Structure-based identification of functional residues in the nucleoside-2′-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, M. E. and Roy, P. (2015) Structure-based identification of functional residues in the nucleoside-2′-O-methylase domain of Bluetongue virus VP4 capping enzyme. FEBS Open Bio, 5(1), pp. 138-146. (doi: 10.1016/j.fob.2015.02.001) (PMID:25834778) (PMCID:PMC4359970)

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Abstract

Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X-ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside-2′-O-methyltransferase (2′-O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2′-O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series ofin vitro biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K-D-K-E were sufficient to abolish 2′-O MTase activityin vitro and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine-N7-methyltransferase activities. Mutations of the surrounding substrate-binding pocket (predicted to recruit cap0) had variable effects onin vitro VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2′-O MTase function for any member of theReoviridae and highlights the significance of K-D-K-E tetrad and surrounding residues for the efficiency of 2′-O MTase activity and in turn, for virus fitness.

Item Type:Articles
Additional Information:This research was funded by National Institutes of Health (NIH), under award number R01AI045000-11A1
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Stewart, Dr Meredith
Authors: Stewart, M. E., and Roy, P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:FEBS Open Bio
Publisher:Elsevier
ISSN:2211-5463
ISSN (Online):2211-5463
Published Online:24 February 2015
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in FEBS Open Bio 5(1):138-146
Publisher Policy:Reproduced under a creative commons license

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