Document Detail

West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5.
MedLine Citation:
PMID:  16912287     Owner:  NLM     Status:  MEDLINE    
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 (m(7)GpppAm). 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 m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) 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 K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) 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.
Debashish Ray; Aaloki Shah; Mark Tilgner; Yi Guo; Yiwei Zhao; Hongping Dong; Tia S Deas; Yangsheng Zhou; Hongmin Li; Pei-Yong Shi
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of virology     Volume:  80     ISSN:  0022-538X     ISO Abbreviation:  J. Virol.     Publication Date:  2006 Sep 
Date Detail:
Created Date:  2006-08-16     Completed Date:  2006-09-25     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  0113724     Medline TA:  J Virol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  8362-70     Citation Subset:  IM    
Wadsworth Center, New York State Department of Health, Albany, 12201, USA.
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MeSH Terms
5' Untranslated Regions / chemistry*,  metabolism
Cell Line
Genome, Viral
Methyltransferases / metabolism*
RNA Caps
Ribose / analogs & derivatives*,  metabolism*
Viral Nonstructural Proteins / metabolism*
Virus Replication
West Nile virus / chemistry,  genetics,  metabolism*,  physiology
Grant Support
Reg. No./Substance:
0/5' Untranslated Regions; 0/NS5 protein, flavivirus; 0/RNA Caps; 0/Viral Nonstructural Proteins; 50-69-1/Ribose; EC 2.1.1.-/Methyltransferases; EC (guanine(N7))-methyltransferase

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