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.
Jacobs
B L
BL
Department of Biological Sciences, University of California, Santa Barbara 93106.
Miyamoto
N G
NG
Samuel
C E
CE
D000818:Animals; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D007372:Interferons / Q000494:pharmacology*; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012087:Reoviridae / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism
D011506:Proteins; D012333:RNA, Messenger; D007372:Interferons