Vaccinia virus morphogenesis is blocked by temperature-sensitive mutations in the F10 gene, which encodes protein kinase 2.
Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts28, ts54, ts61, and ts15) composing a single complementation group have been mapped by marker rescue to the F10 open reading frame located within the genomic HindIII F DNA fragment. Sequencing of the F10 gene from wild-type and mutant viruses revealed single-amino-acid substitutions in the F10 polypeptide responsible for thermolabile growth. Although the ts mutants displayed normal patterns of viral protein synthesis, electron microscopy revealed a profound morphogenetic defect at the nonpermissive temperature (40 degrees C). Virion assembly was arrested at an early stage, with scant formation of membrane crescents and no progression to normal spherical immature particles. The F10 gene encodes a 52-kDa serine/threonine protein kinase (S. Lin and S. S. Broyles, Proc. Natl. Acad. Sci. USA 91:7653-7657, 1994). We expressed a His-tagged version of the wild-type, ts54, and ts61 F10 polypeptides in bacteria and purified these proteins by sequential nickel affinity and phosphocellulose chromatography steps. The wild-type F10 protein kinase activity was characterized in detail by using casein as a phosphate acceptor. Whereas the wild-type and ts61 kinases displayed similar thermal inactivation profiles, the ts54 kinase was thermosensitive in vitro. These findings suggest that protein phosphorylation plays an essential role at an early stage of virion assembly.
Wang
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Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D015247:Deoxyribonuclease HindIII; D005814:Genes, Viral; D005816:Genetic Complementation Test; D006639:Histidine; D006358:Hot Temperature; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009024:Morphogenesis; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D013696:Temperature; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine; C095546:protein kinase 2, Vaccinia virus; D017346:Protein Serine-Threonine Kinases; D015247:Deoxyribonuclease HindIII