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Journal of Virology, September 2006, p. 8362-8370, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00814-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

West Nile Virus 5'-Cap Structure Is Formed by Sequential Guanine N-7 and Ribose 2'-O Methylations by Nonstructural Protein 5

Debashish Ray,1 Aaloki Shah,1 Mark Tilgner,1 Yi Guo,1 Yiwei Zhao,1 Hongping Dong,1 Tia S. Deas,2 Yangsheng Zhou,2 Hongmin Li,1,2 and Pei-Yong Shi1,2*

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

Received 20 April 2006/ Accepted 20 June 2006

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m7GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m7GpppA-capped RNAs to m7GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m7GpppA- and m7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K61-D146-K182-E218 tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K61-D146-K182-E218 motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K182-E218 motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.

* Corresponding author. Mailing address: Wadsworth Center, New York State Department of Health, Albany, NY 12201. Phone: (518) 473-7487. Fax: (518) 473-1326. E-mail: ship{at}wadsworth.org.

Journal of Virology, September 2006, p. 8362-8370, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00814-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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