Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.
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.
Lee
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Edery
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Sonenberg
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D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000494:pharmacology; D010455:Peptides / Q000032:analysis; D017955:Poliovirus / Q000235:genetics*; D014176:Protein Biosynthesis; D039381:RNA Cap-Binding Proteins
D002352:Carrier Proteins; D010448:Peptide Initiation Factors; D010455:Peptides; D039381:RNA Cap-Binding Proteins