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Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells
Abstract:
In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.
Insights
Poliovirus infection inactivates a crucial cap-binding protein complex, halting cellular mRNA translation. This viral mechanism targets the 220-kilodalton polypeptide, preventing cap-specific mRNA interactions.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Poliovirus infection inhibits cellular mRNA translation while viral RNA translation continues.
- This translational control is linked to the inactivation of a cap-binding protein complex.
- The cap-binding protein complex includes 24, 50, and 220-kilodalton polypeptides.
Purpose of the Study:
- To identify the specific component of the cap-binding complex responsible for restoring mRNA translation.
- To elucidate the mechanism by which poliovirus inhibits cellular mRNA translation.
- To determine if the cap-binding protein complex is the sole factor inactivated by poliovirus.
Main Methods:
- Restoration of capped mRNA translation and cap-specific mRNA-protein interaction assays.
- Copurification of activities that restore translation and cap-specific interactions.
- Purification and analysis of cap-binding proteins from infected and uninfected HeLa cells.
- In vivo labeling and m7GDP affinity chromatography.
Main Results:
- The cap-binding protein complex, not just the 24-kilodalton subunit, restores cap-specific mRNA-protein interactions.
- The activity restoring translation and cap-specific interactions copurify, indicating a single mechanism.
- The 220-kilodalton polypeptide of the cap-binding complex is proteolytically cleaved during infection.
- A modified cap-binding complex with cleaved 220-kilodalton fragments is isolated from infected cells.
Conclusions:
- Poliovirus infection specifically targets and inactivates the cap-binding protein complex via cleavage of its 220-kilodalton subunit.
- This cleavage disrupts cap-specific mRNA-protein interactions, leading to the inhibition of cellular translation.
- The cap-binding protein complex is identified as the sole factor inactivated by poliovirus, explaining the translational shutoff.