Mechanism of interferon action: studies on the activation of protein phosphorylation and the inhibition of translation in cell-free systems.

We describe the ability of reovirus messenger RNA (mRNA) to serve as a template for translation and as an activator of protein phosphorylation in cell-free extracts prepared from untreated and from interferon (IFN)-treated mouse fibroblast L cells. In vitro transcribed reovirus mRNA was purified by column chromatography on CF-11 cellulose. This procedure removed trace amounts of double-stranded RNA (dsRNA) [0.01%-0.1%] present in mRNA preparations purified solely by extensive LiCl precipitation. In the absence of added dsRNA, CF-11 cellulose-purified reovirus mRNA did not detectably activate phosphorylation of either ribosome-associated protein P1 or the alpha subunit of protein synthesis initiation factor eIF-2 in S-10 extracts prepared from L cells; the CF-11 cellulose-purified reovirus mRNA was translated more efficiently than was LiCl-purified reovirus mRNA in these extracts. Highly purified CF-11 reovirus mRNA was, however, translated less efficiently by S-10 extracts prepared from IFN-treated L cells than by extracts prepared from untreated L cells, suggesting that the inefficient translation by IFN-treated extracts was an integral property of reovirus mRNA. Increasing the secondary structure of reovirus mRNA by substituting bromouridine (Br-uridine) for uridine in the mRNA caused an increased inhibition of mRNA binding to ribosomes in extracts prepared from IFN-treated as compared to untreated cells. The mechanism of inhibition of translation of CF-11 cellulose-purified reovirus mRNA in IFN-treated systems remains to be established.

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