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Originally published In Press as doi:10.1074/jbc.M002281200 on May 31, 2000

J. Biol. Chem., Vol. 275, Issue 34, 26428-26435, August 25, 2000
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Persistent Yeast Single-stranded RNA Viruses Exist in Vivo as Genomic RNA·RNA Polymerase Complexes in 1:1 Stoichiometry*

Alicia SolórzanoDagger , Nieves Rodríguez-Cousiño, Rosa Esteban, and Tsutomu Fujimura§

From the Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Salamanca 37007, Spain

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.

* This work was supported in part by Grant PB97-1121 from the Dirección General de Enseñanza Superior of the Spanish Ministry of Education.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a fellowship from the Spanish Ministry of Education.

§ To whom correspondence should be addressed: Inst. de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Avda. del Campo Charro s/n, Salamanca 37007, Spain. Tel.: 34-923-120673; Fax: 34-923-224876; E-mail: tfujimura@www-micro.usal.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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