Abstract
The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site (SAMP) also contains a novel pocket (NSP) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the SAMP-adjoining site holding the nucleotides undergoing methylation (SUBP) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2′O and N7 positions, and also displays nucleotide triphosphatase activity.
Similar content being viewed by others
Introduction
The Mononegavirales order groups five families of monopartite, negative-strand RNA viruses many of which are highly pathogenic and/or contagious; the Filoviridae (of which Ebola virus is a representative), the Bornaviridae (Borna disease virus), the Nyamiviridae (midway virus), the Rhabdoviridae (rabies, vesicular stomatitis virus (VSV)) and the Paramyxoviridae (measles virus, human metapneumovirus (hMPV)). These viruses encode a large RNA polymerase (L) (usually >2,000 amino acids) that is crucial to viral replication (Fig. 1a). It has two distinct roles to replicate the RNA genome and to transcribe viral mRNA. As such it not only polymerizes RNA but also synthesizes fully methylated cap structures1. Capping involves the co-transcriptional addition of a guanosine (G) to the first nucleotide (N1) of the nascent RNA chain via a 5′-5′ triphosphate bridge, resulting in a GpppN1- structure. Typically, this is followed by methylation of nitrogen 7 (N7) of G, giving rise to mGpppN1-, and of the 2′-oxygen (2′O) of the N1 ribose (mGpppN1m-). The cap protects mRNAs against 5′-exonucleases and promotes RNA transport and translation, while 2′O-methylation prevents detection by cellular-immunity sensors2,3,4. In Rhabdoviridae, CR-V catalyses cap addition by means of an unconventional polyribonucleotydyl-transferase (PRNTase) reaction where a conserved histidine in CR-V forms a covalent phosphoamide bond with the transcript, resulting in a CR-V-pRNA intermediate. The capped transcript is released after ligation of a Gpp to the pRNA. This mechanism differs from capping in eukaryotes and most other viruses, in which a guanylyltransferase (GTase) forms a phosphoamide bond with Gp, before transferring it to 5′ppRNA2. Paramyxoviridae also contain a PRNTase signature motif in their CR-V domains, suggesting they use the same capping strategy as Rhabdoviridae. In addition, however, paramyxovirus and filovirus L proteins contain a KxxxKxxG sequence (K-K-G motif) at their C termini, reminiscent of a signature motif for eukaryotic GTases, where one of the lysines forms the transient phosphoamide bond with Gp in the capping reaction5. A C-terminal domain of Rinderpest virus L (containing CR-VI and the downstream K-K-G motif) was shown to form such a bond6, leaving open the possibility that Paramyxoviridae use GTase activity for capping.
Cap methylation is catalysed by S-adenosylmethionine (SAM)-dependent methyltransferases (MTases), which position a SAM molecule next to the target atom on the RNA, enabling the direct transfer of a methyl group and converting SAM into S-adenosylhomocysteine (SAH). In 2′O-MTases, a conserved K-D-K-E tetrad potentiates methyl transfer2. As yet, the boundaries of CR-VI, the putative MTase of L, are not well defined, nor is it clear if CR-VI acts on its own or in conjunction with other domains, whether it mediates both N7- and 2′O-methylation and which of these methylations would take place first. Research into the activities of L and the mechanisms underpinning them has historically been hampered by a complete, order-wide absence of high-resolution structures. To redress this, we set out to study L domains of hMPV, a paramyxovirus of the Pneumovirinae subfamily closely related to respiratory syncytial virus (RSV). Like RSV, hMPV is highly contagious and causes respiratory tract disease7. As a part of this study, we expressed CR-VI+, a 406-residue fragment comprising CR-VI and the adjoining ‘+ domain’, the variable region carrying the K-K-G-motif, investigated its MTase activity and solved its crystal structure. Besides sequentially methylating the 2′O and N7 atoms of small capped RNAs, CR-VI+ also 2′O methylates uncapped substrates and displays nucleotide triphosphatase (NTPase) activity. Both the CR-VI domain, which assumes a fairly standard MTase fold, and the K-K-G motif of the (mainly helical) +domain are required for the MTase reactions. Combined, the data provide new insights into the modification of the 5′-ends of transcripts emerging from the polymerase domain of L. This structural information on a mononegavirus L protein, and the new insights in the capping mechanism it provides, should spur the development of novel antiviral drugs against this important group of highly pathogenic viruses.
Results
MTase activities
In vitro, CR-VI+ most effectively binds and methylates synthetic RNAs containing the conserved start sequence of hMPV transcripts, preferring a substrate length of nine nucleotides (Fig. 2). The methylation occurs at the G N7 and N1-2′O positions (Figs 2 and 3a), with 2′O-methylation preceding N7-methylation (Fig. 3a), an uncommon order of events also occurring in VSV8. CR-VI+, moreover, efficiently methylates uncapped RNAs with 5′-phosphate groups (especially pppRNA), primarily at the 2′O atom of N1 since almost no 3H-methyl transfer takes place onto the pppGmGGACAAGU substrate where this atom is blocked (Fig. 2b, green bars). The 2′O-methylation of pppGGGACAAGU represents ∼50% of that observed with GpppGGGACAAGU (which has an extra methylation target in the G N7 atom; Fig. 2b, red bars). Mutagenesis studies show that all K-D-K-E residues (K1673, D1779, K1817 and E1848) are essential for 2′O-methylation, while the aspartic acid (D1779) in particular is important for N7-methylation (Fig. 3b), as was also observed in Flaviviridae MTases9. Other residues, many of which belong to the +domain rather than the core MTase fold, were also found to be essential for the MTase activities (the ‘SUBP’ residues in Fig. 3b, described below).
Structure of the +domain
The crystal structure of CR-VI+ was solved to 2.2 Å resolution in space group P212121 with two molecules, disulphide-linked at residue C1877, in the asymmetric unit, assuming a ‘head-to-toe’ conformation (Supplementary Fig. 1). There are no significant differences between the molecules, which have a slightly twisted, bi-lobed shape, composed of two globular domains, the larger corresponding to CR-VI (residues ∼1,616–1,883) and the smaller to the +domain (1,884–2,005; Fig. 1b). Although composed of only ∼120 residues in Pneumovirinae, the size of the +domain varies greatly within the Mononegavirales order, reaching ∼240 residues in Rhabdoviridae. In hMPV, it consists of six α-helices (α+1–6). Helix α+6 contains the K-K-G motif (Supplementary Fig. 2) and together with α+1 leans over the active cleft of CR-VI. Helix α+4, and to a lesser extent helices α+1, α+5 and α+6, packs down on λ1650–1666, the second half of a long loop (residues 1,635–1,666) that swerves around the CR-VI domain (Fig. 1b and Supplementary Fig. 3; ‘λ’ is used throughout the paper to denote loops). The λ1650–1666 region, which contains a small helix (α-a), acts as a fulcrum allowing the +domain to pivot relative to the CR-VI domain. Helix α+3 varies in length, from 4½ turns (in most crystals) to 6 (in Protein Data Bank (PDB) 4UCY), and the loop between α+2 and α+3 is always disordered (Supplementary Fig. 3). In crystals of the monomeric C1877A mutant (space group P3121), this disordered, unstable region is further enlarged, as helix α+2 completely unfolds and α+3 unwinds to 3½ turns.
Structure of the CR-VI domain
The CR-VI domain shares some peripheral characteristics with the 2′O-MTases of SARS coronavirus (PDB: 2XYQ)10, vaccinia virus (1VP3) (ref. 11) and bluetongue virus (VP4-subunit; 2JHA)12, such as the long N-terminal loop and the position of helix αX. In its active core, however, it better resembles RrmJ-type flavivirus MTases (for example, 3EVF9; Supplementary Fig. 4). Most notably, hMPV and flaviviruses share an unusually long (∼10 residues), flexible β2λ (that is, the loop immediately following the β2-strand), which forms the SAM-binding pocket (SAMP) along with loops β1λ and β4λ, shielding it from the solvent (Fig. 1b, c). In Wesselsbron (flavi-)virus, β2λ is found in closed or open conformations, either packing up against SAM (PDB: 3ELW) or exposing it to the solvent (3EMB)13, changes that may assist SAM uptake and/or SAH expulsion. In CR-VI+, β2λ similarly assumes alternate conformations (Fig. 4c,d). In the ‘closed’ form, the ligand’s ribose group is hydrogen bonded to D1725, and—via a water—to D1722. Loops β1λ and β4λ also show a degree of flexibility. β1λ residue E1697, conserved in paramyxo- and filoviruses, forms a hydrogen bond with the NH2 group of SAM and is essential for both MTase activities (Fig. 3b). However, in the absence of SAM, its side chain either moves into the sub-pocket that normally accommodates the NH2 group, or, more markedly, turns towards the solvent in the direction of β2λ, which in this case assumes the ‘open’ position (Fig. 4d). β4λ forms a side wall of SAMP, and together with αD and β5 also defines a deep, hydrophobic cavity not present in other MTases, termed NSP (or nucleoside-binding pocket). Although a role for NSP has yet to be determined, the pocket binds the adenosine moiety of SAM or ATP, soaked at high (25 mM) concentrations into CR-VI+ crystals, causing β4λ to impinge onto SAMP (Fig. 4c,d; when SAM is used for soaking, one SAM molecule occupies SAMP, and another binds to NSP). GTP was not observed in NSP, but this may reflect a lower soaking concentration (2.5 mM), due to GTP’s poor solubility. With the exception of E1781, the amino acids lining NSP are poorly conserved beyond the Pneumovirinae, and mutating key NSP residues barely affects the MTase activities (Fig. 3b). CR-VI, finally, contains a Zn-finger, which is not conserved beyond the Pneumovirinae subfamily and links the small α–d′ to the rest of the structure (Fig. 1b).
During the preparation of this manuscript, a 3.8-Å structure of VSV L, obtained by electron cryo-microscopy, was published14. The CR-VI (or MTase) part of VSV resembles that of hMPV, but apparently lacks a deep NSP pocket (PDB: 5a22, Fig. 5). The +domain (C-terminal domain) is more elaborate in VSV than in hMPV, containing extra regions N and C terminal of helix α+6. The helix itself, however, appears well conserved, both in length and in position. Although a K-K-G motif is not present in VSV, it does contain an arginine (R2038) strictly conserved among the Rhabdoviridae, which is structurally equivalent to hMPV’s K1995, the second lysine of K-K-G motif.
The absence of a classical cap-binding site
A common feature of MTases involved in cap methylation is a defined cap-binding pocket that binds G with high affinity, enabling subsequent, low-affinity interactions with the triphosphate bridge and the first few nucleotides, thus precluding methylation of uncapped RNAs15,16. In CR-VI+, however, an open, solvent-exposed area is found where this pocket is normally located (Fig. 6a). Moreover, GpppG- or mGpppG binding was not observed in co-crystallization or soaking experiments, suggesting that CR-VI+ has a weak affinity for G at best and that the cap is not required for substrate recognition. This is consistent with CR-VI+ binding capped and uncapped RNAs with similar strength and being able to 2′O-methylate uncapped RNAs (Fig. 2b). Strong binding would also prevent translocation of G into SUBP for N7-methylation. Although a high-affinity cap-binding site is clearly absent from CR-VI+, the low-affinity nucleoside binding to NSP and the convenient location of this pocket relative to SUBP suggest it could provide space for G without forming strong interactions (Supplementary Fig. 5).
The absence of a high-affinity cap-binding pocket appears partly compensated for by the narrowing of the groove that in related MTases accommodates the first few nucleotides of the transcripts, by the overhanging +domain (Figs 4 and 6a). In particular, the site adjoining SAMP, which holds the nucleotides undergoing methylation, has become a more elaborate, but narrower and possibly therefore, higher-affinity substrate-binding pocket (termed SUBP as it accommodates the nucleotide undergoing methylation). Consistently, electron density is found in SUBP following soaking or co-crystallization with GTP, whereas in other MTases added GTP predominantly shows up in the cap-binding pocket. In particular, helix α+6 and the +domain-affiliated λ1650–1666 help shape SUBP through the side chains of K1991 and K1995 (of the K-K-G motif), and of H1659 and R1662, respectively (Fig. 4b). The marked decrease in MTase activity of mutants altered at these residues (Fig. 3b) illustrates the importance of SUBP in correctly presenting the substrate nucleotides to SAM. Nevertheless, the pocket is too spacious for a single nucleotide, and the electron density in SUBP from a number of soaked crystals suggests that bound GTP often assumes more than one orientation. In structures where GTP could be fitted with confidence, the guanosine moiety predominantly interacts with λ1650–1666 residues H1659 and R1662, and with K1991 and (K-D-K-E residue) K1673, which clamp the guanine (Fig. 4b). Unusually for cap-MTases, K1673 is not part of αZ, but instead resides on the small z′ (310)-helix (Fig. 1b,c). Whether any of the observed positions of GTP reflects in vivo binding of N1 (as part of a transcript) is unclear; in MTase–RNA complexes (PDB: 1AV6 (ref. 17), 4N49 (ref. 16)), N1 is situated much closer to the K-D-K-E tetrad (Fig. 6a).
A potential role of CR-VI+ in cap addition
K-K-G residue K1995 corresponds to the Gp binding lysine in the signature motif of eukaryotic GTases, but here is in a loop instead of an α-helix (PDB: 3S24 (ref. 18), 1CKN (ref. 19)) and appears ideally placed to target the phosphate tail of GTP. Although incubation of CR-VI+ with [α-32P]- and [β-32P]-GTP, in the absence of Mg++, resulted in radioactive protein bands on denaturing SDS gels, the level of radioactivity was low, and was not diminished by acid treatment before SDS–polyacrylamide gel electrophoresis (SDS–PAGE), implying it is not due to phosphoamide bond formation. A second, strong argument against a GTase-based capping mechanism in hMPV (and in favour of a PRNTase-based one) is the fact that in the closely related RSV the cap is formed by Gpp ligation to pRNA20.
We observed that CR-VI+ also displays NTPase activity, converting GTP into GDP and ATP into ADP (Fig. 7). GTPase activity, which is required for PRNTase-based capping, was previously reported in Mononegavirales L (ref. 21), but as yet could not be linked to a specific domain within the protein. The reaction observed with CR-VI+, however, is quite slow, possibly because other parts of L, or other co-factors, are needed for full activity. In line with this, we were not able to identify key active-site residues. Using the mutants listed in Fig. 3b, the greatest reductions in GTPase activity were obtained with E1697C (corresponding to the flexible residue at the bottom of SAMP; Fig. 4d) and S1669A (part of SUBP, Fig. 4b) to 54 (±20) and 57 (±16) % of the wild-type activity, respectively (n=3). The deletion of the dipeptide G1645K1646 from the long N-terminal loop resulted in a somewhat more pronounced ∼70% reduction (Supplementary Fig. 6). The NTPase activity was confirmed using crystallized CR-VI+ (Fig. 7).
Discussion
As a GDP is transferred onto a PRNTase-bound pRNA intermediate during cap synthesis in VSV21, the presence of NTPase activity in CR-VI+ would suggest that this domain is involved in cap addition. It is unclear from the structure of VSV L how capping, cap methylation and RNA synthesis are coordinated14, but an involvement of CR-VI+ in cap addition is consistent with the dynamic nature of the multi-domain polymerases of RNA viruses in general, exemplified by flu where the C-terminal two-thirds of PB2 has been shown to be extremely mobile22,23. The presentation of uncapped (but CR-V-linked) pRNA to CR-VI+ would also explain why hMPV L does not require a high-affinity cap-binding site. The integrity of both the PRNTase and K-K-G motifs, moreover, has been shown to be essential for viable mRNA synthesis in human parainfluenzavirus 2 (hPIV-2)5, in line with capping involving a concerted action of CR-V and CR-VI+. Assuming the transcript is presented to CR-VI+ by the PRNTase, 2′O-methylation may actually occur before the cap is added (as a cap is not required for the reaction; Fig. 2b), which would also be consistent with 2′O-methylation preceding N7-methylation. The possibility that 2′O-methylation precedes cap addition was also suggested by the ability of virions of the rhabdovirus spring viremia of carp virus to synthesize uncapped, N1-methylated oligonucleotides24. We did not observe significant methylation of free GTP by CR-VI+ (levels were <5% of that of GpppGGGACAAGU methylation), suggesting G N7-methylation takes place after G is added to the RNA. In addition 2′O-methylated substrates (GpppGmGGACAAGU and pppGmGGACAAGU) show an ∼2-fold higher affinity for CR-VI+, compared with their unmethylated counterparts (Fig. 2b), which presumably reflects an RNA repositioning mechanism allowing the G N7 atom access to the SAM methyl donor in the second methylation step.
In conclusion, CR-VI+ is a dynamic part of the L protein that potentially completes PRNTase-initiated cap addition, and methylates the cap at its 2′O and N7 positions. The SUBP pocket, which is conserved among Paramyxoviridae and Filoviridae (Fig. 6b), is clearly instrumental in key activities of the domain, and thus represents an attractive target for the structure-based design of (potentially broad-spectrum) antiviral compounds.
Methods
Cloning and expression
The sequence encoding CR-VI+ was PCR amplified using primers that added a C-terminal SGHHHHHH-tag to the translation product, from a synthetic hMPV L gene (hMPV isolate 00-1, GenBank: AF371337.2), codon optimized for expression in Spodoptera frugiperda (Sf) cells (Geneart). The amplicon was cloned into pOPIN-E for expression in HEK293T mammalian cells (following transfection using Lipofectamine 2000; Invitrogen), in BL21 Star bacteria (Invitrogen) and in Sf21 (insect cells) following co-transfection with (flashBACULTRA) baculovirus (Oxford Expression Systems) using Cellfectin II (Invitrogen)25. Mutants were generated by PCR, using primers carrying the mutation (Supplementary Fig. 7). The CR-VI+ DNA was used as a template for mutagenesis, except for the K1991Q/K1995Q and K1991Q/K1992Q/K1995Q mutants, which were obtained using the K1995Q DNA. For each mutation, the forward primer was combined with a vector-specific reverse primer (5′-AGTGGTATTTGTGAGCCAGG-3′), and, in a second PCR, the reverse primer was used together with a vector-specific forward primer (5′-CCTTTAATTCAACCCAACAC-3′). The two PCR products thus generated were digested with either BspQI or BtsI (New England Biolabs), restriction enzymes that cut within the CR-VI+-specific primer regions. The amplicons were then rejoined using T4 DNA ligase (New England Biolabs), and the ligation products were PCR amplified using the vector-specific primers for insertion in the pOPIN-E vector.
Purification
Seventy-two hours after infection of the suspension cultures with recombinant baculovirus, Sf21 cells were spun down (1,000g, 7 min, 22 °C) and lysed in one-fourth volume of buffer L (1.5% triton X-100, 5% glycerol, 50 mM arginine, 300 mM KCl, 10 mM imidazole and 20 mM Tris (pH 8.0)). After clarification (10,000g, 25 min, 4 °C), the lysate was incubated with benzonase (Novagen; to 0.2 U ml−1) and NiNTA resin (Qiagen; 0.3 ml l−1 culture) for 5 h at 4 °C, with gentle shaking. The beads were transferred to a 10-ml column and washed with 3 × 10 ml buffer W (20 mM Tris (pH 8.0), 1.5 M NaCl, 10 mM imidazole and 7.5% glycerol) and 1.5 ml of 0.1 M arginine (pH 8.0). Protein was eluted in 0.8 M arginine (pH 8.0). An equal volume of ice-cold 3 M AmSO4 was added to the eluent, and the precipitated protein collected (10,000g, 10 min, 4 °C). Protein pellets were stored at −20 °C. A similar lysis and purification protocol was used to obtain recombinant protein from HEK293T cells. BL21 Star cells were lysed using Bugbuster Protein Extraction Reagent (Novagen). Expression and purification were monitored by SDS–PAGE and western blotting. Blots were developed with a 1/10,000 dilution of an anti-histidine tag antibody (Penta·His Antibody, Qiagen, catalogue number 34660). Supplementary Fig. 1 shows purified protein following SDS–PAGE under reducing and non-reducing conditions. An uncropped western blot showing the unreduced, insect-cell-expressed protein next to a BenchMark (Invitrogen) molecular weight marker is also shown.
Selenomethionine incorporation
Twenty hours following infection of an Sf21 suspension culture (27.8 °C, with an agitation speed of 130 r.p.m./0.25g), cells were collected (70g, 10 min, 22 °C) and resuspended in cysteine- and methionine-free SF900II medium (Gibco) supplemented with dialysed fetal bovine serum (Gibco; 7% v/v) and 150 mg l−1 L-cysteine (Sigma). Following an additional 4 h at 27.8 °C in the shaking incubator, 250 mg l−1 selenomethionine (Sigma) was added. Protein expression was allowed to continue for another 48 h. Proteins were purified as above.
Crystallization, structure solving, refinement and validation
Crystallization was carried out by vapour diffusion at 20.5 °C using 96-well sitting drop plates (Greiner)26. Protein pellets were dissolved in water to 5–6 mg ml−1, and initial crystals were obtained by equilibrating 100 nl of protein with 100 nl of reservoir solution C11 of the PGA-HT screen (Molecular Dimensions; pH 6.5) supplemented with guanidine hydrochloride (to 0.1 M), against 0.1 ml of reservoir. Glycerol was added (to 20% v/v) for cryoprotection. Diffraction data were collected at 100 K on Diamond beamlines I02, I03, I04 and I24 (Harwell, UK), and processed using the Xia2 programme suite27. A single-wavelength anomalous dispersion experiment allowed determination and refinement of the positions of selenium atoms, as well as calculation of the phases with autoSHARP28. An initial model was obtained using MR-ROSETTA29, enabling manual model building using COOT30. Refinement was performed using autoBUSTER31, and validation employed COOT and Molprobity32. Molecular replacement (using PHASER33) was used to solve additional CR-VI+ structures. Refinement statistics are given in Table 1, and a portion of the electron density map is shown in Supplementary Fig. 8.
Synthesis of RNA substrates
RNA sequences were chemically synthesized on a solid support using an ABI 394 synthesizer. After RNA elongation with 2′O-pivaloyloxymethyl phosphoramidite monomers34,35 (Chemgenes, USA), the 5′-hydroxyl group was phosphorylated and the resulting H-phosphonate derivative36 oxidized and activated into a phosphoroimidazolidate derivative to react with either phosphoric acid (for ppRNA synthesis), pyrophosphate (pppRNA)36 or guanosine diphosphate (GpppRNA)37,38. N7-methylation of the purified GpppRNA was performed enzymatically using N7-hMTase37,38. To prepare monophosphate RNA (pRNA), the 5′-H-phosphonate RNA was treated with a mixture of N,O-bis-trimethylacetamide (0.4 ml), CH3CN (0.8 ml) and triethylamine (0.1 ml) at 35 °C for 15 min, and then oxidized with a tert-butyl hydroperoxide solution (5–6 M in decane, 0.4 ml; 35 °C, 15 min). After deprotection and release from the solid support, RNA sequences were purified by IEX-HPLC (>95% pure) and their identity were confirmed by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Fight) spectrometry.
MTase activity assays
These were performed by combining 4 μM CR-VI+ with 0.7 μM of the purified and validated synthetic RNAs, 10 μM SAM and 0.33 μM 3H-SAM (Perkin Elmer) in 40 mM Tris-HCl (pH 8.5) and 1 mM dithiothreitol (DTT). Reactions (at 30 °C) were stopped by a 10-fold dilution in 100 μM ice-cold SAH and the samples were transferred to DEAE filtermats (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate (pH 8.0), twice with water and once with ethanol. They were then soaked with liquid scintillation fluid, allowing the measurement of 3H-methyl transfer to the RNA substrates using a Wallac MicroBeta TriLux Liquid Scintillation Counter13.
Methylation of GTP was determined in the same buffer (minus the 3H-SAM and RNA), using the EPIgeneous methyltransferase assay kit (Cisbio), which measures the generation of SAH, as it competes with d2-coupled SAH for binding to a Lumi4-Tb-labelled anti-SAH antibody, affecting the TR-FRET signal between these compounds. In practice, MTase reactions were stopped after 16 h by addition of the detection reagents, and 1 h later the TR-FRET signal was monitored using a PHERAstar Flashlamp plate reader.
Thin-layer chromatography analysis of cap structures
G*pppGGGACAAGU (in which the asterisk indicates the [32P]-labelled phosphate) was synthesized by incubating pppGGGACAAGU (10 μM) with vaccinia virus capping enzyme (New England Biolabs) in the presence of 1.65 μCi [α-32P]-GTP (Perkin Elmer). The capped RNA was purified by precipitation in 3 M sodium acetate supplemented with 1 μg μl−1 of glycogen (Thermo Scientific), and submitted to methylation by CR-VI+ (as above), after which it was precipitated again (stopping the reactions), and digested with 1 U of Nuclease P1 (US Biologicals) in 30 mM sodium acetate (pH 5.3), 5 mM ZnCl2 and 50 mM NaCl (4 h, 37 °C). The products were spotted onto polyethylenimine cellulose thin-layer chromatography plates (Macherey Nagel), and resolved in two steps, first using 0.65 M LiCl, then 0.45 M (NH2)2SO4 as mobile phase. The radiolabelled caps released by nuclease P1 were visualized using a Fluorescent Image Analyzer FLA3000 (Fuji) phosphor-imager.
Characterization of RNA-CR-VI+ interactions
Using T4 RNA ligase 1 (20 units; New England Biolabs), [5′-32P]-pCp (1.1 μCi) was ligated to the 3′-ends of the RNA substrates (10 μM) in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT and 1 mM ATP (16 °C, overnight). Ligase was removed by RNA precipitation in 3 M sodium acetate supplemented with 1 μg μl−1 of glycogen (Thermo Scientific). The radiolabelled RNA was incubated (15 min, 37 °C) with increasing concentrations of CR-VI+, in 20 mM Tris-HCl (pH 8.5), 1 mM DTT, 10% glycerol and 30 mM NaCl. The reaction mixtures were spotted onto nitrocellulose (GE Healthcare) using a manifold-1 dot-blot apparatus (Whatman) and washed with 20 mM Tris-HCl (pH 8.5), 1 mM DTT and 50 mM NaCl. Membrane-bound RNA was quantified by phosphor-imaging. Dissociation constants (KDs) were determined using Hill slope curve fitting (Prism).
NTPase assay
Samples combining CR-VI+ (4 μM) with 0.5 μCi [α-32P]-GTP or 0.5 μCi [α-32P]-ATP in 40 mM Tris-HCl (pH 8.5), 1 mM DTT and 5 mM MgCl2 were incubated at 37 °C. Reactions were stopped by adding an equal volume of formamide/EDTA gel-loading buffer, and hydrolysis products were separated over a 20% polyacrylamide/8 M urea gel before phosphor-imaging.
Additional information
Accession codes: Coordinates and structure factors are deposited in the Protein Data Bank under accession codes 4UCI, 4UCL, 4UCJ, 4UCK, 4UCZ, 4UCY and 4UD0.
How to cite this article: Paesen, G. C. et al. X-ray structure and activities of an essential Mononegavirales L-protein domain. Nat. Commun. 6:8749 doi: 10.1038/ncomms9749 (2015).
References
Rahmeh, A. A. et al. Molecular architecture of the vesicular stomatitis virus RNA polymerase. Proc. Natl Acad. Sci. USA 107, 20075–20080 (2010).
Decroly, E., Ferron, F., Lescar, J. & Canard, B. Conventional and unconventional mechanisms for capping viral mRNA. Nat. Rev. Microbiol. 10, 51–65 (2012).
Zust, R. et al. Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat. Immunol. 12, 137–143 (2011).
Daffis, S. et al. 2′-O-methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452–456 (2010).
Nishio, M. et al. Human parainfluenza virus type 2L protein regions required for interaction with other viral proteins and mRNA capping. J. Virol. 85, 725–732 (2011).
Gopinath, M. & Shaila, M. S. RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus. J. Gen. Virol. 90, 1748–1756 (2009).
Feuillet, F., Lina, B., Rosa-Calatrava, M. & Boivin, G. Ten years of human metapneumovirus research. J. Clin. Virol. 53, 97–105 (2012).
Ogino, T. & Banerjee, A. K. An unconventional pathway of mRNA cap formation by vesiculoviruses. Virus Res. 162, 100–109 (2011).
Ray, D. et al. West Nile virus 5′-cap structure is formed by sequential guanine N-7 and ribose 2′-O methylations by nonstructural protein 5. J. Virol. 80, 8362–8370 (2006).
Decroly, E. et al. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2′-O-methyltransferase nsp10/nsp16 complex. PLoS Pathog. 7, e1002059 (2011).
Hodel, A. E., Gershon, P. D., Shi, X., Wang, S. M. & Quiocho, F. A. Specific protein recognition of an mRNA cap through its alkylated base. Nat. Struct. Biol. 4, 350–354 (1997).
Sutton, G., Grimes, J. M., Stuart, D. I. & Roy, P. Bluetongue virus VP4 is an RNA-capping assembly line. Nat. Struct. Mol. Biol. 14, 449–451 (2007).
Bollati, M. et al. Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus. J. Mol. Biol. 385, 140–152 (2009).
Liang, B. et al. Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy. Cell 162, 314–327 (2015).
Hodel, A. E., Gershon, P. D., Shi, X. & Quiocho, F. A. The 1.85A structure of vaccinia protein VP39: a bifunctional enzyme that participates in the modification of both mRNA ends. Cell 85, 247–256 (1996).
Smietanski, M. et al. Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation. Nat. Commun. 5, 3004 (2014).
Hodel, A. E., Gershon, P. D. & Quiocho, F. A. Structural basis for sequence-nonspecific recognition of 5′-capped mRNA by a cap-modifying enzyme. Mol. Cell 1, 443–447 (1998).
Chu, C. et al. Structure of the guanylyltransferase domain of human mRNA capping enzyme. Proc. Natl Acad. Sci. USA 108, 10104–10108 (2011).
Hakansson, K., Doherty, A. J., Shuman, S. & Wigley, D. B. X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes. Cell 89, 545–553 (1997).
Barik, S. The structure of the 5′ terminal cap of the respiratory syncytial virus mRNA. J. Gen. Virol. 74, (Pt 3): 485–490 (1993).
Ogino, T. & Banerjee, A. K. Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus. Mol. Cell. 25, 85–97 (2007).
Reich, S. et al. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361–366 (2014).
Hengrung, N. et al. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus. Nature doi:10.1038/nature15525.
Gupta, K. C. & Roy, P. Synthesis of capped and uncapped methylated oligonucleotides by the virion transcriptase of spring viremia of carp virus, a rhabdovirus. Proc. Natl Acad. Sci. USA 78, 4758–4762 (1981).
Berrow, N. S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007).
Walter, T. S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D Biol. Crystallogr. 61, 651–657 (2005).
Winter, G., Lobley, C. M. & Prince, S. M. Decision making in xia2. Acta Crystallogr. D Biol. Crystallogr. 69, 1260–1273 (2013).
Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).
DiMaio, F. Advances in Rosetta structure prediction for difficult molecular-replacement problems. Acta Crystallogr. D Biol. Crystallogr. 69, 2202–2208 (2013).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
BUSTER version 2.9. Global Phasing Ltd. (Cambridge, United Kingdom, (2010).
Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Lavergne, T., Bertrand, J.-R., Vasseur, J.-J. & Debart, F. A base-labile group for 2′-OH protection of ribonucleosides: a major challenge for RNA synthesis. Chem. Eur. J. 14, 9135–9138 (2008).
Debart, F., Lavergne, T., Janin, M., Dupouy, C. & Vasseur, J.-J. in Current Protocols in Nucleic Acid Chemistry Vol. 43, eds Beaucage S.et al. 3.19.11–13.19.27John Wiley & Sons (2010).
Zlatev, I. et al. Chemical solid-phase synthesis of 5′-triphosphates of DNA, RNA, and their analogs. Org. Lett. 12, 2190–2193 (2010).
Thillier, Y. et al. Synthesis of 5′-cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyltransferase. RNA 18, 856–868 (2012).
Barral, K. et al. Development of specific Dengue virus 2′-O- and N7-methyltransferase assays for antiviral drug screening. Antiviral Res. 99, 292–300 (2013).
Acknowledgements
The research was made possible by funding from the European Union Seventh Framework Programme (FP7/2007–2013) under SILVER grant agreement no. 260644 and from an MRC grant (MR/L017709/1). We thank Diamond Light Source for beamtime (proposal mx8423), and the staff of the MX beamlines for assistance. This work was supported by a Wellcome Trust administrative support grant 075491/Z/04.
Author information
Authors and Affiliations
Contributions
G.C.P., A.C., E.D. and J.M.G. conceived and designed the study; G.C.P., A.C. and E.D. performed experiments, using material prepared by C.S., F.D. and J.-J.V.; G.C.P., A.C., E.D, J.M.G. and B.C. analysed data and wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Figures 1-8 (PDF 1224 kb)
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Paesen, G., Collet, A., Sallamand, C. et al. X-ray structure and activities of an essential Mononegavirales L-protein domain. Nat Commun 6, 8749 (2015). https://doi.org/10.1038/ncomms9749
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/ncomms9749
- Springer Nature Limited
This article is cited by
-
Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases
Communications Biology (2023)
-
Cryo-EM structure of the respiratory syncytial virus RNA polymerase
Nature Communications (2020)
-
Reversible methylation of m6Am in the 5′ cap controls mRNA stability
Nature (2017)