NDLI: Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Butcher, Rebecca E. Yap, Thai Leong Vasudevan, Subhash G. Lescar, Julien Lim, Siew Pheng Kroschewski, Helga Wright, Peter J. Davidson, Andrew D.
Description	Author Affiliation: Kroschewski H ( Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD United Kingdom.)
Abstract	The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities required for sequential methylation of the cap structure present at the 5' end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-l-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2'-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-l-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-l-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-l-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-l-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.
ISSN	00219258
e-ISSN	1083351X
Journal	Journal of Biological Chemistry
Issue Number	28
Volume Number	283
Language	English
Publisher	American Society for Biochemistry and Molecular Biology (United States)
Publisher Date	2008-07-11
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Dengue Virus Enzymology Genome, Viral Physiology Methyltransferases Metabolism Mutagenesis Viral Nonstructural Proteins Virus Replication Aedes Amino Acid Substitution Animals Binding Sites Genetics Catalysis Cercopithecus Aethiops Enzyme Stability Mutation, Missense Protein Structure, Tertiary RNA, Viral S-Adenosylmethionine Vero Cells Research Support, Non-U.S. Gov't Biochemistry Molecular Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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