Mechanism of interferon action studies on the mechanism of interferon-mediated inhibition of reovirus messenger RNA translation in cell-free protein synthesis systems from mouse ascites tumor cells

The biochemical mechanism of the interferon-mediated inhibition of reovirus messenger RNA translation in cell-free extracts prepared from mouse ascites tumor cells was investigated. The following results were obtained: (1) Cell-free extracts from interferon-treated cells catalyze the formation of methionyl-X initiation dipeptides in response to reovirus mRNA at a rate and extent only slightly less than that of extracts from untreated cells. Under conditions where the overall translation in vitro of reovirus mRNA is inhibited by 75% or more, the formation of methionyl-X initiation dipeptides is inhibited much less, about 30%. (2) Neither methionyl-X initiation dipeptide formation nor overall translation of reovirus mRNA is significantly affected by the addition of either S-adenosyl-l-methionine or S-adenosyl-l-homocysteine to cell-free systems prepared from either interferon-treated or untreated cells. (3) Reovirus mRNA synthesized in vitro in the presence of S-adenosyl-l-methionine is translated slightly more efficiently than either reovirus mRNA synthesized in the presence of S-adenosyl-l-homocysteine or reovirus mRNA synthesized in the absence of both S-adenosyl-l-methionine and S-adenosyl-l-homocysteine by cell-free systems prepared from untreated ascites cells; by contrast, all of the reovirus mRNA preparations were translated very poorly by cell-free systems prepared from interferon-treated cells. (4) Exogenously added mouse ascites cell transfer RNA partially reverses the inhibition of viral mRNA translation in vitro catalyzed by cell-free systems prepared from interferon-treated cells in response to reovirus mRNA synthesized in the absence or in the presence of either S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. Yeast and rat liver tRNA as well as tRNA from two prokaryotes, Escherichia coli and Streptococcus faecalis, do not significantly stimulate viral mRNA translation in the interferon-treated cell-free system. Translation catalyzed by untreated control extracts is not dependent upon the addition of tRNA. (5) Reovirus [³²P]mRNA recovered from interferon-treated cell-free protein synthesis systems after 15-min incubation possesses a profile similar to that of mRNA recovered from untreated control systems when analyzed by polyacrylamide-agarose-urea gel electrophoresis. These results indicate that the interferon-mediated inhibition of viral mRNA translation in vitro is exerted at a step of polypeptide chain biosynthesis which is subsequent to formation of the first peptide bond and which either directly or indirectly involves the participation of transfer RNA.