Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)₂) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [³H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)₃) was completely blocked by pA(2'p5'A)₂. 2) The dsRNA-dependent phosphorylation of protein P₁ and the α subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 μg/ml). However, high concentrations of dsRNA (>100 μg/ml) drastically reduced the phosphorylation of both P₁ and eIF-2α. Neither P₁ nor eIF-2α phosphorylation was affected by either pA(2'p5'A)₂ or pppA(2'p5'A)₃. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)₃. Whereas pA(2'p5'A)₂ completely reversed the pppA(2'p5'A)₃-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)₂. Under conditions where the pppA(2'p5'A)₃ mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P₁ and eIF-2α phosphorylation. These results suggest that the pppA(2'p5'A)(n)-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.