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J. Biol. Chem., Vol. 280, Issue 40, 33725-33734, October 7, 2005

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Launching of the Yeast 20 S RNA Narnavirus by Expressing the Genomic or Antigenomic Viral RNA in Vivo^*

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

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.

Received for publication, June 16, 2005 , and in revised form, July 25, 2005.

^* This work was supported in part by Grants BMC2001-1065 and BFU2004-00373 from the Spanish Ministry of Education and Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Recipient of a fellowship from the Autonomous Regional Government of Castilla and León (Spain) and the European Fund for Regional Development.

³ Recipient of a contract from the Spanish Research Program "Ramón y Cajal."

¹ To whom correspondence should be addressed: Inst. de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Avda. del Campo Charro s/n, Salamanca 37007, Spain. Tel.: 34-923-120-673; Fax: 34-923-224-876; E-mail: mrosa{at}gugu.usal.es.

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This article has been cited by other articles:

				R. Esteban, L. Vega, and T. Fujimura 20S RNA Narnavirus Defies the Antiviral Activity of SKI1/XRN1 in Saccharomyces cerevisiae J. Biol. Chem., September 19, 2008; 283(38): 25812 - 25820. [Abstract] [Full Text] [PDF]

				T. Fujimura and R. Esteban Interactions of the RNA Polymerase with the Viral Genome at the 5'- and 3'-Ends Contribute to 20S RNA Narnavirus Persistence in Yeast J. Biol. Chem., June 29, 2007; 282(26): 19011 - 19019. [Abstract] [Full Text] [PDF]