Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon

Abstract

We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNA_U) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30_INT). We find now that after the methylation of reo mRNA_U has stopped in S30_INT, the RNA can be reisolated and further methylated in an extract of control cells (S30_C). Thus the impairment of methylation in S30_INT cannot be due to cleavage or irreversible inactivation of reo mRNA_U. Freshly added reo mRNA_U can be methylated in S30_INT in which the methylation of previously added reo mRNA_U has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNA_U. Freshly added reo mRNA_U can be methylated in S30_INT in which the methylation of previously added reo mRNA_U has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNA_U for methylation. The extent of the impairment of reo mRNA_U methylation in S30_INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNA_U is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I)-poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30_INT is the same as in the presence of S30_C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30_INT but not by S30_C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.