title: Polyomavirus hr-t mutant-specific induction of a G2/M cell-cycle arrest that is not overcome by the expression of middle T and/or small T.
abstract: The polyomavirus hr-t class of mutants has served as a major prototype to study the function of middle T + small T in the virus lytic cycle, Biochem. Biophys. Acta 695 (2), 69-95). The properties of these middle T + small T defective mutants were defined by comparisons with "wild-type" strains reconstructed by marker rescue. Similar comparisons in the A2 genetic background have revealed a number of differences, J. Virol. 75, 8380-8389). Here we describe a major divergence in their effects on cell-cycle progression of both permissive mouse NIH3T3 cells and semipermissive Fischer rat FR3T3 cells. Infection of NIH3T3 or FR3T3 cells in serum-rich medium with wild-type A2 (WTA2) or WTA2-derived middle T + small T-defective mutants did not perturb cell cycling, tested up to entry into the third cycle. In contrast, infection with four hr-t mutants analyzed, examined in detail with mutant B2, resulted in an accumulation of cells in G2/M in a dose-dependent and serum-independent manner. The arrest began in the first cell cycle. At multiplicities of infection above 10 PFU/cell, 50-80% of the cell population became arrested by the end of the second cycle. FR3T3 arrested cells detached from the monolayer with a rounded up morphology. Three other hr-t mutants investigated were also found to arrest cells in G2/M. Expression of middle T and/or small T either in trans or in cis did not abrogate this cell-cycle arrest, as demonstrated in the latter case with the middle T + small T expressing strain "wtB2" obtained by repair of the B2 deletion. In FR3T3 cells, the induction of a cell-cycle arrest by wtB2 was accompanied by a severe delay and reduction in neoplastic transformation relative to WTA2 used at equal dose. Mutation(s) in the C-terminal domain of large T antigen, upstream of the site-specific DNA binding activity, is necessary for the cell-cycle block. The possible causes for the cell-cycle block are discussed.
authors.affiliation: Department of Microbiology and Molecular Genetics and Interdepartmental Program in Cell, and Molecular Biology, Michigan State University, East Lansing 48824-1101, USA.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Spink
authors.forename: Michele M
authors.initials: MM
authors.lastname: Fluck
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D016195:G2 Phase; D051379:Mice; D008938:Mitosis; D009154:Mutation; D010940:Plant Proteins; D011120:Polyomavirus / Q000502:physiology*; D051381:Rats; D015534:Trans-Activators; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010940:Plant Proteins; D015534:Trans-Activators; D014157:Transcription Factors