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J. Biol. Chem., Vol. 283, Issue 28, 19410-19421, July 11, 2008

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Mutagenesis of the Dengue Virus Type 2 NS5 Methyltransferase Domain^*

Helga Kroschewski, Siew Pheng Lim, Rebecca E. Butcher^¶, Thai Leong Yap^||, Julien Lescar^||, Peter J. Wright^¶, Subhash G. Vasudevan^**, and Andrew D. Davidson¹

From the Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD United Kingdom, Novartis Institute for Tropical Diseases, 05-01 Chromos, 10 Biopolis Rd., Singapore, 138670, Singapore, ^¶Department of Microbiology, Monash University, Clayton 3168, Australia, ^||School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore, and ^**Duke-NUS Graduate Medical School, 2 Jalan Bukit Merah, Singapore

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.

Received for publication, January 24, 2008 , and in revised form, May 8, 2008.

^* This work was supported in part by a Global Partnering grant from the Novartis Institute for Tropical Diseases, Singapore and grants from the National Health and Medical Research Council of Australia. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed. E-mail: andrew.davidson{at}bristol.ac.uk.

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