2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

Hongping Dong, David C. Chang, Maggie Ho Chia Hua, Siew Pheng Lim, Yok Hian Chionh, Fabian Hia, Yie Hou Lee, Petra Kukkaro, Shee Mei Lok, Peter C. Dedon, Pei-Yong Shi

Research output: Contribution to journalArticle

72 Citations (Scopus)

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 N 6-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.

Original languageEnglish (US)
JournalPLoS Pathogens
Volume8
Issue number4
DOIs
StatePublished - 2012
Externally publishedYes

Fingerprint

Flavivirus
Methyltransferases
Adenosine
Methylation
RNA
Viral RNA
Ribosomal RNA
Viral Genome
Host-Pathogen Interactions
S-Adenosylmethionine
West Nile virus
Replicon
Dengue Virus
Guanosine Triphosphate
Recombinant Proteins
Mutagenesis
Virion
Catalytic Domain
Binding Sites
Genome

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology

Cite this

2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase. / Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee Mei; Dedon, Peter C.; Shi, Pei-Yong.

In: PLoS Pathogens, Vol. 8, No. 4, 2012.

Research output: Contribution to journalArticle

Dong, H, Chang, DC, Hua, MHC, Lim, SP, Chionh, YH, Hia, F, Lee, YH, Kukkaro, P, Lok, SM, Dedon, PC & Shi, P-Y 2012, '2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.', PLoS Pathogens, vol. 8, no. 4. https://doi.org/10.1371/journal.ppat.1002642
Dong, Hongping ; Chang, David C. ; Hua, Maggie Ho Chia ; Lim, Siew Pheng ; Chionh, Yok Hian ; Hia, Fabian ; Lee, Yie Hou ; Kukkaro, Petra ; Lok, Shee Mei ; Dedon, Peter C. ; Shi, Pei-Yong. / 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase. In: PLoS Pathogens. 2012 ; Vol. 8, No. 4.
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