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.