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J. Biol. Chem., Vol. 279, Issue 17, 17013-17018, April 23, 2004

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Synthesis in Vitro of Rabbit Hemorrhagic Disease Virus Subgenomic RNA by Internal Initiation on (–)Sense Genomic RNA

MAPPING OF A SUBGENOMIC PROMOTER^*

Mónica Morales, Juan Bárcena, Miguel A. Ramírez, José A. Boga, Francisco Parra, and Juan M. Torres¶

From the Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain and Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33006 Oviedo, Spain

Rabbit hemorrhagic disease virus (RHDV), a positive-strand RNA virus, is the type species of the Lagovirus within the Caliciviridae. In addition to the genomic RNA of 7.4 kb, a subgenomic mRNA (sgRNA) of 2.2 kb, which is identical in sequence to the 3' one-third of the genomic RNA, is also synthesized in RHDV-infected cells. Numerous RNA viruses make sgRNA for expression of their 3'-proximal genes. A relevant mechanism for viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. In this study, we have investigated in vitro the sgRNA synthesis mechanism using recombinant RHDV RNA-dependent RNA polymerase produced in baculovirus-infected insect cells and synthetic RHDV (–)RNAs of different lengths containing regions located upstream of the subgenomic start site. We report evidences supporting that the sgRNA of RHDV is synthesized in vitro by internal initiation (subgenomic promoter) on (–)RNA templates of genomic length. The deletion mapping of the subgenomic promoter starting from minus-strand genomic length RNA showed that a sequence of 50 nucleotides upstream of the sgRNA start site (+1) is sufficient for full subgenomic promoter activity in an in vitro assay using recombinant RHDV RNA-dependent RNA polymerase. This study reports the first description of a subgenomic promoter in a member of the Caliciviridae.

Received for publication, December 15, 2003

^* This work was supported by Grant BIO2000-0578-C02. 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.

^¶ To whom correspondence should be addressed. Tel.: 34-91-620-23-00; Fax: 34-91-620-22-47; E-mail: jmtorres{at}inia.es.

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