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J. Biol. Chem., Vol. 279, Issue 13, 13215-13223, March 26, 2004

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Bipartite 3'-Cis-acting Signal for Replication in Yeast 23 S RNA Virus and Its Repair^*

Tsutomu Fujimura and Rosa Esteban

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

23 S RNA narnavirus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome is small (2.9 kb) and only encodes its RNA-dependent RNA polymerase. Recently, we have succeeded in generating 23 S RNA virus from an expression vector containing the entire viral cDNA sequence. Using this in vivo launching system, we analyzed the 3'-cis-acting signals for replication. The 3'-non-coding region of 23 S RNA contains two cis-elements. One is a stretch of 4 Cs at the 3' end, and the other is a mismatched pair in a stem-loop structure that partially overlaps the terminal 4 Cs. In the latter element, the loop or stem sequence is not important but the stem structure with the mismatch pair is essential. The mismatched bases should be purines. Any combination of purines at the mismatch pair bestowed capability of replication on the RNA, whereas converting it to a single bulge at either side of the stem abolished the activity. The terminal and penultimate Cs at the 3' end could be eliminated or modified to other nucleotides in the launching plasmid without affecting virus generation. However, the viruses generated regained or restored these Cs at the 3' terminus. Considering the importance of the viral 3' ends in RNA replication, these results suggest that this 3' end repair may contribute to the persistence of 23 S RNA virus in yeast by maintaining the genomic RNA termini intact. We discuss possible mechanisms for this 3' end repair in vivo.

Received for publication, December 17, 2003 , and in revised form, January 5, 2004.

^* This work was supported by Grant BMC2001-1065 from The Spanish Ministry of Science and Technology. 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.

Recipient of a contract from the Spanish Research Program "Ramón y Cajal." To whom correspondence should be addressed: Instituto de Microbiología Bioquiímica CSIC, Universidad de Salamanca, Avda. del Campo Charro s/n Salamanca 37007, Spain. Tel.: 34-923-120673; Fax: 34-923-224876; E-mail address: tfujimura{at}usal.es.

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