Please use this identifier to cite or link to this item: https://doi.org/10.1074/jbc.M800613200
Title: Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain
Authors: Kroschewski, H.
Siew, P.L.
Butcher, R.E.
Thai, L.Y.
Lescar, J.
Wright, P.J.
Vasudevan, S.G. 
Davidson, A.D.
Issue Date: 11-Jul-2008
Citation: Kroschewski, H., Siew, P.L., Butcher, R.E., Thai, L.Y., Lescar, J., Wright, P.J., Vasudevan, S.G., Davidson, A.D. (2008-07-11). Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain. Journal of Biological Chemistry 283 (28) : 19410-19421. ScholarBank@NUS Repository. https://doi.org/10.1074/jbc.M800613200
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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
Source Title: Journal of Biological Chemistry
URI: http://scholarbank.nus.edu.sg/handle/10635/110184
ISSN: 00219258
DOI: 10.1074/jbc.M800613200
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
There are no files associated with this item.

SCOPUSTM   
Citations

56
checked on Feb 19, 2020

WEB OF SCIENCETM
Citations

54
checked on Feb 19, 2020

Page view(s)

57
checked on Feb 22, 2020

Google ScholarTM

Check

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.