Crystal Structure of the RNA Polymerase Domain of the West Nile Virus Non-structural Protein 5*
- Hélène Malet‡1,
- Marie-Pierre Egloff‡1,
- Barbara Selisko‡,
- Rebecca E. Butcher§,
- Peter J. Wright§,
- Michael Roberts¶,
- Arnaud Gruez‡,
- Gerlind Sulzenbacher‡,
- Clemens Vonrhein¶,
- Gérard Bricogne¶,
- Jason M. Mackenzie∥,
- Alexander A. Khromykh∥,
- Andrew D. Davidson** and
- Bruno Canard‡2
- ‡Architecture et Fonction des Macromolécules Biologiques, CNRS, and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France, the §Department of Microbiology, Monash University, Clayton, Victoria 3168, Australia, ¶Global Phasing Ltd., Sheraton House, Cambridge CB3 0AX, United Kingdom, the ∥School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia, and the **Department of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, United Kingdom
- 2 To whom correspondence should be addressed. Tel.: 33-491-82-86-44; Fax: 33-491-82-86-46; E-mail: bruno.canard{at}afmb.univ-mrs.fr.
Abstract
Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-Å resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.
Footnotes
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↵3 The abbreviations used are: DENV, dengue virus; aa, amino acids; a/bNLS, nuclear localization sequence that recognize importin-α; bNLS, nuclear localization sequence that recognize importin-β; BVDV, bovine viral diarrhea virus; DENV-2, dengue virus type 2; ds, double-stranded; HCV, hepatitis C virus; Impα/β, karyopherin-α/β heterodimer, also known as importin-α/β; Impα, karyopherin-α, also known as importin-α; Impβ, karyopherin-β, also known as importin-β; MTase, methyltransferase; NLS, nuclear localization sequence; NS3, non-structural protein 3; NS5, nonstructural protein 5; POL, West Nile virus polymerase; POL1, construct 273-905 of West Nile virus polymerase; POL2, construct 317-905 of West Nile virus polymerase (in complex with calcium ion); POL3, construct 273-882 of West Nile virus polymerase; Phi6, Pseudomonas phage Phi-6; RdRp, RNAdependent RNA polymerase; r.m.s., root mean square; RT, reverse transcriptase; WNV, West Nile virus.
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↵4 B. Coutard, unpublished results.
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The atomic coordinates and structure factors (code 2HCN, 2HCS, and 2HFZ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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↵* This work was supported by SPINE project Grant QLRT-2001-00988, VIZIER integrated project Grant LSHG-CT-2004-511960 of the European Union 6th Framework Programme (FP6), as well as the Conseil Régional de la Région Provence-Alpes-Côte d'Azur. 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.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental material and Fig. 1S.
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↵1 Both authors contributed equally to this work.
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- Received August 1, 2006.
- Revision received January 29, 2007.
