title: Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.
abstract: COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854-5638.
authors.forename: R M
authors.initials: RM
authors.lastname: Lloyd
authors.forename: A J
authors.initials: AJ
authors.lastname: Shatkin
mesh_terms: D000818:Animals; D036022:Capsid Proteins; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D015870:Gene Expression; D007739:L Cells; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D010957:Plasmids; D014176:Protein Biosynthesis; D012330:RNA, Double-Stranded / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D016601:RNA-Binding Proteins; D012087:Reoviridae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; C053179:sigma protein 3, Reovirus; D008715:Methionine; D013762:Tetrahydrofolate Dehydrogenase