Article3 June 2002free access An RNA cap (nucleoside-2′-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization Marie-Pierre Egloff Marie-Pierre Egloff Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Delphine Benarroch Delphine Benarroch Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Barbara Selisko Barbara Selisko Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Jean-Louis Romette Jean-Louis Romette Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Bruno Canard Corresponding Author Bruno Canard Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Marie-Pierre Egloff Marie-Pierre Egloff Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Delphine Benarroch Delphine Benarroch Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Barbara Selisko Barbara Selisko Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Jean-Louis Romette Jean-Louis Romette Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Bruno Canard Corresponding Author Bruno Canard Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France Search for more papers by this author Author Information Marie-Pierre Egloff1, Delphine Benarroch1, Barbara Selisko1, Jean-Louis Romette1 and Bruno Canard 1 1Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Université Aix-Marseille I et II, ESIL, Campus de Luminy, F-13288 Marseille, Cedex 09, France ‡M.-P.Egloff and D.Benarroch contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2757-2768https://doi.org/10.1093/emboj/21.11.2757 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 Å resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2′-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses. Introduction The cap is a unique structure found at the 5′-end of viral and cellular eukaryotic mRNA (Bisaillon and Lemay, 1997; Furuichi and Shatkin, 2000). It is critical for both mRNA stability and binding to the ribosome during translation. mRNA capping is a co-transcriptional modification resulting from a series of three chemical reactions (Shuman, 2001). The 5′-triphosphate of the mRNA is first converted to a diphosphate by an RNA triphosphatase. The second reaction is a transfer of a guanosine monophosphate (GMP) moiety from GTP to the 5′-diphosphate RNA by a guanylyltransferase to yield G5′-ppp5′-N. In a third reaction utilizing S-adenosyl-L-methionine (AdoMet) as the methyl donor, the transferred guanosine moiety is methylated by a (guanine-N7)-methyltransferase (N7MTase) to yield 7MeG5′-ppp5′-N (cap 0 structure). A second methylation reaction catalysed by a (nucleoside-2′-O-)-methyltransferase (2′OMTase) occurs on the first nucleotide 3′ to the triphosphate bridge to yield 7MeG5′-ppp5′-NMe (cap 1 structure). Adjacent nucleotides 3′ to the first one can also be 2′-O-methylated to various extents. The order of the methyltransfer reactions is variable, and in some viruses GTP is methylated at its N7 position before being transferred to the RNA 5′-end (Ahola and Kaariainen, 1995; Furuichi and Shatkin, 2000). Few enzymes involved in the RNA capping pathway have been structurally characterized. The crystal structures of two cellular RNA triphosphatases from yeast and mouse have been determined at 2.0 and 1.6 Å resolution, respectively. These structures provide a mechanistic insight into the first reaction in the RNA capping pathway catalysed by prototypic enzymes of the metal-dependent and -independent RNA triphosphatase family, respectively (Lima et al., 1999; Changela et al., 2001). Structural insights into the DNA virus PBCV-1 (Chlorella virus) guanylyltransferase in complex with GTP have revealed mechanistic determinants of the second reaction of the RNA capping pathway (Hakansson et al., 1997). The crystal structure of the double-stranded RNA Reovirus core at 3.6 Å resolution comprises a subunit of the λ2 protein as the sole example of a structurally defined RNA virus guanylyltransferase (Reinisch et al., 2000). It revealed that the guanylyltransferase domain adopts a different fold from that of the Chlorella virus enzyme, but provided no structural information about GTP binding. The crystal structure of three RNA cap MTases involved in the third reaction of the RNA capping pathway has been determined. They are the 2′OMTase VP39 of the double-stranded DNA vaccinia virus (Hodel et al., 1996), and the N7MTase and 2′OMTase domains of core protein λ2 of Reovirus, a double-stranded RNA virus (Reinisch et al., 2000). RNA cap MTases share a common fold with a vast family of AdoMet-dependent MTases comprising small-molecule MTases, protein MTases, DNA (adenine-N6-), (cytosine-5-) and (cytosine-N4-) MTases, rRNA (adenine-N6-) MTases and rRNA 2′OMTases (Fauman et al., 1999; Bugl et al., 2000; Wang et al., 2000). Although they methylate different substrates, they all share a common catalytic core domain consisting of a seven-stranded β-sheet surrounded by six α-helices. Depending on the size of the substrate, target recognition domains might be appended to the catalytic core (Fauman et al., 1999). Alternatively, the MTase core domain can be integrated in a multidomain protein as is the case in protein λ2 of Reovirus (Reinisch et al., 2000), or in a protein complex. The only common feature of AdoMet-dependent MTases at the sequence level is a short motif involved in AdoMet binding (Ingrosso et al., 1989). Vaccinia virus VP39 represents the sole structurally characterized viral MTase for which 2′-O-methyltransfer specificity has been ascertained (Barbosa and Moss, 1978). The crystal structure of the Reovirus λ2 protein showed the physical organization of two structurally related AdoMet-dependent mRNA MTase domains (Reinisch et al., 2000). Based on the spatial arrangement of the subdomains and the known methylation pathway, Reinisch et al. (2000) proposed an assignment of the N7MTase and 2′OMTase activities to respective domains of λ2; however, a sequence- and structure-based analysis of MTase substrate specificities suggested that this assignment should be swapped around (Bujnicki and Rychlewski, 2001). This proposition cannot be tested due to the difficulty of isolating enzymatically active λ2 protein. Many viruses replicate in the cytoplasm of their eukaryotic host. Since cellular RNA capping is localized in the nucleus, these viruses often encode their own capping enzymes while relying on the host translation machinery for gene expression. Although RNA cap structures originating from viral or cellular enzymes are often identical, the physical organization of genes, subunit composition, structure, and catalytic mechanisms of enzymes from the capping apparatus differ significantly in fungi, metazoans, protozoa and viruses (Shuman, 2001). This diversity makes RNA capping an attractive target for drug design. The group Flaviviridae comprises important human pathogens such as West Nile, Dengue and Yellow Fever viruses. Their single-stranded RNA genome is of positive polarity, and is capped with a cap 1 structure (Chambers et al., 1990). Little is known about the components of the flavivirus RNA capping machinery. RNA triphosphatase activity has been described for West Nile virus only, and has been mapped to the C-terminus of non-structural protein 3 (NS3) (Wengler, 1993). The guanylyltransferase has not yet been identified, and sequence analysis has revealed the presence of the characteristic motif of AdoMet-dependent MTases within the N-terminal domain of NS5, the viral RNA-dependent RNA polymerase (Koonin, 1993). Flaviviruses are emerging mosquito-born agents that are expanding their distribution across the world. Dengue virus, an agent responsible for haemorrhagic fever, infects more than 50 million people every year, with an increasing incidence in the world's tropical regions (Rigau-Perez et al., 1998; Isturiz et al., 2000). Likewise, the recent introduction of West Nile virus in North America may be an important milestone in the evolving history of this virus, as exemplified by outbreaks in the New York area (Anderson et al., 1999) and the subsequent geographic extension of its endemic zone (Enserink, 2001). Several regions of Europe re-witnessed West Nile virus infection in the late 1990s (Hubalek and Halouzka, 1999; Lvov et al., 2000). As inhibitors of viral replicases currently meet with clinical success, the capping and polymerization apparatus of flaviviruses remains a poorly defined but attractive therapeutical target. In this report we present the crystal structure at 2.4 Å resolution of the N-terminal MTase domain of Dengue virus NS5 in complex with the product of the methylation reaction, S-adenosyl-L-homocysteine (AdoHcy). Enzymatic analysis and the structural conservation of characteristic amino acid residues in the active site demonstrate that this domain functions as a 2′OMTase. In addition, we found a GTP-binding site located on an N-terminal appendage of the MTase domain. This site demonstrates novel folding and achieves specific binding of guanine in an original manner. Results Structure determination and model quality Based on sequence alignments, hydrophobicity profiles and secondary structure predictions, we delineated a putative globular domain at the N-terminus of NS5. This domain comprised the MTase signature within a consensus MTase domain (Fauman et al., 1999) and preceeded the known nuclear localization sequence (NLS; Forwood et al., 1999) (Figure 1A). Cloning of the corresponding DNA fragment of Dengue virus type 2 (New Guinea strain) in several expression vectors was performed, and tested for protein expression in Escherichia coli. The isolated domain was overexpressed as a soluble protein (His6-tagged) and subsequently purified (see Materials and methods). The 33-kDa recombinant protein comprised residues 1–296 and was named NS5MTaseDV (protein NS5, MTase domain, Dengue virus). It crystallized with lithium sulfate as precipitant (see Materials and methods) in space group P3121, with cell parameters a = b = 111.6 Å, c = 56.2 Å. The crystal structure of NS5MTaseDV was determined using multi-wavelength anomalous dispersion (MAD). A bound Hg2+ ion was the anomalous scatterer. Like most structurally characterized MTases (Fauman et al., 1999), NS5MTaseDV is monomeric both in solution and in the crystal. An atomic model consisting of residues 7–267 was built in the 2.8 Å experimental electron density map and refined to a resolution of 2.4 Å. The remaining amino acids (268–296) were not observed in the electron density map, although examination of the crystal packing showed that there would be enough space to accommodate these missing residues. Mass spectrometry analysis of dissolved NS5MTaseDV crystals showed that the C-terminus missing in the structure is present in the crystallized protein. Thus, the C-terminal part appears to be flexible, probably due to the absence of the polymerase domain of NS5. The crystallographic R-factor is 23.6% for the data between 30 and 2.4 Å. Most of the residues (88%) lie in the most favoured region of the Ramachandran plot. Data collection and refinement statistics are reported in Table I. Figure 1.General features of NS5MTaseDV. (A) Predicted domain structure of Dengue protein NS5. The putative N-terminal methyltransferase domain is shown in grey. The position of the AdoMet-binding motif I (residues 77–86) described by Koonin (1993) is highlighted in black. The region of the C-terminal polymerase domain containing motifs I to VIII of positive-strand RNA virus RNA polymerases (Koonin, 1991) is marked in grey. Motifs A to D, shared by RNA-dependent polymerases (Poch et al., 1989), are shown in black. AdoMet, S-adenosyl-L-methionine; NLS, nuclear localization sequence; Pol, polymerase. (B) Crystal structure of NS5MTaseDV in complex with AdoHcy. A ball-and-stick representation is used for AdoHcy, whereas NS5MTaseDV is drawn as a ribbon. The N-terminal subdomain of NS5MTaseDV (residues 7–54) is coloured red. The core subdomain (residues 55–222) has a typical AdoMet-dependent MTase topology and is coloured yellow. The C-terminal part of NS5MTaseDV (residues 223–267) is coloured cyan. The figure was generated using MOLSCRIPT (Kraulis, 1991) and rendered using RASTER3D (Merrit and Murphy, 1994). (C) Sequence alignment of flavivirus NS5MTases coloured according to the ribbon representation of NS5MTaseDV in (B). NS5MTase domains from Dengue virus type 2 New Guinea isolate (D2V), West Nile virus New York isolate (WNV) and Yellow Fever 17D (YFV) were aligned using Clustal_W (Thompson et al., 1994) and rendered using ESPript (Gouet et al., 1999). Secondary structures (α-helices and β-strands) of subdomains 1, 2 and 3 are indicated above the alignment and coloured in red, yellow and cyan, respectively. Helices and strands are named using Greek letters inside the core domain (subdomain 2), and roman letters outside (subdomains 1 and 3). Amino acids involved in GTP binding (see text) are indicated by a grey star below aligned sequences, and amino acids interacting with AdoHcy are indicated by a pink sphere. Download figure Download PowerPoint Table 1. Crystallization, data collection, structure solution and refinement statistics Data set Hg(CN)2 remote Hg(CN)2 peak Hg(CN)2 inflection Native GDPMP soak Data collection Resolution range (Å)a 30–2.8 (2.95–2.8) 30–2.8 (2.95–2.8) 30–2.8 (2.95–2.8) 30–2.4 (2.53–2.4) 30–2.9 (3.06–2.9) Wavelength (Å) 0.83211 1.00474 1.00850 0.933 0.933 Cell parameters (Å) a = b = 111.6, c = 56.2 a = b = 112.2, c = 56.5 Unique reflectionsb 10 159 10 220 10 198 16 248 9310 I/(σ(I)) 14.7 15.7 15.6 17.4 16.5 Rsym (%)a,c, a,c 6.1 (30.4) 5.5 (24.5) 8.1 (30.5) 5.1 (35.8) 4.1 (27.7) Completeness (%)a,b, a,b 99.7 (99.7) 99.9 (99.9) 99.8 (99.8) 99.6 (99.6) 99.9 (99.9) Multiplicitya,b, a,b 4.9 (3.9) 7.0 (6.2) 7.0 (6.1) 5.5 (5.2) 4.1(4.1) MAD analysis No. of sites 1 Phasing power (acentrics/centrics) 0.91/0.77 0.61/0.47 Rcullis (acentrics/centrics) 0.87/0.86 0.96/0.94 Rcullis anomalous 0.89 0.95 FOMmlphare (30–2.8 Å) 0.36 FOMdm (30–2.4 Å) 0.87 Refinement statistics Resolution range (Å) 30–2.4 30–2.9 No. of reflections (F>0) 16 049 10 251 Rcrystd 23.6 21.9 Rfreee 25.0 25.0 R.m.s. deviations Bonds (Å) 0.009 0.008 Angles (°) 1.485 1.418 a Values indicated in parentheses are for the highest resolution shell. b Unique reflections, completeness and multiplicity assuming that F+ and F− are not equivalent. c Rsym = Σ|I − |/Σ I, where is the average intensity over symmetry equivalent reflections. d Rcryst = Σ||Fobs| − |Fcalc||/Σ|Fobs|. All data were used with no sigma cut-off. Summation is over the data used for refinement. e Rfree = Σ||Fobs| − |Fcalc||/Σ|Fobs|, where Fobs are test set amplitudes (5% of the data) not used in the refinement. Description of the structure and MTase fold identification NS5MTaseDV has an overall globular fold with approximate dimensions of 55 × 45 × 40 Å, and can be subdivided into three subdomains (Figure 1B and C). The core subdomain 2 (in yellow, residues 55–222) folds into a seven-stranded β-sheet surrounded by four α-helices. This core structure closely resembles the catalytic domain of other AdoMet-dependent MTases (Fauman et al., 1999) and will be discussed later. Appended to the core are an N-terminal extension (subdomain 1 in red, residues 7–54) and a C-terminal extension (subdomain 3 in cyan, residues 223–267). The N-terminal subdomain comprises a helix–turn–helix motif followed by a β-strand and an α-helix. The C-terminal subdomain consists of an α-helix and two β-strands, and is located between subdomains 1 and 2. The consensus topology of the catalytic domain of Ado Met-dependent MTases, compared with NS5MTaseDV and the closely related mRNA cap MTase VP39, is shown in Figure 2. The consensus topology consists of a twisted mixed β-sheet comprising seven strands flanked by three α-helices on each side of the sheet (Fauman et al., 1999). The NS5MTaseDV core subdomain 2 adopts the depicted consensus fold with two major differences (Figures 1B and 2). First, helix B between β-strands 2 and 3 is virtually absent. It consists of only one turn immediately following β-strand 2, as in the DNA MTase M.TaqI (Schluckebier et al., 1997; Fauman et al., 1999). Secondly, helix C between β-strands 3 and 4 is completely missing in NS5MTaseDV, as it is again in M.TaqI, in another DNA MTase M.HhaI (O'Gara et al., 1996), in the protein MTase CheR (Djordjevic and Stock, 1997) and in a small-molecule MTase glycine-N-MTase (Fu et al., 1996). After β-strand 4, NS5MTaseDV matches the MTase consensus fold exactly. Comparing the NS5MTaseDV core domain with existing MTase structures, the nearest structural neighbours are the prokaryotic E.coli MTase FtsJ (Bugl et al., 2000) (r.m.s.d. of 2.4 Å for the 155 Cα atoms defined as structurally equivalent using the DALI server; Holm and Sander, 1993), M.TaqI (Schluckebier et al., 1997), the C-terminal domain of the fibrillarin homologue Mj0687 from the archaebacteria Methanococcus jannaschii (Wang et al., 2000) and the vaccinia virus MTase VP39 (Hodel et al., 1996). A comparison of the topologies of VP39 and NS5MTaseDV is discussed in detail below. Figure 2.Topological diagram of the consensus fold of AdoMet-dependent MTases (Fauman et al., 1999), NS5MTaseDV and VP39 (Hodel et al., 1996). Triangles represent strands and circles represent helices. The positions of the N- and C-termini are represented by squares. The elements of the consensus AdoMet-dependent MTase fold are coloured in light grey. Those that are replaced by different elements (loops or β-strand) in NS5MTaseDV or VP39 are dashed. AdoMet or AdoHcy bound at the C-terminus of the β-sheet is shown in black. The shape and dimension indicate which secondary structural elements are included in its contact zone. N- or C-terminal appendages of the MTase core in both NS5MTaseDV and VP39 are shown in black and dark grey, respectively. Download figure Download PowerPoint All the proteins mentioned above display a central cleft between β-strands 1 and 4. This cleft has been described as the active site where AdoMet and the substrate bind, and where the methyltransfer occurs (Fauman et al., 1999). Such a cavity is also present in the NS5MTaseDV structure (Figure 3A). During the refinement procedure, the Fo − Fc map revealed a residual strong density in this putative active site pocket. An AdoHcy molecule, a co-product of the methyltransfer reaction, was identified, modelled in this density and refined (Figure 3B). As neither AdoMet nor AdoHcy was added during crystallization, AdoHcy originated from E.coli and co-purified with NS5MTaseDV. The binding of AdoHcy relies on a network of hydrogen bonds (Figure 3B) and van der Waals interactions, similar to the described consensus interactions of MTase proteins (Fauman et al., 1999). The adenine is accommodated within a hydrophobic pocket defined by the side chains of Thr104, Lys105, Val132 and Ile147, and stabilized by hydrogen bonds with a side-chain oxygen of Asp131, and main-chain atoms of Lys105 and Val132 (all residues from β-strands 2, 3 and 4; see also Figure 1C). The ribose moiety is bridged via a water molecule to main-chain atoms of Gly106 and Glu111 (not shown), and to the side-chain oxygen of Thr104. The ribose is also hydrogen-bonded to a sulfate ion, which originates from the crystallization buffer and is conserved in the two crystal structures presented here. The remainder of the AdoHcy molecule is stabilized by hydrogen bonds to two well ordered water molecules, the side chain oxygen of Ser56 and Tyr219, and the main-chain nitrogens of Gly86 and Trp87 (residues from helices X and A). Figure 3.The AdoMet/AdoHcy-binding site. (A) General view of the position of AdoHcy within NS5MTaseDV. The solvent-accessible surface of NS5MTaseDV was calculated and is displayed using GRASP (Nicholls et al., 1991). It has been coloured according to electrostatic potential. Potential values range from −25 kT (red) to 0 (white) to +25kT (blue), where k is the Boltzman constant and T is the temperature. The AdoHcy molecule is shown in stick representation. The area indicated by the red dotted square delineates the sector shown in detail later in Figure 7B. (B) Stereo diagram of the AdoMet/AdoHcy-binding site in NS5MTaseDV. Carbons are displayed in yellow, oxygens in red, nitrogens in cyan and sulfur in green. The Fo − Fc difference map, contoured at 3σ, was calculated at 2.4 Å resolution from a model in which the ligand was omitted. It is clear from the electron density map that the molecule is a co-product of the methyltransfer, i.e. the AdoHcy molecule. Main hydrogen bonds between NS5MTaseDV and AdoHcy are indicated by black dotted lines. Also shown is a sulfate ion, originating from the crystallization conditions. This sulfate ion is hydrogen-bonded to the 2′- and 3′-oxygen of the AdoHcy ribose. For clarity, two water molecules and Tyr219, which are also interacting via hydrogen bonds, are not represented. Download figure Download PowerPoint Methyltransferase activity assay The enzymatic MTase activity of NS5MTaseDV was assayed by following the transfer of a radiolabelled methyl group from AdoMet to various RNA substrates using a filter-binding assay. Capped and non-capped short RNA substrates (GpppACCCCC, 7MeGpppACCCCC and ppp ACCCCC) were used as methyl acceptors. As shown in Figure 4A, the protein is able to transfer a methyl group from AdoMet to the capped RNA subtrates GpppACCCCC and 7MeGpppACCCCC, but not to the non-capped substrate pppACCCCC. Methyltransfer to capped RNA occurs even when the N7 position of the guanine is already methylated. Figure 4.MTase activity. (A) Assay of MTase activity. The extent of methyltransfer from Ado[methyl-3H]Met to three different RNA substrates (pppACCCCC, GpppACCCCC and 7MeGpppACCCCC) by 5 μg of NS5MTaseDV is plotted as a function of time. Data points represent the averages of three independent experiments and are presented as percentage of [methyl-3H] incorporation. The plateau of 100% incorporation represents a concentration of 1.5 μM transferred methyl groups in the reaction at the final reaction time. (B) Identification of the nucleoside methylated by NS5MTaseDV. RNAs incubated in the presence of Ado[methyl-3H]Met and purified recombinant NS5MTaseDV were treated with phosphodiesterase and alkaline phosphatase, and analysed using thin-layer chromatography. The experiment was performed independently twice. The figure shows a qualitative analysis of one chromatogram. Indicated positions of marker nucleosides [N7-methylated guanosine, (7MeG), guanosine (G), adenosine (A) and 2′-O-methylated adenosine (A2′OMe)] were determined under UV light. The radiolabelled products were analysed as described in Materials and methods. Download figure Download PowerPoint In order to characterize the methylated nucleoside(s), the reaction mixture was treated with phosphodiesterase, which cleaves both RNA and cap structure, and alkaline phosphatase to render the nucleoside components. Separation of the reaction products using thin-layer chromatography (Figure 4B) shows that most of the radioactivity co-migrates with 2′-O-methylated adenosine (A2′OMe), and not with N7-methylated guanosine (7MeG). These results demonstrate that, under our experimental conditions, methylation occurs exclusively at the 2′-O position of the second nucleotide. They do not exclude, however, that the N7 position of the guanine would be methylated by NS5MTaseDV under conditions found in the replication complex in vivo. We conclude that NS5MTaseDV is the 2′OMTase of the Dengue virus. The physical coupling of this domain to the polymerase domain should be relevant to the coordination of the initiation of genomic (+) RNA synthesis and RNA capping. Comparative analysis of NS5MTaseDV in relation to AdoMet-dependent MTases The large family of AdoMet-dependent MTases shows a high degree of structural homology that is not reflected at the amino-acid sequence level. It is thus difficult to establish relationships between existing AdoMet-dependent MTases (Fauman et al., 1999). For DNA MTases of different origins and specificities (methyltransfer to C5-cytosine, N6-adenosine and N4-cytosine) it has been possible to define nine conserved motifs that are involved in AdoMet binding and in catalysis of methyltransfer (Malone et al., 1995). Within the group of RNA MTases, comparable sequence conservation can only be found in specific subfamilies. For example, cellular and double-stranded DNA virus N7MTases show conservation at the sequence level that is not shared by RNA viruses (Bujnicki et al., 2001). Nevertheless, the existence of three-dimensional structures of RNA MTases allows a comparative analysis of these enzymes even if they present a high degree of diversity with respect to origin and specificity. Figure 5A shows a structure-based sequence alignment of the MTase core domain of NS5MTaseDV with rRNA MTase FtsJ (Bugl et al., 2000), the C-terminal rRNA MTase domain of Mj0697 (Wang et al., 2000), mRNA MTase VP39 (Hodel et al., 1996), mRNA MTase domains I and II of Reovirus protein λ2 (Reinisch et al., 2000) and the rRNA MTase ErmC′ (Bussiere et al., 1998). M.TaqI (Schluckebier et al., 1997) was included as a representative of the DNA MTases and a close structural neighbour of NS5MTaseDV. The positions of conserved DNA MTase motifs I–X (except for motif IX, which is conserved for C5-cytosine DNA MTases only; Posfai et al., 1989) are indicated. As expected, calculating the homology within the secondary structural elements present in NS5MTaseDV (134 residues spanning the β-strands and α-helices), we could not detect significant sequence conservation within the group of RNA MTases. NS5MTaseDV shares between 10 and 19% identity with the seven MTases listed in Figure 5A. After searching for conserved residues between all listed MTases within the nine motifs defined for DNA MTases (Figure 5A), sequence conservation was found in motif I, the universal AdoMet-binding motif (Koonin, 1993), and in motif III, which is also involved in AdoMet binding (Malone et al., 1995). Within the group of RNA MTases, we detected conservation of residues K61, D146, K181 and E217 (NS5MTaseDV numbering, marked in red in Figure 5A) for NS5MTaseDV, and FtsJ, VP39 and MTaseI of the λ2 protein of Reovirus. These residues are within motifs X, IV, VI and VIII defined for DNA MTases and, with the exception of motif X, are primarily involved in catalysis (Malone et al., 1995). Figure 5.Comparative analysis of NS5MTaseDV. (A) Structure-based sequence alignment of the MTase core domain of NS5MTaseDV with rRNA MTase FtsJ (Bugl et al., 2000), the C-terminal rRNA MTase domain of Mj0697 (Wang et al., 2000), mRNA MTase VP39 (Hodel et al., 1996), mRNA MTase domains I and II of