title: Vaccinia virus mutants with alanine substitutions in the conserved G5R gene fail to initiate morphogenesis at the nonpermissive temperature.
abstract: The initial characterization of the product of the vaccinia virus G5R gene, which is conserved in all poxviruses sequenced to date, is described. The G5 protein was detected in the core fraction of purified virions, and transcription and translation of the G5R open reading frame occurred early in infection, independently of DNA replication. Attempts to delete the G5R gene and isolate a replication-competent virus were unsuccessful, suggesting that G5R encodes an essential function. We engineered vaccinia virus mutants with clusters of charged amino acids changed to alanines and determined that several were unable to replicate at 40 degrees C but grew well at 37 degrees C. At the nonpermissive temperature, viral gene expression and DNA replication and processing were unperturbed. However, tyrosine phosphorylation and proteolytic cleavage of the A17 membrane protein and proteolytic cleavage of core proteins were inhibited at 40 degrees C, suggesting an assembly defect. The cytoplasm of cells that had been infected at the nonpermissive temperature contained large granular areas devoid of cellular organelles or virus structures except for occasional short crescent-shaped membranes and electron-dense lacy structures. The temperature-sensitive phenotype of the G5R mutants closely resembled the phenotypes of vaccinia virus mutants carrying conditionally lethal F10R protein kinase and H5R mutations. F10, although required for phosphorylation of A17 and viral membrane formation, was synthesized by the G5R mutants under nonpermissive conditions. An intriguing possibility is that G5 participates in the formation of viral membranes, a poorly understood event in poxvirus assembly.
authors.affiliation: Laboratory of Viral Diseases, National Institutes of Health, 4 Center Dr., MSC 0445, Bethesda, MD 20892-0445, USA.
authors.forename: Flavio G
authors.initials: FG
authors.lastname: da Fonseca
authors.forename: Andrea S
authors.initials: AS
authors.lastname: Weisberg
authors.forename: Maria F
authors.initials: MF
authors.lastname: Caeiro
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D017124:Conserved Sequence; D003593:Cytoplasm / Q000648:ultrastructure; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D020043:Genes, Essential; D005814:Genes, Viral; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009024:Morphogenesis; D009154:Mutation; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D013696:Temperature; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000254:growth & development*; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000378:metabolism; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics; D019065:Virus Assembly / Q000235:genetics
chemical_list: C081257:A17L protein, Vaccinia virus; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C091945:H5R protein, Vaccinia virus; D008565:Membrane Proteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C095546:protein kinase 2, Vaccinia virus; D017346:Protein Serine-Threonine Kinases