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Journal of Virology, April 2007, p. 3891-3903, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02704-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Structure and Function of Flavivirus NS5 Methyltransferase

Yangsheng Zhou,^1,2, Debashish Ray,^1, Yiwei Zhao,¹ Hongping Dong,¹ Suping Ren,^1,2 Zhong Li,¹ Yi Guo,¹ Kristen A. Bernard,^1,2 Pei-Yong Shi,^1,2* and Hongmin Li^1,2*

Wadsworth Center, New York State Department of Health,¹ Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201²

Received 7 December 2006/ Accepted 22 January 2007

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m⁷GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA m⁷GpppA m⁷GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m⁷GpppA-RNA can be readily methylated to m⁷GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K₆₁-D₁₄₆-K₁₈₂-E₂₁₈ motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

Published ahead of print on 31 January 2007.

These authors made equal contributions.

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