title: 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.
abstract: RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7)GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore.
authors.forename: Hongping
authors.initials: H
authors.lastname: Dong
authors.forename: David C
authors.initials: DC
authors.lastname: Chang
authors.forename: Maggie Ho Chia
authors.initials: MH
authors.lastname: Hua
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.forename: Yok Hian
authors.initials: YH
authors.lastname: Chionh
authors.forename: Fabian
authors.initials: F
authors.lastname: Hia
authors.forename: Yie Hou
authors.initials: YH
authors.lastname: Lee
authors.forename: Petra
authors.initials: P
authors.lastname: Kukkaro
authors.forename: Shee-Mei
authors.initials: SM
authors.lastname: Lok
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
mesh_terms: D000241:Adenosine / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007313:Insecta; D008745:Methylation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000201:enzymology / Q000235:genetics; D014902:West Nile virus / Q000201:enzymology* / Q000235:genetics; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012359:tRNA Methyltransferases; D000241:Adenosine