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. 2012;8(4):e1002642.
doi: 10.1371/journal.ppat.1002642. Epub 2012 Apr 5.

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

Hongping Dong  1 , 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
Affiliations

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

Hongping Dong et al. PLoS Pathog. 2012.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Internal methylation of RNA by flavivirus NS5 and MTase domain.
(A) The principle of scintillation proximity assay (SPA). CMP-biotinylated RNA was methylated by enzyme using [3H-methyl]-SAM. The biotinylated RNA containing 3H-methyl is captured by streptavidin-coated SPA scintillation beads, leading to a signal that can be measured using a MicroBeta counter. (B) SPA analysis of internal methylation of flaviviral RNAs. Uncapped pppA-RNAs, representing the 5′-terminal 190 nt of WNV genome or the 5′-terminal 211 nt of DENV genome, were methylated by indicated recombinant proteins. The combination of protein and pppA-RNA for each reaction is depicted. (C) SPA analysis of RNA cap methylations. GpppA-RNA or m7GpppA-RNA, representing the first 190 nt of WNV genome or the first 211 nt of DENV genome, was methylated using the indicated MTases. Average results and standard deviations from three independent experiments are shown.
Figure 2
Figure 2. Optimal conditions for internal methylation for DENV-4 NS5 MTase.
SPA-based methylation assays were performed using uncapped pppA-RNA substrate (representing the first 211 nt of DENV genome). The reaction mixtures were incubated for 1 h at room temperature. Optimal pH, temperature, NaCl concentration, MgCl2 concentration, and MnCl2 concentration were obtained by titrating individual parameter while keeping other parameters at the optimal levels. Average results and standard deviations were obtained from three independent experiments.
Figure 3
Figure 3. 2′-O methylation of internal adenosine.
(A) Incorporation of 3H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [3H-methyl]-SAM. After the methylation reaction, the un-incorporated [3H-methyl]-SAM was removed by RNeasy kit. The amount of 3H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A)12, (Am)12, and (m6,m6A)12. All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m6,m6A indicates that the amino N6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′-O-methyladenosine triphosphate (AmTP), or N6 methyl adenosine triphosphate (m6ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m6A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.
Figure 4
Figure 4. Mass spectrometric analysis of Am in MTase-treated polyA and in DENV genomic RNA.
(A) Extracted ion chromatogram of ions with m/z 282.1187 from the LC-QTOF scan of putative Am in hydrolysates of DENV-4 MTase-treated polyA and of standard Am. (B,C) CID spectra of the parent ion m/z 282.1187 representing standard Am (B) and putative Am in hydrolysates of DENV-4 MTase treated polyA species (C). The inset shows the assignment of structures for the CID spectra. (D,E) LC-MS/MS quantification of Am in WT DENV-1 genomic RNA (D) and MTase E217A mutant DENV-1 genomic RNA (E); the solid and dashed lines represent technical replicates. The different retention time for Am in panel A (∼24.5 min) compared to panels D and E (∼4.4 min) is the result of different HPLC flow rates used for the two studies.
Figure 5
Figure 5. Comparison of internal methylation efficiencies between DENV RNAs and host ribosomal RNAs.
(A) Full-length (FL) and 3′ truncated RNAs of DENV-1. pppAG-RNAs, representing the FL and a set of 3′ terminally truncated DENV-1 RNAs, were in vitro synthesized. Numbers indicate nucleoside positions of DENV-1 genome (GenBank accession number U88535). (B) Internal methylation analysis. An equal mass (0.5 µg) of FL and truncated DENV-1 RNAs, and human ribosomal 18 S and 28 S RNAs was treated with DENV MTase in the presence of [3H-methyl]-SAM. The reactions were purified through an RNeasy column to remove unincorporated [3H-methyl]-SAM. The purified RNAs were quantified for internal methylation by a MicroBeta counter. Average results from three experiments are shown; error bars represent standard deviations.
Figure 6
Figure 6. Mutagenesis analysis of DENV-4 MTase.
(A) Co-crystal structure of DENV MTase showing SAH (yellow stick) and GMP (pink stick). (B) Surface presentation of DENV MTase depicting mutated amino acids. Mutated residues in the K-D-K-E motif, SAM-binding pocket, RNA-binding site, and GMP-binding pocket are shown in yellow, blue, red, and green, respectively. The images were produced using DENV-2 MTase structure (PDB code: 1L9K) and PyMOL. (C) Effects of mutations of DENV-4 MTase on internal methylation. Biotinylated pppA-RNA (representing the first 211 nt of DENV genomic RNA) was incubated with WT or various mutant MTases in the presence of [3H-methyl]-SAM. The reactions were quantified for [3H-methyl]-incorporation using SPA analysis. The methylation efficiencies of mutant MTases were compared with that of the WT MTase (set at 100%). Averages of three independent experiments are shown. Error bars indicate standard deviations.
Figure 7
Figure 7. Effects of internal methylation on flavivirus RNA translation and replication.
(A) Replicon analysis. Top panel depicts the procedures to prepare replicon RNAs with and without internal adenosine methylations. Bottom panel shows the effects of internal Am modification on viral RNA translation and synthesis. Both DENV-1 and WNV luciferase replicons were used in the analysis. Specifically, equal amounts (2 µg) of replicon RNAs with and without internal Am modifications were electroporated into BHK-21 cells. The transfected cells were assayed for luciferase activities at indicated time points. For each time point, relative luciferase activities were compared between the replicons with internal Am and the replicon without internal Am (set at 100%). Average results and standard deviations from three experiments are presented. (B) RT-PCR analysis. The transfected cells described in (A) were extracted for total cellular RNA at indicated time points. Equal amounts of total cellular RNA (3 µg) were subjected to RT-PCR quantification using primers targeting viral NS5 gene. actin, a host housekeeping gene, was included as a control. The RT-PCR products were analyzed on a 1% agarose gel. One of the three representative experimental results is presented. (C) Effects of internal Am modification on the replication of genome-length RNA. DENV-1 genome-length RNAs with or without internal Am modifications were prepared as depicted in (A). Equal amounts of RNAs with or without internal Am modifications were transfected into BHK-21 cells, and compared for their specific infectivities and virus yields at indicated time points post transfection. (D) Effect of 2′-O-methylation on viral polymerase activity. An RNA elongation assay was used to compare the RdRp activities between RNA templates with and without 2′-O-methyladenosine. RNA sequences of primer/template are shown (left panel). Incorporation of 3H-UTP in to the biotinylated RNA primer in the presence of DENV-4 NS5 was measured (right panel). Average results and standard deviations from three independent experiments are shown.
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