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Journal of Virology, June 2009, p. 5671-5682, Vol. 83, No. 11
0022-538X/09/$08.00+0     doi:10.1128/JVI.00261-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of Arterivirus Nonstructural Protein 11 Reveals the Nidovirus-Wide Conservation of a Replicative Endoribonuclease ^,

Danny D. Nedialkova,¹ Rachel Ulferts,² Erwin van den Born,^1, Chris Lauber,¹ Alexander E. Gorbalenya,^1, John Ziebuhr,^2, and Eric J. Snijder^1,*

Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands,¹ Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, The Queen's University of Belfast, Belfast, United Kingdom²

Received 6 February 2009/ Accepted 5 March 2009

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

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* Corresponding author. Mailing address: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, LUMC E4-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31 71 5261657. Fax: 31 71 5266761. E-mail: e.j.snijder{at}lumc.nl

Published ahead of print on 18 March 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

Present address: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.

A.E.G., J. Z., and E.J.S. contributed equally to the study.

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Journal of Virology, June 2009, p. 5671-5682, Vol. 83, No. 11
0022-538X/09/$08.00+0     doi:10.1128/JVI.00261-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.