2′-O methylation of internal adenosine by flavivirus NS5 methyltransferase

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 (m7gpppam) 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. © 2012 dong et al.