Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2.
A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.
O'Neill
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Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Racaniello
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D015075:2-Aminopurine / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002472:Cell Transformation, Viral; D003609:Dactinomycin / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017955:Poliovirus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis / Q000187:drug effects; D014764:Viral Proteins / Q000096:biosynthesis; D014779:Virus Replication
D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D012367:RNA, Viral; D014764:Viral Proteins; D003609:Dactinomycin; D015075:2-Aminopurine