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The alpha subunit of eucaryotic initiation factor 2 is phosphorylated in mengovirus-infected mouse L cells
J DeStefano1, E Olmsted, R Panniers
1Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3125.
Abstract:
Infection of mouse L cells with mengovirus resulted in the activation of a protein kinase (PK) that selectively phosphorylated the small, 38,000-molecular-weight alpha subunit of eucaryotic initiation factor 2 (eIF-2) in vitro. The mengovirus-activated kinase was detected in vitro approximately 3 h after virus adsorption. The ratio of phosphorylated to unphosphorylated eIF-2 also increased in vivo between 3 and 7 h after adsorption. The virus-activated kinase fractionated with the ribosomal pellet and had a high affinity for DEAE-cellulose and Mono Q ion-exchange columns. Gel electrophoresis of the kinase activity eluting from the Mono Q column and silver staining of the gel revealed only one protein band with a molecular mass of 70 kilodaltons. The optimal assay conditions for the mengovirus-activated kinase paralleled those of the double-stranded RNA-activated PK (dsRNA-PK). Lysates from infected cells contained elements capable of activating partially purified dsRNA-PK. These elements were identified as double-stranded RNA by their sensitivity to double-stranded RNase. The phosphorylation of the alpha subunit of eIF-2 coincided with the synthesis of dsRNA in infected cells, suggesting that the mengovirus-activated kinase is the dsRNA-PK. The phosphorylation of the alpha subunit of eIF-2 correlated with the global inhibition of protein synthesis that occurs at late times after infection.
Insights
Mengovirus infection activates a specific protein kinase (PK) that phosphorylates eukaryotic initiation factor 2 (eIF-2). This phosphorylation, linked to double-stranded RNA synthesis, inhibits protein synthesis in infected cells.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Mengovirus infection in mouse L cells triggers cellular responses.
- Protein synthesis regulation is crucial during viral infections.
Purpose of the Study:
- To investigate the protein kinase activated by mengovirus infection.
- To identify the substrate and regulatory mechanisms of this kinase.
- To understand its role in viral-induced protein synthesis inhibition.
Main Methods:
- In vitro kinase assays using mengovirus-infected cell lysates.
- Analysis of protein phosphorylation patterns of eukaryotic initiation factor 2 (eIF-2).
- Biochemical purification of the activated kinase using ion-exchange chromatography.
- Identification of activating factors via nuclease sensitivity assays.
Main Results:
- A protein kinase (PK) activated by mengovirus infection was identified.
- This PK selectively phosphorylated the alpha subunit of eIF-2.
- The kinase activity correlated with the presence of double-stranded RNA (dsRNA) and its optimal assay conditions mimicked the dsRNA-activated PK (dsRNA-PK).
- Phosphorylation of eIF-2 coincided with dsRNA synthesis and global protein synthesis inhibition.
Conclusions:
- The mengovirus-activated kinase is likely the dsRNA-PK.
- dsRNA synthesis during mengovirus infection leads to eIF-2 alpha subunit phosphorylation.
- This phosphorylation event is responsible for the late-stage inhibition of protein synthesis.