Related Experiment Videos
Frog virus 3-induced translational shut-off: activation of an eIF-2 kinase in virus-infected cells
1Department of Microbiology, University of Mississippi Medical Center, Jackson 39216.
Abstract:
Infection of susceptible fathead minnow or Friend erythroleukemia cells with either infectious or heat-inactivated frog virus 3 led to the rapid inhibition of cellular protein synthesis. As seen in other cells, translational shut-off was accompanied by the dissociation of polysomes, but not the degradation of irreversible inactivation of cellular mRNAs. In addition, lysates from cells infected with heat-inactivated FV3 showed a reduced capacity to synthesize protein and to form 43S pre-initiation complexes in vitro. These results indicate that the in vitro systems accurately reflected in vivo events, and suggest that translational shut-off occurred prior to the union of the 40S ribosomal subunit and the [eIF-2.GTP.Met tRNAi] ternary complex. To determine the basis for the translational block, lysates from mock- and FV3-infected cells were assayed in vitro for their ability to phosphorylate the alpha subunit of eIF-2. In contrast to lysates from mock-infected cells, lysates from cells infected with heat-inactivated or infectious FV3 readily phosphorylated the alpha subunit of eIF-2. Since phosphorylation of the alpha subunit of eIF-2 inhibits its catalytic utilization during polypeptide chain initiation, these findings suggest that translational shut-off mediated by FV3 may be due to activation of a kinase that selectively phosphorylates this key initiation factor.
Insights
Frog virus 3 (FV3) infection rapidly halts protein synthesis by inhibiting translation initiation. FV3 activates a kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2), blocking protein production.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Viral infections can disrupt host cell functions.
- Protein synthesis is crucial for cellular activity and viral replication.
- Translation initiation is a key regulatory step in protein synthesis.
Purpose of the Study:
- To investigate the mechanism by which frog virus 3 (FV3) inhibits cellular protein synthesis.
- To identify the specific stage of translation affected by FV3 infection.
- To determine if FV3 alters the activity of key translation initiation factors.
Main Methods:
- Infection of fathead minnow and Friend erythroleukemia cells with FV3.
- Analysis of polysome dissociation and mRNA integrity.
- In vitro assays to assess protein synthesis capacity and pre-initiation complex formation.
- In vitro kinase assays to measure eIF-2 alpha subunit phosphorylation.
Main Results:
- FV3 infection rapidly inhibited cellular protein synthesis and caused polysome dissociation without mRNA degradation.
- Lysates from FV3-infected cells showed reduced protein synthesis and 43S pre-initiation complex formation in vitro.
- FV3 infection led to increased phosphorylation of the eIF-2 alpha subunit, mediated by an activated kinase.
Conclusions:
- FV3-induced translational shut-off occurs at the level of translation initiation, prior to ternary complex formation.
- Activation of a kinase that phosphorylates eIF-2 alpha is the likely mechanism for FV3-mediated translational inhibition.
- This mechanism highlights a strategy used by viruses to control host cell protein synthesis.