Biosynthesis of reovirus-specified polypeptides: Effect of methylation on the efficiency of reovirus genome expression in Vitro

Abstract

The effect of methylation on the transcription of reovirus genome RNA and the translation of reovirus messenger RNA in vitro was investigated. The following results were obtained: (1) Purified reovirions converted to cores by chymotrypsin treatment catalyzed the transcription of reovirus RNA at comparable rates in the presence of either S-adenosyl- l-methionine (SAdoMet) or S-adenosyl- l-homocysteine (SAdoHcy). Methylated virus reovirus RNA synthesized in the presence of SAdoMet possessed an electrophoretic pattern on agarose-urea-polyacrylamide gels indistinguishable from unmethylated mRNA synthesized in the presence of SAdoHcy. (2) The relative level of SAdoMet-mediated methylation of the four s adn three m class reovirus message species was comparable for methylated mRNA; SAdoHcy at an SAdoHcy:SAdoMet ratio of 350:1 reduced the methylation by more than 98%. (3) Methylated reovirus mRNA was translated by the wheat cell-free system in the presence of either SAdoMet or SAdoHcy with comparable efficiency. Unmethylated mRNA was translated about 50% as efficiently as methylated mRNA in the presence of SAdoMet; in the presence of SAdoHcy, unmethylated mRNA. (4) Polyacrylamide-gel electrophoresis revealed that the σ, μ, and ψ reovirus-specified polypeptides synthesized in vitro in responce to unmethylated mRNA were qualitatively indistiniguishable from those synthesized in response to methylated mRNA, although quantitatively the amount of reovirus polypeptides synthesized with methylated mRNA was much greater. (5) Chemical removal of the 5′-terminal 7-methylguanosine residue of methylated reovirus mRNA by β-elimination reduced the translational activity of the methylated mRNA by about 75%; the translational activity of unmethylated mRNA was reduced only slightly by treatment under identical conditions. (6) Unmethylated reovirus mRNA did not competitively inhibit the translation of methylated mRNA but, rather, appeared to have a cooperative effect. (7) The translation of methylated reovirus mRNA was sensitive to inhibition by 7-methylguanosine 5′-monophosphate (7mGMP), whereas the translation of unmethylated reovirus mRNA was insensitive to 7mGMP; the translational activities of comparable concentrations of methylated and unmethylated mRNA were nearly identical in the presence of 1 m M or greater 7mGMP. (8) The binding of unmethylated reovirus mRNA to wheat ribosomes was detectable by sucrose density centrifugation; however, the binding of methylated mRNA was significantly more efficient than unmethylated mRNA. These results indicate that methylation has no significant effect on the transcription of reovirus RNA in vitro but that the efficiency of translation of reovirus mRNA catalyzed by the wheat system is greatly increased by methylation of the viral mRNA although methylation does not appear to be an obligatory requirement for translation.