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Inhibition of polyoma gene expression in transformed mouse cells by hypermethylation
Abstract:
The evolution of mouse cells transformed by a recombinant plasmid containing the genome of the tsA mutant of polyoma virus (Py) cloned at the BamHI site into the plasmid pML, whose sequences therefore interrupt the Py late region, has been studied. Clones of transformed cells were selected at 39 degrees (nonpermissive temperature for large T antigen). Under these conditions viral DNA integration is stable and the cells display a uniformed transformed phenotype. Also studied in detail was the evolution of one of these cell lines (A4) upon shift to a temperature permissive for large T-Ag function (33 degrees); immediately after shift, 90% of the population became intensely positive for T-Ag and a considerable amount of free-viral DNA was produced, accompanied by a clear cytopathic effect. Surviving cells proliferated actively after 4 weeks at 33 degrees and showed a decreased expression of large T-Ag (only 2-3% of the population was T-Ag positive by immunofluorescence), a drastic reduction in the amount of free-viral DNA produced, but no apparent change in the pattern of integration of Py DNA in the host chromosomes. Analysis of the high-molecular-weight DNA with the restriction enzymes HpaII and MspI revealed that the cytosines in the recognition sequences of these enzymes were methylated. Accordingly, treating the cells with 5-Azacytidine, a methylation inhibitor, results in the expression of viral T-Ags in more than 80% of the cell population. Analysis of DNA transcription revealed a dramatic reduction of virus-specific poly(A)+ mRNA in the methylated cells; in addition, the phenotype of the 33 degrees A4 populations was much less transformed than that of the original cultures. The block of Py expression by methylation is not complete; approximately 2% of the cells remain T-Ag positive and viral transcription is not completely suppressed. This could be explained by an incomplete methylation which randomly leaves unmethylated sequences essential for Py gene expression, or by the fact that methylation is not sufficient to block transcription completely. Possible mechanisms underlying this type of evolution are discussed.
Insights
Mouse cells transformed with polyoma virus (Py) DNA show changes in viral gene expression and cell phenotype. DNA methylation silences viral gene expression, but this effect can be reversed with a methylation inhibitor.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Mouse cells were transformed with a recombinant polyoma virus (Py) plasmid.
- Transformed cells were initially selected at a non-permissive temperature (39°C) for viral T-antigen function.
- Cell line A4 was further studied after shifting to a permissive temperature (33°C).
Purpose of the Study:
- To investigate the evolution of mouse cells transformed by polyoma virus.
- To understand the role of viral DNA integration and expression in transformed cell phenotype.
- To explore the impact of DNA methylation on viral gene silencing and cell evolution.
Main Methods:
- Selection of transformed cell clones at different temperatures.
- Analysis of viral DNA integration and free viral DNA production.
- Immunofluorescence for T-antigen detection.
- Restriction enzyme digestion (HpaII, MspI) to assess DNA methylation.
- Treatment with 5-Azacytidine, a methylation inhibitor.
- Analysis of virus-specific mRNA transcription.
Main Results:
- At 33°C, initial high T-antigen expression and viral DNA production decreased over time in cell line A4.
- Surviving cells showed reduced T-antigen positivity and viral DNA, with stable integration patterns.
- DNA methylation was identified as a mechanism for silencing viral gene expression.
- 5-Azacytidine treatment reactivated viral T-antigen expression and transcription.
- Methylated cells exhibited a less transformed phenotype with reduced viral mRNA levels.
Conclusions:
- DNA methylation plays a significant role in silencing polyoma virus gene expression in transformed mouse cells.
- Reactivation of viral gene expression is possible via inhibition of DNA methylation.
- The observed evolution suggests a dynamic interplay between viral DNA, host cell, and epigenetic modifications.
- Incomplete methylation or insufficient suppression by methylation could explain residual viral activity.