Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase.
Deletion of the vaccinia virus K3L gene, a homologue of the alpha subunit of protein synthesis initiation factor 2, has been reported to reduce the ability of the virus to grow in interferon-treated cells (Beattie, E., Tattaglia, J., and Paoletti, E. (1991) Virology 183, 419-422). Purified recombinant K3L gene product, pK3r, has potent effects on activation of double-stranded (ds) RNA-dependent, initiation factor-2 alpha (eIF-2 alpha)-specific protein kinase (PKR) in in vitro reactions. Recombinant pK3 prevents the inhibition of protein synthesis by dsRNA in a cell-free translation system from rabbit reticulocytes at levels equal to, or lower than, the level of endogenous eIF-2 alpha. In the cell-free translation system, pK3r exerts its effects at all dsRNA concentrations tested, by preventing phosphorylation of eIF-2 alpha. In addition, pK3r reduces the autophosphorylation of immunopurified PKR, as well as its ability to phosphorylate the alpha subunit of purified eIF-2. At 400 mM NaCl, in vitro translated [35S]methionine-radiolabeled pK3 can be co-immunoprecipitated with human PKR, using a monoclonal antibody to PKR. This tight binding is consistent with a role for pK3 as a pseudosubstrate for the kinase, and identifies the amino-terminal 30% of eIF-2 alpha as the domain recognized by the eIF-2 alpha-specific protein kinases. In addition, the tight binding opens up the possibility of using binding assays to identify functional domains within the kinase and pK3. Recombinant pK3 also prevents activation of the heme-sensitive eIF-2 alpha-specific protein kinase, eIF-2 alpha-PKh, in both cell-free translation systems as well as in partially purified preparations. This suggests some similarity between the eIF-2 alpha binding domains of the two eIF-2 alpha specific protein kinases.
Carroll
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Center of Marine Biotechnology, Baltimore, Maryland 21202.
Elroy-Stein
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Moss
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Jagus
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D017353:Gene Deletion; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D012156:Reticulocytes / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics* / Q000254:growth & development; D014764:Viral Proteins / Q000494:pharmacology*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; C081646:pK3 protein, Vaccinia virus; D011494:Protein Kinases; D019892:eIF-2 Kinase