA.org:uu-90156DiVA: diva2:162400
Available from: 2003-02-26 Created: 2003-02-26 Last updated: 2013-06-13Bibliographically approved
In thesis
1. Studies on the Molecular Biology of the Mouse Pneumotropic Polyomavirus
Open this publication in new window or tab >>Studies on the Molecular Biology of the Mouse Pneumotropic Polyomavirus
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 48 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1227
Molecular biology, murine pneumotropic virus, Kilham mouse polyomavirus, large T antigen, enhancer, regulatory region rearrangement, gene expression regulation, DNA replication, transposable elements, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:uu:diva-3326 (URN)91-554-5541-7 (ISBN)
Public defence
2003-03-19, C4:305, BMC, Uppsala, 09:15
Available from: 2003-02-26 Created: 2003-02-26Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Magnusson, Göran
By organisation
Department of Medical Biochemistry and Microbiology
In the same journal
Journal of Virology
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 173 hits
ReferencesLink to record
Permanent link

Direct link