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1 Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK
2 Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK
3 Department of Virology, Telethon Institute for Child Health Research, University of Western Australia, Perth, WA 6008, Australia
4 CNRS, UMR2472, IFR 115, Virologie Moléculaire et Structurale, 91198 Gif sur Yvette, France
5 INRA, UMR1157, Virologie Moléculaire et Structurale, 91198 Gif sur Yvette, France
Correspondence
Jonathan M. Grimes
jonathan{at}strubi.ox.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Coordinates and structure factors are deposited with the Protein Data Bank: MT1, 2px2; MT2, 2px4; MT3, 2px5; MT-GTP, 2px8; MT-GTPG, 2pxa; and MT-GTPA, 2pxc.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Shatkin, 1976). This not only protects mRNA against exonucleases, but is required for the efficient initiation of translation and is involved in processes such as splicing (Gu & Lima, 2005; Parker & Song, 2004). Cap formation is sequential: firstly the 
-phosphate of the terminal base is removed by an RNA triphosphatase, secondly an RNA guanylyltransferase (GTase) couples guanosine monophosphate to the 
-phosphate of the terminal base via a 5'–5' linkage and thirdly, a methyltransferase (N7MTase) methylates the N7 position of the terminal guanosine. Subsequently the ribose 2'-OH position of the second, third and even fourth base (counting from the start of the cap) may be methylated by an 2'-OMTase. The methylation reactions use S-adenosyl-L-methionine (AdoMet) as a methyl donor, generating, as a by-product, S-adenosyl-L-homocysteine (AdoHcy).
Eukaryotic viruses have come up with a variety of ways to produce capped mRNA. Some, including the lentiviruses and herpesviruses, hijack the host capping machinery, others, such as influenza viruses and hantaviruses, ‘snatch’ cap structures from capped cellular mRNAs, whilst viruses like orbiviruses, orthopoxvirus and flaviviruses encode their own capping enzymes (reviewed in Gale et al., 2000; Schneider & Mohr, 2003). Little is known about the components of the capping machinery of flaviviruses, such as dengue virus and West Nile virus. The capped positive-sense, single-stranded RNA genome is translated as a single polyprotein that is processed by cellular and viral proteases into three structural and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Chambers et al., 1990). Although the guanylyltransferase has yet to be identified, the other activities required for cap formation have been mapped to non-structural proteins: RNA triphosphatase activity to NS3 (Bartelma & Padmanabhan, 2002; Kuo et al., 1996; Warrener et al., 1993; Wengler, 1993), 2'-OMTase activity to the N-terminal domain of the RNA-dependent RNA polymerase, NS5 (Egloff et al., 2002; Ray et al., 2006), and, most recently, N7MTase activity to the same domain (Ray et al., 2006). The crystal structure of the NS5 MTase domain of dengue virus type 2 (Egloff et al., 2002) shows structural similarity to the vaccinia virus VP39 and orthoreovirus 
2 2'-OMTase domains (Bujnicki & Rychlewski, 2001; Hodel et al., 1996; Reinisch et al., 2000). However, crystal soaking experiments with a non-hydrolysable guanosine triphosphate (GTP) analogue and ribavirin 5'-triphosphate, a competitive inhibitor (Benarroch et al., 2004), showed binding at a site distinct from the ‘conventional’ cap-binding site observed previously in several cap-binding proteins (Calero et al., 2002; Fechter & Brownlee, 2005; Hodel et al., 1997; Marcotrigiano et al., 1997). Conventional binding sandwiches the m7 guanosine in between two aromatic side chains, achieving ring stacking. In contrast, in the dengue virus MTase structure the nucleotide analogue was stacked against a single aromatic side chain (F25) in a shallow depression on the surface of the protein, some 10 Å (1 nm) from the equivalent m7GTP-binding site in vaccinia virus VP39, so it remained unclear as to how methylation of either N7 or 2'-O groups might occur and, to date, no structural information has been available on the binding of cap analogues or capped RNA, to a flavivirus NS5.
We report here the 2 Å (0.2 nm) resolution crystal structure of the NS5 MTase domain of Murray Valley encephalitis virus (MVEV). MVEV is a member of the Japanese encephalitis group of flaviviruses, which includes the West Nile virus. In addition, a number of complexes of this domain with nucleotides and cap analogues have been determined at approaching 2 Å (0.2 nm) resolution, in an attempt to understand cap binding and throw light on the mechanism of both N7 and 2'-O methylation. In particular, a crystallographic dimer of the MTase domain in complex with GpppA suggests a paradigm for the coordination of the sequential methylation of the terminal guanosine N7 and the second ribose 2'-OH position.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Berrow et al., 2007)], adding a C-terminal KHHHHHH tag to the protein (Table 1). Constructs were PCR amplified using the KOD HotStart polymerase (Merck Biosciences), gel purified using QIAquick 96 plates (Qiagen) and used for In-Fusion cloning with linearized pOPINE vector. For small-scale expression screening, the recombinant vectors were used to transform Escherichia coli Rosetta(DE3)pLysS. Expression screening was performed in 3 ml GS96 (Q-biogene) cultures in 24-well blocks, supplemented with 50 µg carbenicillin ml–1, 35 µg chloramphenicol ml–1 and 1 % glucose. Cultures were grown to an OD600 
0.6 at 37 °C, shaken at 220 r.p.m., the temperature was dropped to 20 °C, IPTG was added to 0.5 mM final concentration and incubated for 16–20 h. Cell pellets were processed on a Qiagen biorobot 8000 using magnetic Ni-NTA beads (Qiagen) to extract his-tagged proteins from the soluble fraction. For large-scale expression, a fresh colony was picked to inoculate an overnight starter culture in Luria–Bertani broth containing 50 µg carbenicillin ml–1, 35 µg chloramphenicol ml–1 and 1 % glucose at 37 °C, shaken at 220 r.p.m. Then 1 litre of GS96 (Q-Biogene) containing 50 µg carbenicillin ml–1, 35 µg chloramphenicol ml–1 and 1 % glucose was inoculated with 20 ml of starter culture and grown to an OD 0.6 at 37 °C, shaken at 220 r.p.m., at which point the temperature was dropped to 20 °C, IPTG was added to 0.5 mM final concentration, and incubation continued for 16–20 h at 20 °C.
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). Reductive methylation of lysine residues was employed as an aid to crystallization, using the method described elsewhere (Walter et al., 2006). The extent of methylation was assessed by electrospray mass spectrometry.
Crystallization.
Three NS5 constructs (1–293, 1–291 and 1–269) yielded protein of sufficient quality for crystallization trials (Table 2). The methylated and unmethylated proteins were concentrated to 3 mg ml–1 (NS51–293 native), 2.6 mg ml–1 (NS51–293 methylated), 6 mg ml–1 (NS51–291 native and methylated), 9 mg ml–1 (NS51–269 native) and 6 mg ml–1 (NS51–269 methylated). Crystallization trials were set-up in the presence or absence of 0.5 mM AdoHcy and monitored using the OPPF crystallization and imaging pipeline (Mayo et al., 2005; Walter et al., 2005). Useful crystals of NS51–269 were only obtained for methylated protein. These appeared after 24 h in a large number of conditions in the Hampton PEG/Ion screen and Hampton crystal screens 1 and 2 (Hampton Research). Crystallization conditions for crystals used to collect data are given in Table 2 (all X-ray data were collected at the ESRF, Grenoble, France). In addition data were collected for m7GTP (Sigma-Aldrich) soaked methylated NS51–269 and for methylated NS51–269 co-crystallized with a cap analogue at 2 : 1 molar ratio [P1–P3 diguanosine triphosphate (GpppG); Sigma]. Subsequently, it was found that non-methylated NS51–269 in the presence of 0.5 mM AdoMet co-crystallized with either GpppA or m7GpppA (New England Biolabs) (cap analogue to protein ratio 2 : 1). The best crystals were with GpppA analogue, for which a dataset was collected.
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) (Table 2
). Crystals were flash frozen in 25 % glycerol diluted using the crystallization solution. In addition for cap analogue soaking experiments, the analogue was present in the cryo-solution at a concentration no less than that in the soaking solution. Crystals of the first NS5 MTase–AdoHcy complex (form 1) belonged to space group P21 with unit cell dimensions a=46.4 Å (4.64 nm), b=68.3 Å (6.83 nm), c=82.0 Å (8.2 nm), =98. ° and contained two molecules in the crystallographic asymmetric unit. The structure was solved by molecular replacement using the structure of dengue virus NS5 MTase domain [Protein Data Bank (PDB) id. 1L9K] with CNS (Brunger et al., 1998). The model was automatically rebuilt with ARP/WARP (Perrakis et al., 1999) and refinement at 2 Å (0.2 nm) completed using REFMAC (Murshudov et al., 1997). The current model, comprising 256 residues, has an R-factor of 0.200 (R-free 0.273) and good stereochemistry (93.6 % of residues lie in the most favoured region of the Ramachandran plot, Table 2). The remaining five structures were solved by molecular replacement from these coordinates and refined using similar procedures (Table 2). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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] failed to yield crystals, so five shorter constructs differing in the choice of the C terminus were assessed (Table 1). These constructs were truncated on the basis of RONN disorder predictions (Yang et al., 2005) and/or the presence of regions of lower sequence conservation amongst the flaviviruses. MVEV NS51–269 maps to the portion of the dengue virus NS5 MTase domain visible in the study by Egloff et al. (2002). Small-scale expression screening demonstrated that most constructs were expressed in E. coli, albeit with significantly differing levels of soluble expression (compare NS51–269 and NS51–272, Table 1). Crystallization trials with NS51–291, NS51–293 and NS51–269 yielded no crystals suitable for diffraction analysis, either in the presence or absence of AdoHcy. However, methylation of NS51–269 (adding two methyl groups to accessible lysine residues and to the N terminus) yielded crystals in a variety of conditions in the presence of AdoHcy. Mass spectrometry analysis revealed that 18 out of the 21 potential sites had been methylated. In contrast, despite their improved appearance, crystals of methylated NS51–291 and NS51–293 remained unsuitable for diffraction analysis. Subsequent experiments showed that non-methylated NS51–269 could be crystallized in the presence of both a cap analogue (GpppA) and AdoMet.
Overall architecture and comparison with related structures
Three crystal forms of the MTase domain of NS5 in complex with AdoHcy have been solved, giving five independent views of the molecule. All five molecules are essentially identical in structure [pairwise root-mean-square deviations (rmsds) between 0.3 (0.03) and 0.5 Å (0.05 nm) for all C
atoms], and amongst known structures they are most similar to the MTase domain of dengue virus NS5 [248 Cs matched with rmsd 0.7 Å (0.07 nm), using program SHP (Stuart et al., 1979), Fig. 1]. The structure has the characteristic class I methyltransferase fold (Martin & McMillan, 2002; Schubert et al., 2003), with N-terminal (helices A1–A3, Fig. 1) and C-terminal (helix A4-end) extensions (nomenclature of Egloff et al., 2002). As in the dengue virus MTase domain, helix B, between strands 2 and 3 is essentially a single turn and C (which would lie between 3 and 4), is entirely absent. One difference between the dengue virus and MVEV structures is that the end of helix A3 and the following loop (43–49) is ordered in only two of the five MVEV MTase domain structures (where it is stabilized by either crystal contacts or the presence of a cap analogue).
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A and 1, which interacts with the amino acid portion of AdoMet, and an acidic loop connecting 2 with B, interacting with the ribose hydroxyls (Fig. 1a). The adenosine ring is bound in a hydrophobic pocket, sandwiched between I147 and the aliphatic component of K105. Although K105 is involved in binding AdoHcy, it is clear from the electron density map that it has been methylated. The conformations of the bound AdoHcy molecule and interacting side chains are essentially identical between the dengue virus and MVEV MTase domains, with the exception of two side chains that are perturbed in dengue virus by the presence of a sulphate ion, which interacts with the ribose hydroxyls (in MVEV residue E111 rearranges to hydrogen bond to the 3' ribose hydroxyl group and H110 moves to coordinate the 2'-O group). The KDKE tetrad, characteristic of 2'-OMTase active sites is conserved in MVEV MTase (K61, D146, K182, E218), and superposes well on the equivalent residues of the 2'-OMTases of dengue virus (NS5; PDB id. 1L9K), vaccinia virus (VP39; PDB id. 1AV6) and reovirus (PDB id. 1EJ6, A434–A691).
Structurally, the MVEV MTase domain is more similar to 2'-OMTase domains than to N7MTase domains; however, functional studies suggest that flavivirus NS5 possesses both activities (Ray et al., 2006). Superposition of this domain and the Ecm1 N7MTase (from the microsporidian parasite Encephalitozoon cuniculi, PDB id. 1Rl1; Fabrega et al., 2004) aligns the conserved seven-stranded core of the class I methyltransferases [matching 188 Cs with 2.4 Å (0.24 nm) rmsd, Fig. 2]. The AdoMet substrate is similarly positioned in the two structures, although the contacting helices and loops differ. A significant difference between the two structures is an extra domain (175–229 in the Ecm1 numbering scheme) inserted into the class I fold in Ecm1 at residue 185 in MVEV NS5. This small domain appears to stabilize the binding of the cap structure, closing around the GTP so that it is positioned to accept the methyl group from the AdoMet substrate. The electron density maps for the co-crystal structure of Ecm1 N7MTase with m7GpppG revealed only GTP, suggesting a preferential binding for unmethylated GTP (Fabrega et al., 2004).
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). The guanosine stacks against the residue F24 with the methylated N7 exposed to solution and not directly in contact with the protein. Base specificity is achieved through electrostatic interactions between the N2 of the base and the carbonyl oxygens of residues L16 and M19. The ribose hydroxyl groups are contacted by residues N17 and K13. The latter forms hydrogen bonds with both hydroxyls (the electron density map shows no evidence of methylation), whereas N17 only contacts the 2'-O group. In addition the conformation of the loop containing residues S150 and P152 that packs against the sugar is identical in both proteins. Additional contacts are observed between residues R28, S215, R213 and the triphosphate group.
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). The base and ribose groups of the first guanine are bound in an identical manner to m7GTP, but the conformation of the triphosphate groups differs slightly, presumably due to the disorder in the second guanine. The R213 contact becomes, here, a water-mediated hydrogen bond and the S215 contact is lost altogether.
Complex with GpppA and AdoMet: a model for methylation
Unlike the other nucleotide/cap analogue complexes, the GpppA–AdoMet co-crystal structure reveals a crystallographic dimer of the methyltransferase domain, with two GpppA molecules bound per monomer (see Fig. 3c). One (GA1) binds at the same position as GpppG and m7GTP and the second (GA2) binds adjacent to it, interacting with positively charged residues near the AdoMet-binding cleft. The two molecules interact, with the adenosine base of GA1 stacked against the guanosine base of GA2. GA1 and GA2 are further stabilized by a complex set of twofold interactions generated by the crystallographic twofold axis, in particular for GA2, which lies adjacent to this axis. In total this association sandwiches eight bases between the two monomers and effectively spans the two crystallographically related GTP-binding sites (details of the interactions are provided in Fig. 3c).
Surprisingly, although AdoMet is bound in an identical fashion to that described above for the product AdoHcy, none of the moieties in either GA1 or GA2 that would be methylated by either the N7 or 2'-O methyltransferase activities are in a catalytically competent position. The closest 2'-O group to the AdoMet belongs to GA1, but is over 10 Å (1 nm) from the leaving methyl group. Comparison with vaccinia virus VP39 complexed to capped RNA (Hodel et al., 1998) suggests that helix A3 binds the incoming RNA, which might explain the binding of the second GpppA in a similar position. Why only a single, partly ordered GpppG was observed is not clear, but could be related to the adenosine-specific effects on binding/stacking, or possibly because methylated protein was used in the NS51–269–GpppG complex, whereas for the GpppA complex native protein was used (although no lysines interact with GA2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Moure et al., 2006). However, a recent biochemical study suggests that NS5 of flaviviruses can perform, sequentially, both N7 and 2'-O methylation, but only in the presence of appropriate viral RNA (Ray et al., 2006). Our analysis confirms the binding site for the m7GTP component of the cap structure observed by Egloff et al. (2002), and a complex with GpppG reveals that the second guanosine moiety is disordered. Although we have been unable to capture a catalytically relevant binding state, the presence of eight bases lining the dimer twofold interface in the GpppA complex suggests a model for the interaction of viral RNA, which explains why it is required for activity and also suggests a novel mechanism for coordinating the sequential capping. We note that our dimer differs significantly from that observed by Egloff et al. (2002) (superposing the one of the two monomers of the dengue virus MTase domain and MVEV domains leads to a 12 ° difference in orientation for their dimeric partners); furthermore PISA (Krissinel & Henrick 2005) suggests that, unlike the dengue virus MTase dimer, the GpppA dimer interface is not extensive enough to be considered physiologically relevant. However, there are intriguing similarities with the observations of Egloff et al. (2002), whose dimer interface was cemented by sulphate ions, which have similar properties to the phosphate groups of RNA. Our model for the sequential methylation N7 and 2'-O groups of the capped RNA is illustrated schematically in Fig. 4. We propose that RNA with an unmethylated cap locks into the MTase dimer interface. As it enters, the N7 group of the terminal guanosine is methylated by the first AdoMet molecule encountered. As the cap is pushed further in, the terminal methylated guanosine binds at the GTP site (Phe-24), and the second AdoMet molecule is then positioned to methylate the 2'-O group of the second ribose. The products, fully methylated capped RNA and AdoHcy, are then released, allowing substrate AdoMet to rebind, priming the dimer for the next dual methylation cycle. Note that, due to the symmetry of the molecular dimer, cap can enter in either direction (presumably from either polymerase domain) and still be methylated in the correct sequence. Despite the attractive features of this paradigm, there are still many outstanding questions, thus whilst NS5 in solution is monomeric, it is not clear what the oligomeric state of NS5 is during replication. There are also additional complexities to the capping process, for instance dengue virus NS3 and NS5 form a replication complex (Yon et al., 2005). One of the domains of NS3 has RNA stimulated nucleoside triphosphatase and 5' triphosphatase activity and may play a role in cap synthesis; however, the molecular species responsible for the guanylyltransferase activity remains unknown. In addition, the proper positioning for the first, N7, reaction may be facilitated by the recognition of a specific 5' RNA element adjacent to the cap (in dengue virus named SLA), by the NS5 polymerase domain (Filomatori et al., 2006).
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ACKNOWLEDGEMENTS |
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Received 4 December 2006;
accepted 20 April 2007.
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H. Dong, S. Ren, B. Zhang, Y. Zhou, F. Puig-Basagoiti, H. Li, and P.-Y. Shi West Nile Virus Methyltransferase Catalyzes Two Methylations of the Viral RNA Cap through a Substrate-Repositioning Mechanism J. Virol., May 1, 2008; 82(9): 4295 - 4307. [Abstract] [Full Text] [PDF]
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. Surrounding protein is shown in cartoon representation coloured as in Fig. 1(a)