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De Novo Initiation of RNA Synthesis by the Arterivirus RNA-Dependent RNA Polymerase

Nancy Beerens,¹ Barbara Selisko,² Stefano Ricagno,^2, Isabelle Imbert,² Linda van der Zanden,¹ Eric J. Snijder,^1* and Bruno Canard^2*

Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands,¹ Centre National de la Recherche Scientifique and Universites d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolecules Biologiques, AFMB-CNRS-ESIL, 13288 Marseille Cedex 9, France²

Received 18 March 2007/ Accepted 19 May 2007

All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3' end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3'-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.

Published ahead of print on 30 May 2007.

Present address: Department of Biomolecular Sciences and Biotechnology and CNR-INFM, University of Milan, I-20131 Milan, Italy.

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• van Hemert, M. J., de Wilde, A. H., Gorbalenya, A. E., Snijder, E. J. (2008). The in Vitro RNA Synthesizing Activity of the Isolated Arterivirus Replication/Transcription Complex Is Dependent on a Host Factor. J. Biol. Chem. 283: 16525-16536 [Abstract] [Full Text]