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Cellular mobile genetic elements in the regulatory region of the pneumotropic mouse polyomavirus genome: structure and function in viral gene expression and DNA replication
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.
2003 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 77, no 6, 3477-3486 p.Article in journal (Refereed) Published
Abstract [en]

DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR. The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length. Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type. A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent. There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini. Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells. In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters. In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations. Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter. Analysis by Southern blotting and quantification using dot blots showed that approximately 103 copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment. These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.

Place, publisher, year, edition, pages
2003. Vol. 77, no 6, 3477-3486 p.
National Category
Medical and Health Sciences Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90157DOI: 10.1128/​JVI.77.6.3477-3486.2003PubMedID: 12610123OAI: oai:DiVA.org:uu-90157DiVA: diva2:162401
Available from: 2003-02-26 Created: 2003-02-26 Last updated: 2013-05-15Bibliographically 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.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1227
Keyword
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
Identifiers
urn:nbn:se:uu:diva-3326 (URN)91-554-5541-7 (ISBN)
Public defence
2003-03-19, C4:305, BMC, Uppsala, 09:15
Opponent
Available from: 2003-02-26 Created: 2003-02-26Bibliographically approved

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