title: West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5.
abstract: 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.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, 12201, USA.
authors.forename: Debashish
authors.initials: D
authors.lastname: Ray
authors.forename: Aaloki
authors.initials: A
authors.lastname: Shah
authors.forename: Mark
authors.initials: M
authors.lastname: Tilgner
authors.forename: Yi
authors.initials: Y
authors.lastname: Guo
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Zhao
authors.forename: Hongping
authors.initials: H
authors.lastname: Dong
authors.forename: Tia S
authors.initials: TS
authors.lastname: Deas
authors.forename: Yangsheng
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hongmin
authors.initials: H
authors.lastname: Li
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D016679:Genome, Viral; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D012315:RNA Caps; D012266:Ribose / Q000031:analogs & derivatives* / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D014902:West Nile virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D020121:5' Untranslated Regions; C096041:NS5 protein, flavivirus; D012315:RNA Caps; D017361:Viral Nonstructural Proteins; D012266:Ribose; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase