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Mutations in polyomavirus middle T antigen affecting tumorigenesis
C Gelinas1, B Schaffhausen, B Bockus
1Department of Microbiology, University of Sherbrooke, Quebec, Canada.
Abstract:
P155 is a polyomavirus mlt mutant with normal transforming ability but impaired tumorigenic potential. The mutation, a 12-bp deletion (nucleotides 1348-1359), removes amino acids 372 to 375 from middle T and affects its ability to function in tumorigenesis (C. Gelinas, S. Masse, and M. Bastin, 1984, J. Virol. 51, 242-246). We used deletion loop mutagenesis to introduce point mutations within the wild-type sequence spanned by the P155 deletion. A mutant phenotype resembling that of P155 could be produced by as little as one alanine to valine substitution at residue 373. The mutants were impaired in their ability to induce tumors in rats but they could still transform established cell lines or primary fibroblasts in culture. To define the biochemical defect, we examined the mutant middle T antigen both for association with pp60c-src, the cellular src gene product, as well as its pattern of phosphorylation. No obvious differences explaining the phenotype were observed. The mutant middle T associated with, and activated pp60c-src, but exhibited a slightly altered pattern of phosphorylation, presumably because of additional sites on the middle T protein.
Insights
A polyomavirus mutant (P155) transforms cells normally but shows reduced tumor formation. Specific mutations in middle T antigen impair tumorigenesis without affecting pp60c-src association or activation.
Area of Science:
- Virology
- Molecular Biology
- Oncogenesis
Background:
- Polyomavirus middle T antigen is crucial for cell transformation and tumor development.
- The P155 mutant exhibits normal transforming ability but diminished tumorigenic potential due to a specific deletion in middle T antigen.
Purpose of the Study:
- To investigate the role of specific amino acid residues within the P155 deletion in middle T antigen's function.
- To elucidate the biochemical mechanisms underlying the impaired tumorigenic potential of P155 mutants.
Main Methods:
- Deletion loop mutagenesis was employed to introduce point mutations into the wild-type middle T antigen sequence.
- Mutant middle T antigens were analyzed for their ability to transform cells in culture and induce tumors in rats.
- Biochemical assays assessed middle T antigen association with and activation of cellular src kinase (pp60c-src) and its phosphorylation patterns.
Main Results:
- A single amino acid substitution (Ala to Val at residue 373) mimicked the P155 phenotype, impairing tumor induction while retaining transforming ability.
- Mutant middle T antigens associated with and activated pp60c-src.
- Mutant middle T antigens displayed slightly altered phosphorylation patterns compared to wild-type.
Conclusions:
- The region encompassing amino acids 372-375 in polyomavirus middle T antigen is critical for tumorigenesis but not for cell transformation or pp60c-src activation.
- Altered phosphorylation patterns of middle T antigen may contribute to its reduced tumorigenic potential.