Mechanism of interferon action Interferon-mediated inhibition of reovirus mRNA translation in the absence of dettectable mRNA degradation but in the presence of protein phosphorylation

Abstract

The effect of treating mouse fibroblast L cells with interferon on the ability of nonpreincubated and micrococcal nuclease-treated extracts prepared from them to catalyze reovirus messenger RNA translation, protein phosphorylation, and messenger RNA degradation was investigated both in the presence and the absence of exogenously added double-stranded RNA (dsRNA) and transfer RNA (tRNA). The following results were obtained: (1) Phosphorylation of ribosome-associated protein P, and protein synthesis initiation factor eIF-2α was enhanced in both nonpreincubated and micrococcal nuclease-treated extracts prepared from interferon-treated cells. The enhancement in phosphorylation was dependent upon the presence of reovirus dsRNA, although the apparent dsRNA requirement could also be fulfilled by reovirus mRNA at concentrations of mRNA typical of those used in cell-free protein synthesizing systems. (2) By contrast, the degradation of reovirus [ 3H]mRNA was enhanced in extracts prepared from interferon-treated cells when nonpreincubated, but not when micrococcal nuclease-treated and supplemented with ethyleneglycol-bis(β-aminoethyl ether)- N, N′-tetraacetic acid (EGTSA) and 2′-deoxythymidine-3′,5′-diphosphate (pTp). The enhancement in degradation observed in nonpreincubated extracts lacking EGTA and pTp was dependent upon the presence of dsRNA and the absence of tRNA; enhanced dsRNA-dependent degradation was not detectable in the presence of tRNA. The limited degradation of reovirus [ 3H]mRNA, in both the presence and the absence of dsRNA and tRNA, was comparable in EGTA- and pTp-supplemented micrococcal nuclease-treated extracts prepared from either interferon-treated or untreated cells. The addition of 2′,5′-oligoadenylate enhanced degradation in nonpreincubated extracts but not in EGTA- and pTp-supplemented micrococcal nuclease-treated extracts (3) Reovirus mRNA was translated poorly by interferon-treated as compared to untreated systems in both the nonpreincubated and the micrococcal nuclease-treated extracts in the absence of reovirus dsRNA. The presence of dsRNA further decreased the translation by interferon-treated systems. The efficiency of endogenous cellular mRNA translation by untreated as compared to interferon-treated systems was comparable in nonpreincubated extracts, both in the presence and absence of dsRNA. The addition of mouse ascites tRNA stimulated translation in both untreated and interferon-treated systems, both in nonpreincubated and micrococcal nuclease-treated extracts. These results suggest that the translation of reovirus mRNA in interferon-treated systems is inhibited both in the presence and absence of interferon-mediated mRNA degradation, and that protein phosphorylation may play a primary role in regulating translation in interferon-treated cells.