Impairment of reovirus mRNA methylation in extracts of interferon treated Ehrlich ascites tumor cells: Further characteristics of the phenomenon

The authors 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 Ehrlich ascites tumor cells (S30(INT)). The authors 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 not due to the depletion of S adenosylmethionine (the methyl donor), the accumulation of S denosylhomocysteine (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 mRNA(U) 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.