title: Persistent yeast single-stranded RNA viruses exist in vivo as genomic RNA.RNA polymerase complexes in 1:1 stoichiometry.
abstract: Yeast narnavirus 20 S and 23 S RNAs encode RNA-dependent RNA polymerases p91 and p104, respectively, but do not encode coat proteins. Both RNAs form ribonucleoprotein complexes with their cognate polymerases. Here we show that these complexes are not localized in mitochondria, unlike the closely related mitoviruses, which reside in these organelles. Cytoplasmic localization of these polymerases was demonstrated by immunofluorescence and by fluorescence emitted from green fluorescent protein-fused polymerases. These fusion proteins were able to form ribonucleoprotein complexes as did the wild-type polymerases. Fluorescent observations and cell fractionation experiments suggested that the polymerases were stabilized by complex formation with their viral RNA genomes. Immunoprecipitation experiments with anti-green fluorescent protein antibodies demonstrated that a single polymerase molecule binds to a single viral RNA genome in the complex. Moreover, the majority (if not all) of 20 S and 23 S RNA molecules were found to form complexes with their cognate RNA polymerases. Since these viral RNAs were not encapsidated, ribonucleoprotein complex formation with their cognate RNA polymerases appears to be their strategy to survive in the host as persistent viruses.
authors.affiliation: Departamento de Microbiologia y Genética, Instituto de Microbiologia Bioquimica, Consejo Superior de Investigaciones Cientificas/Universidad de Salamanca, Salamanca 37007, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Solorzano
authors.forename: N
authors.initials: N
authors.lastname: Rodríguez-Cousiño
authors.forename: R
authors.initials: R
authors.lastname: Esteban
authors.forename: T
authors.initials: T
authors.lastname: Fujimura
mesh_terms: D015152:Blotting, Northern; D003593:Cytoplasm / Q000821:virology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008928:Mitochondria / Q000821:virology; D012328:RNA Viruses / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000821:virology*
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases