title: 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.
abstract: 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.
mesh_terms: 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*
chemical_list: 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