Mechanism of interferon action. Increased phosphorylation of protein synthesis initiation factor eIF-2 alpha in interferon-treated, reovirus-infected mouse L929 fibroblasts in vitro and in vivo.
The effect of interferon (IFN) treatment and virus infection on the phosphorylation both in vitro and in vivo of the alpha subunit of protein synthesis initiation factor eIF-2 (eIF-2 alpha) was examined in mouse fibroblast L929 cells. The [gamma-32P]ATP-mediated in vitro phosphorylation of eIF-2 alpha catalyzed by cell-free extracts prepared from IFN-treated, uninfected cells was dependent upon exogenously added double-stranded RNA (dsRNA). However, the dsRNA requirement for eIF-2 alpha phosphorylation in vitro was eliminated by prior infection of cells with reovirus Dearing strain virions but not with defective top component particles. The enhanced phosphorylation in vitro of eIF-2 alpha and ribosome-associated protein P1 depended in a similar manner upon the multiplicity of virus infection. The extent of phosphorylation in vivo of eIF-2 alpha prepared from L929 cells was also examined by utilizing two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting techniques. About 5-10% of the eIF-2 alpha was typically phosphorylated in vivo in untreated, mock-infected cells, whereas 25-30% was phosphorylated in IFN-treated, reovirus-infected cells. An intermediate extent of eIF-2 alpha phosphorylation, routinely between 15 and 20%, was observed with either IFN treatment or reovirus infection alone. The integrity of eIF-4A and eIF-4B was also examined by two-dimensional electrophoresis and immunoblotting, and no significant alterations in molecular size or charge heterogeneity were detected when these factors were prepared from IFN-treated, reovirus-infected cells as compared to untreated, uninfected cells.
D000818:Animals; D002472:Cell Transformation, Viral; D015852:Eukaryotic Initiation Factor-2; D039601:Eukaryotic Initiation Factor-4A; D039642:Eukaryotic Initiation Factors; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology*; D007700:Kinetics; D007739:L Cells / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012087:Reoviridae / Q000235:genetics*
D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D007370:Interferon Type I; D010448:Peptide Initiation Factors; D011506:Proteins; C031799:eIF-4B; D039601:Eukaryotic Initiation Factor-4A