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Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus
Abstract:
Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.
Insights
Vesicular stomatitis virus infection inhibits protein synthesis by impairing translation initiation factors. Supplementing with eukaryotic initiation factor 2 (eIF-2) and other factors partially restored cellular mRNA translation and significantly boosted viral mRNA translation.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Vesicular stomatitis virus (VSV) infection of mouse L cells leads to a shutdown of host protein synthesis.
- This inhibition is linked to a defect in the translation of both cellular and viral messenger RNAs (mRNAs) in infected cell lysates.
Purpose of the Study:
- To investigate the role of specific eukaryotic initiation factors (eIFs) in overcoming the translation inhibition caused by VSV infection.
- To identify the specific defects in translation initiation machinery within VSV-infected L cells.
Main Methods:
- Preparation of cell lysates from VSV-infected and mock-infected mouse L cells.
- Assay of protein synthesis stimulation using rabbit reticulocyte initiation factors, including eukaryotic initiation factor 2 (eIF-2), guanine nucleotide exchange factor (GEF), and fractions containing eIF-4B, eIF-4F, and eIF-4A.
- Quantification of endogenous cellular and viral mRNA translation in response to factor addition.
Main Results:
- Eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF) strongly stimulated protein synthesis in infected cell lysates, with maximal effects when combined with eIF-4B and eIF-4F fractions.
- While cellular mRNA translation increased twofold, viral mRNA translation showed significantly higher stimulation (M protein 8-fold, NS 5-fold, N 2.5-fold, G 12-fold).
- eIF-4B, eIF-4F, and eIF-4A fractions also stimulated translation in the presence of eIF-2 in infected lysates, suggesting a deficiency in mRNA binding.
Conclusions:
- VSV-infected L cell lysates exhibit defects in the catalytic utilization of eIF-2.
- These lysates are also deficient in mRNA binding protein activity, contributing to the overall inhibition of protein synthesis.
- Specific initiation factors can partially restore cellular and enhance viral mRNA translation, highlighting key points of viral-mediated translational control.