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A kinase able to phosphorylate exogenous protein synthesis initiation factor eIF-2 alpha is present in lysates of
Abstract:
Infection of mouse L929 cells by mengovirus resulted in the expression of a kinase activity that selectively phosphorylated the small, 38,000-molecular-weight subunit of eucaryotic initiation factor 2 and histone H2. This kinase activity was independent of host cell RNA synthesis and was located in the postribosomal supernatant (S-100 fraction) early after infection (up to 3 h). At later times after infection (5 h), kinase activity was also associated with the polysome fraction. The kinase present in the S-100 fraction bound strongly to DEAE-cellulose, its peak activity eluting at 0.5 M KCl. Kinase activity was independent of the presence of exogenous double-stranded RNA, and KCl at concentrations greater than 0.1 M inhibited eucaryotic initiation factor 2 phosphorylation. The 67,000-molecular-weight phosphoprotein activated in interferon-treated cells by double-stranded RNA was not detected by standard phosphorylation assays in lysates from mengovirus-infected cells. Labeling of this protein in vivo during 5 h of infection was also not detected. The DEAE-cellulose-purified mengovirus kinase inhibited protein synthesis in reticulocyte lysates, and the inhibition was not reversible by high concentrations of poly(I).poly(C).
Insights
Mengovirus infection activates a specific kinase that phosphorylates eukaryotic initiation factor 2, inhibiting protein synthesis. This viral kinase differs from host cell interferon-induced pathways.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Mengovirus infection in L929 cells triggers cellular responses.
- Eukaryotic initiation factor 2 (eIF2) is crucial for protein synthesis.
- Viral infections can modulate host cell translation machinery.
Purpose of the Study:
- To characterize the kinase activity induced by mengovirus infection.
- To determine the substrate specificity and localization of the mengovirus-induced kinase.
- To investigate the kinase's role in protein synthesis inhibition.
Main Methods:
- Cell culture and viral infection (mengovirus, L929 cells).
- Kinase assays using eIF2 and histone H2 as substrates.
- Fractionation of cell lysates (S-100, polysome).
- DEAE-cellulose chromatography for kinase purification.
- Protein synthesis assays in reticulocyte lysates.
Main Results:
- Mengovirus infection induced a kinase phosphorylating eIF2 and histone H2.
- The kinase was initially in the S-100 fraction, later associated with polysomes.
- The purified kinase inhibited protein synthesis in reticulocyte lysates.
- This activity was independent of double-stranded RNA and distinct from interferon-induced pathways.
Conclusions:
- Mengovirus encodes or induces a kinase that selectively targets eIF2.
- This viral kinase plays a role in inhibiting host protein synthesis.
- The mechanism differs from the double-stranded RNA-activated protein kinase (PKR) pathway.