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Inhibition of binding to initiation complexes of nascent reovirus mRNA by double-stranded RNA-dependent protein kinase.

ABSTRACT

A coupled, cell-free system for the transcription and translation of reovirus mRNA was developed. Activated reovirions were incubated with reticulocyte lysate and an appropriate energy mix. Active transcription was obtained, but protein synthesis was inhibited after a short lag even by low concentrations of reovirions. This inhibition was abolished by the addition of the kinase inhibitor 2-aminopurine. With this addition, the synthesis of viral proteins could be detected in reaction mixtures containing nuclease-treated reticulocyte lysate. The binding of nascent reovirus mRNA to 80S initiation complexes measured after 2 min of incubation was greatly inhibited, whereas the binding of cellular mRNA added to the same reaction mixtures for the next 2 min was not inhibited. The inhibition of reovirus mRNA binding could not be explained by the synthesis of defective templates, since most of the mRNA could be bound to 80S complexes after the addition of 2-aminopurine. These results indicate that the binding of nascent reovirus mRNA was preferentially inhibited by a protein kinase. Reovirions preincubated with reticulocyte lysate could phosphorylate initiation factor eIF-2. This phosphorylation was inhibited by the addition of high concentrations of double-stranded RNA, which are inhibitory for the eIF-2 kinase present in elevated levels in reticulocyte lysate and in interferon-treated cells. These results indicate that the translation of viral mRNA may be preferentially inhibited in interferon-treated cells by the eIF-2 kinase activated by viral transcriptional complexes containing double-stranded RNA.

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