DNA Methylation and Herpes Simplex Virus

Infection of rat embryo (RE) cells with herpes simplex vims type 2 (HSV-2) inhibits the methylation of newly synthesised cellular DNA. This hypomethylation may be an important step in the transformation of cells by HSV-2 as various cell strains also show reduced levels of methylation in comparison to the parent RE cells. The mechanism of HSV infection induced hypomethylation and in addition, the relationship between this hypomethylation and the hypomethylation of transformed cell strains was investigated during the course of this work. DNA methylase activity, as measured in an in vitro assay, is unaltered following HSV-2 infection. There is no increase in the rate of degradation of the methyl group donor, SAM and the ratio of SAM/SAH is also unaltered in infected cells. Moreover, cellular DNA in infected cells retains the ability to accept methyl groups. These results suggest, therefore, that despite the inhibition of DNA methylation, RE cells infected with HSV-2 appear to retain all the factors necessary for DNA methylation to take place. The possibility that HSV infection inhibits DNA methylase activity in vivo by altering the intracellular ionic environment was investigated However, we were unable to mimic infection induced hypomethylation by treating cells with high (0.24 M) concentrations of NaCl or with a variety of ionophores. Immunofluoresence studies using anti-methylase antibodies showed that DNA methylase concentrates around the nuclear periphery following infection with HSV-2. This is in contrast to the sites of DNA replication which are distributed throughout the nucleus and this differential distribution leads us to speculate that the inhibition of DNA methylation following infection occurs as a result of DNA methylase being distanced from its substrate, newly synthesised DNA. In support of this theory we have demonstrated that a viral mutant which does not cause an inhibition of DNA methylation when infected into RE cells similarly does not cause the redistribution of DNA methylase. The mechanism of this redistribution is still unknown but we hypothesise that DNA methylase may migrate to the nuclear periphery by association with cellular chromatin which undergoes nuclear margination and condensation at early times after infection. RE cells can be transformed with the Bgl II n fragment of HSV-2 and these cloned cell strains have a genome-wide reduction in the level of methylation as compared to the parent RE cells. However, we have no evidence for inhibition of methylation within 48 hours following the transfection of RE cells with the Bgl II n or neighbouring fragments. This suggests that an inhibition of methylation is not the primary event in the transformation of RE cells by fragments of HSV-2 although it may be important in the maintenance of the transformed state