Structure and Function of Flavivirus NS5 Methyltransferase�
Yangsheng Zhou,1,2† Debashish Ray,1† Yiwei Zhao,1 Hongping Dong,1 Suping Ren,1,2 Zhong Li,1Yi Guo,1 Kristen A. Bernard,1,2 Pei-Yong Shi,1,2* and Hongmin Li1,2*
Wadsworth Center, New York State Department of Health,1 and Department of Biomedical Sciences,School of Public Health, State University of New York,2 Albany, New York 12201
Received 7 December 2006/Accepted 22 January 2007
The plus-strand RNA genome of flavivirus contains a 5� terminal cap 1 structure (m7GpppAmG). Theflaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, tocatalyze both guanine N-7 and ribose 2�-OH methylations during viral cap formation. Representativeflavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generateGpppA 3 m7GpppA 3 m7GpppAm. The 2�-O methylation can be uncoupled from the N-7 methylation,since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinctmethylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a singlebinding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNAshould be repositioned to accept the N-7 and 2�-O methyl groups from SAM during the sequentialreactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to theSAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site duringcap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2�-O cap methylationsrequire distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggestingthat the two reactions use different mechanisms. In the context of complete virus, defects in bothmethylations are lethal to WNV; however, viruses defective solely in 2�-O methylation are attenuated andcan protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylationactivity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target forflavivirus therapy.
Eukaryotic mRNAs possess a 5� cap structure that is co-transcriptionally formed in the nucleus. mRNA capping is es-sential for mRNA stability and efficient translation (13, 39).Most animal viruses that replicate in cytoplasm encode theirown capping machinery to produce capped RNAs. RNA cap-ping generally consists of three steps in which the 5� triphos-phate end of nascent RNA transcript is first hydrolyzed to a 5�diphosphate by an RNA triphosphatase, then capped withGMP by an RNA guanylyltransferase, and finally methylatedat the N-7 position of guanine by an RNA guanine-methyl-transferase (N-7 MTase) (15). Additionally, the first and sec-ond nucleotides of many cellular and viral mRNAs are furthermethylated at the ribose 2�-OH position by a nucleoside 2�-OMTase, to form cap 1 (m7GpppNm) and cap 2 (m7GpppNmNm)structures, respectively (13). Both N-7 and 2�-O MTases useS-adenosyl-L-methionine (SAM) as a methyl donor and gen-erate S-adenosyl-L-homocysteine (SAH) as a by-product. Theorder of capping and methylation varies among cellular andviral RNAs (13).
The genus Flavivirus comprises approximately 70 viruses,many of which are important human pathogens, including fourserotypes of dengue virus (DENV), yellow fever virus (YFV),
St. Louis encephalitis virus, and West Nile virus (WNV) (23).The flavivirus genome is a single-stranded RNA of positive(i.e., mRNA sense) polarity. The 5� end of the genome con-tains a type 1 cap followed by a conserved dinucleotide se-quence 5�-AG-3� (7, 41). The single open reading frame of theflavivirus genome encodes a polyprotein, which is processed byviral and cellular proteases into three structural proteins andseven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A,NS4B, and NS5) (23). Of the four enzymes required for syn-thesis of flavivirus m7GpppAm cap structure, the RNAtriphosphatase and 2�-O MTase have been, respectively,mapped to NS3 (20, 42) and NS5 (9). We recently showed thatWNV NS5 carries both guanine N-7 and ribose 2�-O MTaseactivities (34). The guanylyltransferase for flavivirus cappingremains elusive.
Flavivirus NS5 consists of an N-terminal MTase and a C-terminal RNA-dependent-RNA polymerase (RdRp) domain(1, 16, 28). The structure of DENV-2 MTase suggests thatflavivirus NS5 MTase belongs to a family of SAM-dependentMTases (9). Most of the MTases within this family, includingboth N-7 and 2�-O RNA MTases such as Encephalitozooncuniculi (Ecm1) N-7 MTase and vaccinia virus 2�-O MTaseVP39 (10, 18), share a common core structure referred to as a“SAM-dependent MTase fold,” composed of an open �/�/�sandwich structure (11, 24). Structure and sequence compari-sons of the 2�-O MTases suggest that a conserved K-D-K-Etetrad forms the active site for the 2�-O methyl transfer reac-tion (9). Using Ala substitution, we recently showed that allresidues within the K61-D146-K182-E218 tetrad of the WNVMTase are essential for 2�-O methylation activity, whereas
* Corresponding author. Mailing address: Wadsworth Center, NewYork State Department of Health, 120 New Scotland Ave., Albany,NY 12208. Phone for Pei-Yong Shi: (518) 473-7487. Fax: (518) 473-1326. E-mail: [email protected]. Phone for Hongmin Li: (518) 486-9154. Fax: (518) 408-2190. E-mail: [email protected].
† These authors made equal contributions.� Published ahead of print on 31 January 2007.
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D146 is more critical than the other three residues for N-7methylation. In addition, we found that methylations of gua-nine N-7 and ribose 2�-O of the WNV cap structure are se-quential, with N-7 preceding 2�-O methylation (34). The WNVMTase represents a unique system to study how a single en-zyme catalyzes two distinct cap methylations.
Here we report that, similar to the WNV, MTases fromother flaviviruses also sequentially methylate viral RNA cap atguanine N-7 and ribose 2�-O positions, indicating that it is ageneral mechanism for flaviviruses to encode the NS5 MTasewith dual methylation activities for an efficient synthesis of theviral RNA cap. By contrast, the crystal structure of the WNVMTase in complex with SAH shows only a single SAM-bindingsite. Thus, the 5� cap of flavivirus RNA must evidently berepositioned to accept two methyl groups from SAM duringmethylations. Biochemical and mutagenesis analyses suggestthat the WNV MTase methylates the N-7 and 2�-O positionsusing two distinct mechanisms. In the context of full-lengthWNV, a mutation (D146A) defective in both the N-7 and 2�-Omethylations is lethal to the virus. Mutant viruses inactive for2�-O but not N-7 methylation (K61A, K182A, or E218A) areattenuated in cell culture and in mice and can be used toprotect mice from challenge with wild-type WNV.
MATERIALS AND METHODS
Cloning, expression, and purification of WNV, DENV-1, and YFV MTases.The WNV MTase domain containing the N-terminal 300 amino acids of NS5 wasprepared for crystallization and enzyme assays (34). A QuikChange II XL site-directed mutagenesis kit (Stratagene) was used to engineer mutations into theK61-D146-K182-E218 motif of the WNV MTase. DNA fragments representing theN-terminal 262 and 264 amino acids of DENV-1 and YFV NS5, respectively,were PCR-amplified from a DENV-1 replicon cDNA (33) and a YFV infectiouscDNA clone (3) and cloned into plasmid pET26b(�) (Novagen) at NdeI andXhoI sites. The DENV-1 and YFV MTases containing a C-terminal His6 tagwere expressed and purified through a Ni-nitrilotriacetic acid column followed bya gel filtration 16/60 Superdex column (Amersham). Briefly, Escherichia colistrain Rosetta 2(DE3)pLysS (Novagen) bearing the expression plasmid wasgrown at 37°C to 0.8 absorbance optical density at 600 nm (OD600), induced with0.5-mM isopropyl-�-D-thiogalactopyranoside (IPTG) at 15°C for 12 h, and har-vested by centrifugation. Cell pellets were resuspended and sonicated in a lysisbuffer containing 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, and 5 mM �-mercap-toethanol. After centrifugation, the lysate supernatant was applied to a Ni-nitrilotriacetic acid column, washed with the lysis buffer supplemented with 20mM imidazole, and eluted with lysis buffer containing 300 mM imidazole. Theproteins were then concentrated and subjected to size exclusion chromatographyby a gel filtration 16/60 Superdex column (Amersham). For crystallization, thepurified WNV MTase was concentrated to 5 to 10 mg/ml in a buffer composedof 10 mM HEPES, pH 7.5, 0.5 M NaCl, and 5 mM dithiothreitol (DTT).
Crystallization, X-ray data collection, and structure determination and re-finement. Crystals of the WNV MTase domain were grown at room temperaturein hanging drops, by mixing 2 �l of protein solution with an equal volume ofreservoir solution containing 13 to 15% polyethylene glycol 4000, 5% isopropa-nol, and 0.1 M sodium citrate, pH 5.6. Crystals appeared within a week and grewto maximal size in a month.
The crystals belong to space group P1. Prior to data collection, all crystals weretransferred to a reservoir solution containing 25% glycerol, and then flashed-cooled under a nitrogen stream at 100 K, and stored in liquid nitrogen. Diffrac-tion data were collected at 100 K at a beam line X4A of the national synchrotronlight source (Brookhaven National Laboratory). All of the data were processedand scaled using HKL2000 (30).
With the structure of the DENV MTase (PDB code 1L9K) used as a searchmodel, clear solutions were found using AmoRe (26). Structural refinementwas carried out using Crystallography & NMR System (5). Noncrystallo-graphic symmetry restraints were applied to the main chain atoms throughoutthe refinement. At 2.8-Å resolution, the final Rcryst is 25.8%, with an Rfree of33.3% (Table 1).
Atomic coordinates have been deposited in the Protein Data Bank as entry2OY0.
RNA cap methylation assays. Vaccinia virus capping enzyme (Ambion) wasused to prepare 32P-labeled RNA substrates, G*pppA- and m7G*pppA-RNA(representing the 5�-terminal 190 nucleotides of the WNV genome; an asteriskindicates that the following phosphate is 32P labeled) for the N-7 and 2�-Omethylation assays, respectively. Since the optimal pH values for the N-7 and2�-O methylation are at 7 and 10 (see details in Fig. 3), respectively, the N-7methylation was performed in 50 mM Tris, pH 7.0, 50 mM NaCl, and 2 mM DTTat 22°C for 5 or 30 min as indicated; the 2�-O methylation was incubated in 50mM glycine, pH 10, and 2 mM DTT at 22°C for 1 h. The other components ofthe reaction mixtures were identical to those previously described (34). Timecourse experiments for YFV and DENV-1 MTase (see Fig. 2E) were performedin the 2�-O methylation buffer because this buffer could support both N-7 and2�-O methylations, whereas the N-7 methylation buffer could barely support the2�-O methylation. Control m7G*pppAm-RNA was prepared by incubating thevaccinia virus VP39 protein in a 20-�l reaction mixture (50 mM Tris-HCl pH 8.0,5 mM DTT, 10 �M SAM, 15,000 cpm of m7G*pppA-RNA, and 30 pmol ofrecombinant VP39) for 1 h at 37°C. The methylation reaction mixtures weredigested with nuclease P1 or tobacco acid pyrophosphatase (TAP) and analyzedon polyethyleneimine cellulose thin-layer chromatograph (TLC) plates (JTBaker) (34).
Construction of mutant WNV cDNA. Mutant cDNA plasmids of full-lengthWNV were constructed using a modified infectious cDNA clone pFLWNV (38)and a shuttle vector. The shuttle vector was created by digestion of the pFLWNVplasmid with restriction enzymes KpnI and XbaI, followed by ligation of theresulting 3.2-kb fragment (representing nucleotide position 7762 to the 3� end ofthe genome; GenBank no. AF404756) into the predigested pcDNA3.1 (�) vec-tor. For each K-D-K-E mutant, the specific mutation was first introduced into theshuttle vector using an overlapping PCR-mediated mutagenesis. The mutationsin the shuttle vectors were confirmed by DNA sequencing. The mutated DNAfragments from the shuttle vectors were then swapped into the pFLWNVthrough unique restriction enzymes SpeI and XbaI (representing nucleotide
TABLE 1. X-ray data collection and structure refinement statistics
Parameter Result
Data collectionSpace group ...........................................................................P1Cell dimensions
RefinementResolution limits (Å) ............................................................39.1–2.8No. of reflections...................................................................15,957Rwork (%) ................................................................................25.8Rfree (%) ..................................................................................33.3No. of non-H atoms
Protein ................................................................................4,208Water ..................................................................................256AdoHcy...............................................................................2
Average B (Å2) ......................................................................26.5Geometry
rmsd bond length (Å) .......................................................0.008rmsd bond angle (°)...........................................................1.4
position 8022 to the 3� end of the genome). The mutant pFLWNV was againverified by DNA sequencing.
In vitro transcription, RNA transfection, IFA, and real-time RT-PCR. Ge-nome-length RNAs were in vitro transcribed and transfected into BHK cells(37). After transfection, viral protein synthesis was monitored by immunofluo-rescence assay (IFA) using the WNV-immune mouse ascites fluid (AmericanType Culture Collection) and goat anti-mouse immunoglobulin G conjugatedwith Texas Red as the respective primary and secondary antibodies (38). Forviral RNA quantification, the transfected cells were thoroughly washed withphosphate-buffered saline twice. Total cellular RNA was extracted using RNeasykits (QIAGEN) and quantified with a NanoDrop spectrophotometer. Equalamounts of extracted RNA (100 ng) were measured for viral RNA by real-timereverse transcription (RT)-PCR (7500 RT-PCR system; Applied Biosystems)using a primer/probe set targeting the NS5 gene (36). Full-length RNA tran-scribed from an infectious cDNA clone of WNV (38) was used as a reference forquantification of the real-time RT-PCR.
Specific infectivity assay and virus growth kinetics. For specific infectivityassays, BHK cells were electroporated with wild-type and mutant genome-lengthRNAs. The transfected cells were adjusted to 1.0 � 107 cells per ml of culturemedium. One milliliter each of a series of 1:10 dilutions of the transfected cellswas seeded onto nearly confluent Vero cells (6 � 105 cells per well were seededin a six-well plate 3 days in advance). The seeded cells were allowed to attach tothe plates for 7 to 10 h before the first layer of agar was added (31). A secondlayer containing neutral red was added after incubation of the plates for 2 days.Plaques were counted at 12 to 18 h after addition of the second layer of agar. Thespecific infectivity was calculated as the number of PFU per microgram oftransfected RNA. For viral growth curves, Vero and C6/36 cells grown in 12-wellplates were infected with WNV (0.1 multiplicity of infection [MOI]). After 1 hincubation, the virus inocula were removed. The cells were washed twice withphosphate-buffered saline and cultured with 1 ml of fresh medium per well. Theculture medium was collected at indicated time points, stored at 80°C, and thenassayed for virus titers using standard plaque assays on Vero cells (33).
In vivo virulence analysis in mice. Six- to seven-week-old, female C3H/HeN(C3H) mice (Taconic) were inoculated subcutaneously (s.c.) in the left rearfootpad with a 10-�l inoculum, using a 30-gauge needle and 100-�l glass syringe(Hamilton, Reno, NV). Four mice were inoculated with diluent alone, and eightmice per group were inoculated with 10, 103, or 105 PFU of wild-type, mutantK61A, or mutant K182A WNV. All mice were observed for clinical disease dailyfor the entire study and weighed daily for at least 14 days postinoculation andthen three times weekly for the duration of the study. Clinical signs includedruffled fur, ataxia, weakness, and weight loss. Morbidity was defined as clinicalsigns for at least 2 days and/or greater than 9% weight loss. Mice that exhibitedsevere disease were euthanized. On day 28 postinoculation, surviving mice werebled, and sera were tested for WNV-specific antibodies by enzyme-linked im-munosorbent assay (32). On day 34 after the initial inoculation, surviving micewere challenged by intraperitoneal inoculation with 106 PFU of wild-type WNVin 100 �l, using a 25-gauge needle and 1-ml syringe. Mice were monitored asdescribed above for 28 days postchallenge. All studies were approved by theInstitutional Animal Care and Use Committee of the Wadsworth Center andfollowed criteria established by the National Institutes of Health. Survival curveswere analyzed with a Logrank test using GraphPad Prism (GraphPad Software,Inc.). A chi-squared test (Microsoft Office Excel) was used to compare morbiditydata.
RESULTS
Crystal structure of the WNV MTase. We recently showedthat the WNV MTase carries both guanine N-7 and ribose2�-O MTase activities (34). However, no N-7 MTase activitywas detected for the DENV-2 MTase in a previous study (9).To exclude the possibility that the discrepancy results fromstructural differences between the two flavivirus MTases, wehave crystallized the WNV NS5 MTase domain (amino acids[aa] 1 to 300). The crystal structure was determined to 2.8-Åresolution using the molecular replacement method (Fig. 1Aand Table 1). The electron density maps were of good quality(Fig. 1B). The WNV NS5 MTase forms homodimers in thecrystals. In the final model, each chain of the homodimercontains 262 residues (aa 6 to 267) and one SAH molecule.
The SAH molecule could have originated from E. coli andcopurified with the WNV MTase, since no SAH was addedduring any step of the protein purification or crystallization.Similar observations have been reported for many SAM-de-pendent MTases, including the DENV-2 MTase (9). In addi-tion to the SAH, 256 water molecules were included in the finalmodel. No density was observed for the N-terminal residues 1to 5, the C-terminal residues 268 to 300, and the His tag (fusedto the N terminus of the MTase), presumably due to disorder.Similar disorder has also been found in the crystal structure ofthe DENV-2 MTase, in which the C-terminal 29 residues weremissing from the structure (9).
Structure comparison of the WNV and DENV-2 MTases.The core of the WNV MTase domain displays a structural foldshared by nearly all SAM-dependent MTases (Fig. 1A). TheWNV MTase contains 8 �-helices and 12 �-strands that areorganized into three domains: the N-terminal domain, the coredomain, and the C-terminal domain. The overall structure ofthe WNV MTase is similar to that of the DENV-2 MTase (9),with a root-mean-squared deviation (rmsd) of 0.62 Å (Fig. 1C).In addition, SAH, the by-product of the methyl transfer reac-tion, binds into similar pockets of the two enzymes, indicatingthat the two enzymes have similar SAM-binding sites (Fig. 1Cto E).
Between the two structures, four regions, residues 36 to 51,107 to 110, 173 to 177, and 245 to 252, showed the greateststructural differences, with rmsd values of 1.8 Å, 1.2 Å, 2.1 Å,and 1.9 Å, respectively (Fig. 1C). Three of the four structuredifferences are, at least in part, due to a one-residue insertionat each of positions 51, 173, and 251 for the WNV MTase,compared to the DENV-2 MTase (see sequence alignment inFig. 2A). In contrast, all residues are identical at loop 107 to110 in the two proteins. Loop 173 to 177 is located at thesurface opposite to that of the SAM-binding site, while loop245 to 252 is far from the SAM donor site and is structurallyhindered from access to the SAM site by the helix �2 (Fig. 1C).Therefore, the structural differences in these two regions maynot be of significance to the biological functions of the MTases.
In contrast to the loops formed by aa 173 to 177 and 245 to252, residues 36 to 51 form the helix �3 and a short loop (Fig.1C). These residues do not contact the SAH. However, theycould participate in binding of the RNA substrate, since theyare at the edge of the potential RNA binding pocket (seebelow). Loop 107 to 110 is lined at one side of the adenosinebase of the bound SAH. As a consequence, the ND1 atom ofHis-110 of the WNV MTase forms a hydrogen bond with theribose 2�-OH of the SAH molecule (Fig. 1B), whereas theHis-110 in the DENV-2 MTase structure does not. Interest-ingly, although the SAH-contacting residues are essentiallyidentical in the WNV and DENV-2 MTases, the SAH binds tothe two enzymes in slightly different conformations. Indeed,with only a 0.2-Å difference at the C� positions of the SAHmolecules in the two structures,the rmsd for the adenosinebases in the two structures is about 0.83 Å. The adenosine baseof the SAH molecule in the WNV MTase structure does notbind as deeply as does that in the DENV-2 structure. Never-theless, structure differences at the SAH-binding site lead todifferent surface appearance for the two proteins. We notedthat the WNV MTase has an enclosed SAM-binding site,whereas the SAM-binding site in the DENV-2 MTase is much
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FIG. 1. Crystal structure of the WNV MTase and comparison with the DENV-2 MTase. (A) Ribbon representation of the crystal structure of theWNV MTase. The MTase structure is colored as follows: N-terminal domain, red; MTase core, green; C-terminal domain, cyan. The bound SAH isshown in ball-and-stick representation with atom colors as follows: carbon, yellow; oxygen, red; nitrogen, blue; sulfur, green. (B) A representative omit(Fo-Fc) electron density map (magenta) showing the bound SAH and its interactions with the MTase residues. Hydrogen bonds are shown as orangedashed lines. (C) Superposition of the crystal structures of the DENV-2 (2) (pink) and WNV (cyan and red) MTases. Ribavirin (occupying the putativeGTP cap-binding site for the 2�-O methylation) (see Fig. 8) and SAH are shown in ball-and-stick representation. The loops of the WNV structure thatshow significant differences relative to the DENV-2 structure are colored in red. (D and E) Solvent-accessible molecular GRASP (27) surfacerepresentation of the electrostatic potential of the WNV (D) and DENV-2 (E) MTases, showing the putative RNA substrate binding site. The surfaceis colored blue for positive (15 kT), red for negative (15 kT) and white for neutral, where k is the Boltzmann constant and T is the temperature (27).In panels D and E, ribavirin and SAH are in stick representation with atom colors as follows: oxygen, red; nitrogen, blue; carbon, white; sulfur, green.
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more open (Fig. 1D and E). The structure differences at thisregion between the two MTases are, at least in part, due to theconformation differences of residues H110 and E149 that arelined at opposite sites along the bound SAH. Alternatively,
conformational differences at loop 107 to 110 may contributeas well. The open and closed configurations in the two MTasestructures could represent two distinct states for SAM binding,with the closed state to ensure a tight SAM binding, whereas
FIG. 2. Flavivirus NS5 sequentially methylates guanine N-7 and ribose 2�-OH of the viral RNA cap. (A) Sequence alignment of flavivirusMTases. The MTase sequences of WNV, DENV-1, DENV-2, and YFV are derived from GenBank accession numbers AF404756, DVU88535,U87411, and YFU17-66, respectively. The alignment was performed using GCG software (Genetics Computer Group). Identical amino acidsamong all MTases are shaded. The conserved K61-D146-K182-E218 residues mutated in this study are indicated by an asterisk. The exact sequencesof the recombinant DENV-1 and YFV MTases (not including the C-terminal His tag) are shown. (B) Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis analysis of recombinant MTase proteins of YFV, DENV-1, and WNV. (C) Guanine N-7 methylation activity. Substrate G*pppA-RNA was methylated with the indicated MTases or no enzyme (Mock) in the presence of cold SAM in the N-7 assay buffer for 30 min. The reactionmixtures were then digested by TAP and analyzed on a TLC plate followed by autoradiography. (D) Ribose 2�-O methylation activity. m7G*pppA-RNA was methylated by the indicated MTases and digested by nuclease P1. The nuclease P1-resistant cap structures were then analyzed on a TLCplate. (E) Time course analyses of the DENV-1 and YFV MTase activities. 32P-labeled G*pppA-RNA was methylated by DENV-1 (left panel)and YFV MTase (right panel) in the 2�-O methylation buffer for the indicated times, digested with nuclease P1, and analyzed by TLC andautoradiography. The 2�-O methylation buffer is optimal for 2�-O methylation and also supports N-7 methylation. The positions of the origin andthe migrations of G*p, m7G*p, G*pppA, m7G*pppA, and m7G*pppAm molecules are indicated on the left.
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the open state facilitates the release of SAH, the reactionby-product.
The DENV-2 MTase structure contains a novel GTP-bind-ing site (occupied by ribavirin in Fig. 1C and E), which wassuggested to be a cap-binding site (9). Surface comparisonshowed that the putative cap recognition site in WNV is muchmore open than that in DENV-2 (Fig. 1D and E). Despite thestructural differences, a common feature of the two enzymes isa highly positively charged surface adjacent to the SAM andGTP binding sites but not in other regions (Fig. 1D and E).This positively changed region is likely the site for binding ofthe capped RNA substrates. Structure superposition of theVP39 2�-O and Ecm1 N-7 MTases in complex with their RNAsubstrates (10, 18) onto the WNV MTase indicated that theRNA substrates are lined along with the positively chargedregion (data not shown).
Sequential methylations at the N-7 and 2�-O positions of theRNA cap by the DENV-1 and YFV MTases. To demonstratethat NS5 from other flaviviruses also possesses N-7 and 2�-OMTase activities, we prepared recombinant MTase domains ofYFV and DENV-1 (Fig. 2A and 2B). For detection of N-7methylation, we incubated capped RNA substrate, G*pppA-RNA (the asterisk indicates that the following phosphate is 32Plabeled), representing the 5�-terminal 190 nucleotides of theWNV genome, with each of the YFV, DENV-1, and WNVMTases in the presence of SAM. The reaction products weredigested by TAP to release m7G*p (product) and G*p (sub-strate). TLC analysis of the reactions showed that the WNV,DENV-1, and YFV MTases could convert G*pppA-RNA tom7G*pppA-RNA (Fig. 2C). For detection of 2�-O methyl-ation, substrate m7G*pppA-RNA was methylated by the re-combinant flavivirus MTases and cleaved by nuclease P1 torelease m7G*pppAm (product) and m7G*pppA (substrate).The results showed that, similar to VP39 (a known guanineN-7-dependent 2�-O MTase as a positive control) (18), YFV,DENV-1, and WNV MTases could methylate m7G*pppA-RNA to m7G*pppAm-RNA (Fig. 2D).
Kinetic analyses were performed on substrate G*pppA-RNA to examine the order of cap methylations mediated bythe DENV-1 (Fig. 2E, left panel) and YFV (Fig. 2E, rightpanel) MTases. For both enzymes, m7G*pppA was first de-tected at 1 min, reached a maximum at 5 to 15 min, andgradually declined at later time points. Concurrently, the dou-ble-methylated m7G*pppAm was first detected at 15 min andincreased until 60 min. The kinetics of the methylation pattern
derived from the DENV-1 and YFV MTases was similar tothat derived from the WNV MTase (34). These results suggestthat flavivirus NS5 functions similarly to methylate both N-7 and2�-O positions of the cap structure, in the order of GpppA 3m7GpppA3 m7GpppAm.
Distinct conditions required for flavivirus N-7 and 2�-OMTase activities. Using the WNV MTase as a model, wedetermined the optimal conditions for the N-7 and 2�-O capmethylations (Fig. 3). Both activities reached maximums whenperformed at 22°C, whereas the optimal pH and concentra-tions of MgCl2 and NaCl differed. The N-7 methylation re-quired neutral pH 7.0 and 50 to 100 mM NaCl; MgCl2 inhib-ited the activity. In contrast, the 2�-O methylation requiredhigh pH 10 and 5 to 10 mM MgCl2; NaCl inhibited the activity.Similar results were obtained for the DENV-1 and YFVMTase (data not shown). These data demonstrate that optimalN-7 and 2�-O methylations of flavivirus cap require distinctbiochemical conditions.
Differential roles of the K-D-K-E motif in N-7 and 2�-OMTase activities. A K-D-K-E motif conserved among various2�-O MTases was suggested to catalyze an SN2-reaction-mediated 2�-O methyl transfer (17, 18). Structural alignmentof the WNV MTase with the VP39 tertiary complex (MTase,short RNA with cap, and SAH) (18) showed that the K-D-K-E tetrad is nearly superimposable between the two en-zymes (Fig. 4A). We mutated the K61-D146-K182-E218 motifof the WNV MTase to determine the elements required forthe N-7 and 2�-O methylations. Each amino acid was mu-tated to a residue having an electronic charge either similarto or different from that of the wild type. Analysis of themutant MTases showed that all substitution within the K61-D146-K182-E218 motif abolished the 2�-O methylation (Fig.4B). An increase in enzyme amount or incubation time didnot improve the 2�-O activity (data not shown). In contrast,mutations of the tetrad residues exhibited different effectson N-7 methylation (Fig. 4C). In 5-min reactions, mutationsof D146 showed the most dramatic effects on N-7 activity: allthree mutants (D146A, D146N, and D146K) were inert in N-7methylation (Fig. 4C). Mutations of K182 had less severeeffects on N-7 methylation: K182E, K182N, and K182Ashowed 67%, 57%, and 42% of the wild-type activity level,respectively. For residue K61, no N-7 methylation was de-tected for mutant K61E, whereas the N-7 activities of mu-tants K61R and K61A were reduced to 87% and 70% of thewild-type, respectively. For amino acid E218, mutants E218K,
FIG. 3. Optimization of conditions for the WNV N-7 and 2�-O cap methylations. Reactions for N-7 (F) and 2�-O methylation (E) containedsubstrate G*pppA-RNA and m7G*pppA-RNA and were incubated for 5 and 60 min, respectively. The optimal conditions were determined byindividually titrating pH (A), temperature (B), MgCl2 (C), and NaCl (D), while keeping the other three parameters constant at the optimal levels.The reaction mixtures were then treated with nuclease P1, analyzed on TLC plates, and quantified by PhosphorImager analysis. For eachparameter, relative activities were presented using the optimal level as 100%. Average results from two independent experiments are shown.
3896 ZHOU ET AL. J. VIROL.
E218D, and E218A contained 5%, 55%, and 76% of thewild-type N-7 activity, respectively. For mutants that weredefective in N-7 methylation, a longer incubation time, up to30 min, resulted in 4% and 22% of wild-type activity for
K61E and D146A, respectively, but not for D146N and D146K(Fig. 4C, bottom panel). Overall, the results demonstratethat the exact K-D-K-E motif is required for the 2�-O meth-ylation, but not for the N-7 MTase activity.
FIG. 4. Methylation activities of the WNV K-D-K-E mutant MTases. (A) Superposition of the active site residues K-D-K-E of the VP39 andWNV MTases. SAH and K-D-K-E residues of the WNV MTase are in ball-and-stick representation with carbon colored yellow. The carbon atomsof Gppp-RNA and K-D-K-E residues from VP39 are colored in pink. Colors for other atoms are as follows: phosphate, cyan; sulfur, green; oxygen,red; nitrogen, blue. (B and C) Mutant MTases (1 �g) containing the indicated substitutions were assayed for 2�-O (B) and N-7 (C) methylationactivities. The experimental details are described in Materials and Methods. 32P-labeled markers, m7G*pppA and G*pppA, are indicated on top(B). The relative conversions for 2�-O methylation (m7G*pppA to m7G*pppAm in panel B) and for N-7 methylation (G*pppA to m7G*pppA in panelC) were calculated by comparing the products generated from the mutant MTases with that produced from the wild-type protein (set at 100%). For N-7methylation (C), the reaction mixtures were incubated for 5 min (top panel) or 30 min (TLC data not shown); the relative activities between the mutantsand wild type for both incubation times are summarized (bottom panel). Average results from two to three experiments are shown.
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RNA replication, protein synthesis, and virus production in cellstransfected with genome-length RNAs with K-D-K-E mutations. Toexamine the biological relevance of the above mutagenesisresults, we performed Ala substitutions of the MTase K61-D146-K182-E218 tetrad in an infectious cDNA clone of WNV(38). A number of parameters were used to compare the rep-lication efficiency between the wild-type and mutant RNAs.Initially, we measured the specific infectivity of the genome-length RNAs. After transfection into BHK cells (day 3 to 4posttransfection [p.t.]), mutant K61A, K182A, E218A, and wild-type RNAs showed specific infectivity values of 3.4 � 103,1.3 � 103, 6.1 � 103, and 5.9 � 104 PFU per microgram oftransfected RNA, respectively (Fig. 5A). The specific infec-tivity values for mutant K61A, K182A, and E218A RNA were17-, 45-, and 10-fold lower than that of the wild-type RNA,respectively. No PFU was detected in the specific infectivityassay for mutant D146A RNA.
RNA and protein syntheses, as well as virion production,were compared for cells transfected with the wild-type andmutant RNAs. After transfection of BHK cells with equalamounts of RNA, similar levels of viral RNA were detected upto 18 h p.t. using real-time RT-PCR. From 26 to 40 h p.t., theamounts of wild-type and mutant K61A and E218A RNAs in-creased substantially and the level of mutant K182A RNA re-mained low, whereas the level of D146 RNA decreased dra-matically (Fig. 5B). For viral protein synthesis, IFAs showedthat IFA-positive cells transfected with the wild-type RNA
appeared earlier than did those transfected with the mutantRNAs, with the following order of appearance: wild-type,E218A, K61A, and K182A (Fig. 5C). No IFA-positive cells weredetected in cells transfected with the D146 RNA up to 72 h p.t.,the latest time point tested (Fig. 5C). Concurrently, cells trans-fected with wild-type and mutant RNAs began to secrete vi-ruses at different time points p.t.: 13 h for wild type and E218A(no virus was detected at 8 h p.t.), 18 h for K61A, and 26 h forK182A (Fig. 5D). No infectious virus was detected from theD146A RNA-transfected cells, up to 40 h p.t. (Fig. 5D). Theresults demonstrate that full-length RNAs containing the K61-D146-K182-E218 mutations are attenuated or defective in pro-duction of infectious viruses.
Characterization of K-D-K-E mutant WNV in cell culture.Mutant viruses recovered from the transfected cells exhibitedplaque sizes smaller than that of the wild-type WNV (Fig. 6A,upper panel). The relative plaque size among the wild-type andmutant viruses correlated well with the reduction in specificinfectivity (compare Fig. 6A and 5A). Continuous culturing ofthe D146A RNA-transfected cells began to produce detectablevirus on day 8 p.t.; the recovered viruses displayed a plaque sizesimilar to that of the wild-type virus (Fig. 6A). Sequencing ofthe recovered viruses showed a reversion of the mutated D146Ato the wild-type D residue, whereas mutant K61A, K182A, andE218A viruses retained the engineered changes (Fig. 6A, lowerpanels) and the small-plaque morphology, even after four pas-sages of the viruses in Vero cells for over 12 days (data not
FIG. 5. Comparison of specific infectivity, RNA replication, protein synthesis, and virus production in cells transfected with genome-length RNA containing K-D-K-E mutations. Equal amounts of wild-type and mutant genome-length RNAs were transfected into BHK cellsand assayed for specific infectivity (A). At indicated time points, viral RNA replication, protein expression, and virus production weremonitored by real-time RT-PCR (B), IFA (C), and plaque assay (D), respectively. For specific infectivity, an average result of fourindependent experiments is shown (A). One representative result of two independent experiments is shown for real-time RT-PCR (B), IFA(C), and viral yield production (D). Viral RNA synthesis (B) is presented as RNA copy number per 100 ng of total cellular RNA, usingfull-length WNV RNA transcribed from an infectious cDNA clone (38) as a reference. Virus production (D) in the supernatants oftransfected cells was quantified by plaque assays on Vero cells.
3898 ZHOU ET AL. J. VIROL.
shown). The results demonstrate that K61A, K182A, and E218Amutant viruses are stable, whereas the virus recovered from theD146A-transfected cells contained a reversion of the mutationto the wild-type sequence.
Growth kinetics of the wild-type and mutant viruses werecompared in mammalian Vero and mosquito C6/36 cells. Afterinfection of Vero cells at an MOI of 0.1, mutant viruses gen-erated lower viral titers than did the wild-type virus during theinitial 36 h postinfection (p.i.) (Fig. 6B). At 48 h p.i. and later,titers of the mutant viruses were similar to or slightly higherthan that of the wild-type virus. In C3/36 cells, mutant virusesproduced lower viral titers than did the wild-type virus at alltested time points (Fig. 6C). Since the D146A RNA-derivedvirus had reverted to the wild type, the growth curve of therevertant virus has not been included in Fig. 6B and 6C. Over-all, the results demonstrate that within the K-D-K-E motif,residues K61, K182, and E218 are important, but not essential,for the WNV reproduction, whereas residue D146 is essentialfor the viral life cycle in cell culture.
A D146E mutation of the K-D-K-E motif supports the WNVreplication and the N-7 methylation. The essential role of D146
of the WNV MTase in the viral life cycle prompted us to askwhether viral isolates containing an amino acid other than D atposition 146 of the MTase could be selected in cell culture. Wereasoned that the quick reversion of the mutated D146A to itswild-type D146 described above was due to a single nucleotidechange from GAC (Asp) to GCC (mutated nucleotide under-lined; Ala). To minimize such reversion, we prepared anothergenome-length RNA, D146A2, which contained a two-nucle-otide change from GAC (Asp) to GCA (Ala). Transfection ofthe two-nucleotide mutant D146A2 RNA into BHK cellsyielded viruses. Viruses collected on day 5 p.t. showed homog-
enous small plaques (Fig. 7A, upper panel). On day 9 p.t., theviral population exhibited a mixed-plaque phenotype: largeplaques, similar to the plaques of wild-type virus, emergedfrom the background of small plaques. Sequencing of the vi-ruses showed that the engineered GCA (Ala) had beenchanged to GAA (Glu) in the small-plaque virus, and thewild-type GAC (Asp) was recovered in the large-plaque virus(Fig. 7A). No other mutations were found in the complete NS5coding sequence (data not shown). These results showed astepwise reversion of A3E3D at position 146 of the NS5gene after the D146A2 RNA was transfected into cells.
The stepwise reversion of MTase A1463E1463D146 in virusindicated that amino acid E could functionally substitute forthe essential D of the K-D-K-E motif. To test this hypothesis,we prepared recombinant D146E MTase (Fig. 7B) and assayedfor the N-7 (Fig. 7C) and 2�-O methylation activities (Fig. 7D).Compared with the wild-type enzyme, the D146E mutant re-tained 33% of the N-7 activity (in a 5-min reaction) but com-pletely lost the 2�-O methylation activity. The results suggestthat amino acid E could replace D146 to maintain the N-7MTase activity and, consequently, support virus replication.
The K-D-K-E mutant viruses were attenuated and can pro-tect mice from later challenge with wild-type WNV. We com-pared the virulence of the wild-type and MTase mutant WNVin an adult C3H mouse model, by inoculating 10, 103, or 105
PFU s.c. We chose K61A and K182A mutant viruses in themouse study because K61A and E218A mutant viruses repli-cated to a similar level, whereas the replication level of K182Amutant virus was more attenuated in cell culture (Fig. 5). Table2 shows the morbidity, mortality, average survival time, andinfection rate. For the mutant viruses, no mortality was ob-served at any dose; morbidity was only observed for mice
FIG. 6. Plaque morphology and growth kinetics of the wild-type and K-D-K-E mutant viruses. (A) Plaque morphologies of wild-type andmutant viruses on Vero cells. A single- or double-nucleotide change was engineered into the genome-length RNA to introduce an Ala substitutionwithin the K-D-K-E motif. Wild-type and K61A (AAA to GCA; mutated nucleotide underlined), K182A (AAG to GCG), and E218A (GAG toGCG) mutant viruses were harvested on day 5 p.t. “Mutant” D146A (GAC to GCC) virus was recovered starting on day 8 p.t. Plaques weredeveloped at 96 h p.i. Sequences of the mutated regions, derived from the harvested viruses, are presented below the plaque assay plates. (B andC) Growth kinetics of the wild-type, K61A, K182A, and E218A viruses were compared in Vero cells (B) and C6/36 cells (C) by infecting cells withan MOI of 0.1, followed by quantification of viral titers using plaque assay on Vero cells.
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inoculated with mutant K61A WNV at 105 PFU (38%). Incontrast, 88 to 100% morbidity and 50 to 100% mortality wereobserved for mice inoculated with the wild-type WNV. Thedifferences in survival of mice inoculated with the mutant andwild-type viruses were statistically significant (survival analysis,P value 0.05). All mice that were inoculated with wild-typeor mutant viruses at 103 or 105 PFU were productively infectedwith WNV, as demonstrated by seroconversion. At the low
dose of 10 PFU, the infection rate was similar for both wild-type and K182A mutant viruses (63 to 75%), but it was de-creased for the K61A mutant (13%), suggesting that the K61Amutation is more detrimental to replication in vivo.
Surviving mice were challenged with 106 PFU of the wild-type WNV intraperitoneally. Remarkably, all mice that wereseropositive after the initial inoculation exhibited no morbid-ity. In contrast, the control group (mice that were previouslyinoculated with diluent only) showed 100% morbidity and 50%mortality after challenge. Statistical analysis revealed that micethat were inoculated initially with 103 or 105 PFU of virus weresignificantly protected from the WNV-associated disease com-pared to the control group (chi-squared test, P value 0.005).These results demonstrate that the K61A and K182A viruses areattenuated and that both mutant viruses protect mice fromsubsequent challenge with the wild-type WNV.
DISCUSSION
We previously found that the NS5 protein of the flavivirusWNV carries both guanine N-7 and ribose 2�-O MTase activ-ities (34). To investigate whether the N-7 and 2�-O activitiesare unique to the WNV NS5, we extended our studies toadditional representative flaviviruses. We have demonstratedthat the MTases of other representative flaviviruses, DENV-1and YFV, similar to that of WNV, sequentially catalyze theguanine N-7 and ribose 2�-O methylations involved in forma-tion of the viral RNA cap (Fig. 2). Therefore, it is highly likely
FIG. 7. A D146E mutation of the K-D-K-E motif could support the WNV replication and the N-7 MTase activity. (A) A genome-length RNAcontaining a double-nucleotide mutation D146A2 (GAC to GCA) was transfected into BHK cells. Viruses in culture fluids were harvested on day5 and 9 p.t. and assayed for plaque morphologies on Vero cells. The large plaques derived from day 9 p.t. were amplified and are shown. For eachvirus, the sequences of the mutated NS5 regions were presented below the plaque morphology. (B) Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis analysis of the D146E mutant. (C and D) The D146E MTase was assayed for the N-7 (C) and 2�-O methylation activities (D) usingsubstrate G*pppA-RNA and m7G*pppA-RNA, respectively. The relative activities between the wild-type (set as 100%) and mutant D146E arepresented below the TLC images in panels C and D. The N-7 reactions in panel C were incubated in N-7 methylation buffer for 5 min. The positionsof the origin and the G*pppA, m7G*pppA, and m7G*pppAm molecules are indicated on the left of the TLC plates.
TABLE 2. K-D-K-E mutant viruses are attenuated in micecompared to wild-type WNVa
Inoculum Dose(PFU)
Morbidity(no. sick/
total)
Mortality(no. dead/
total)
AST(SD)b
Infectionrate (no.infected/
total)c
Diluent NA 0/4 0/4 NA 0/4WNV NS5 K61A 10 0/8 0/8 NA 1/8WNV NS5 K61A 103 0/8 0/8 NA 8/8WNV NS5 K61A 105 3/8 0/8 NA 8/8WNV NS5 K182A 10 0/8 0/8 NA 6/8WNV NS5 K182A 103 0/8 0/8 NA 8/8WNV NS5 K182A 105 0/8 0/8 NA 8/8WNV wild type 10 7/8 5/8 10.6 (1.9) 5/8WNV wild type 103 8/8 4/8 10.3 (1.3) 8/8WNV wild type 105 8/8 7/8 9.0 (1.2) 8/8
a Six-week old C3H female mice were inoculated s.c. in the left rear footpadand monitored for weight loss and clinical signs. NA, not applicable.
b AST, average survival time (in days) calculated for mice that died only.c Surviving mice were bled on day 28 postinoculation and tested for WNV-
specific antibody by enzyme-linked immunosorbent assay. Positive WNV infec-tion was defined as either seropositivity or death consistent with WNV disease.
3900 ZHOU ET AL. J. VIROL.
that flaviviruses encode the NS5 MTase as a general mecha-nism for dual methylations of the viral RNA cap.
The MTase domain from DENV-2 was previously reportedto have only 2�-O, but not N-7, methylation activity (9). Thediscrepancy was due to the use of different RNA substrates inthe two studies; a nonviral GpppA(C)5 substrate was used inthe DENV-2 study. We recently found that distinct RNA ele-ments are required for flavivirus RNA cap methylation events(7a); the cross activities of cap methylation between DENV-1and YFV MTases and WNV RNA could result from the con-served 5� terminal nucleotides and the RNA structure sharedby the flaviviruses (4). The physical linkage of the MTase to theRdRp domain in a single NS5 protein, together with the se-lective recognition between the MTase domain and the 5�terminus of viral RNA, could confer specificity for viral capmethylation during flavivirus RNA synthesis. Similarly, the Lprotein from viruses in the order Mononegavirales also containsfunctionally discrete cap-MTase and RdRp domains (8, 14,29); capping of cellular mRNAs is achieved through directbinding of the cellular capping apparatus to the RNA poly-merase II elongation complex (6, 25, 43).
Our biochemical analysis suggests that flavivirus MTasemethylates the RNA cap at both the N-7 and 2�-O positions, inthe order GpppA3 m7GpppA3 m7GpppAm (Fig. 2E). Thechemistry of the ribose 2�-O methylation is distinct from that ofthe guanine N-7 methylation (10, 18). For N-7 methylation, thestructure of Ecm1 N-7 MTase suggests an in-line mechanism:no direct contact is observed between the enzyme and eitherthe attacking nucleophile N-7 atom of guanine, the methylcarbon of SAM, or the leaving group sulfur of SAH. Instead,the catalysis is achieved through the close proximity and ge-ometry of the two substrates (10). In contrast, for 2�-O meth-ylation, structural and mutagenesis studies of VP39 and RrmJ2�-O MTase suggest an SN2 reaction of methyl transfer: theconserved K-D-K-E tetrad mediates deprotonation of the tar-get 2�-OH, which nucleophilically attacks the methyl moiety ofSAM to accomplish the methyl transfer (17, 18). In this study,we show that flavivirus WNV and DENV-2 MTases exhibit asimilar structure, with a core fold shared by many SAM-de-pendent MTases (11). Although flavivirus MTase catalyzes twodistinct methylation reactions, only a single SAH-bindingpocket was found in the MTase structure (Fig. 1). In addition,one clustered and positively charged surface, which presum-ably accounts for the binding of the negatively charged phos-phate backbone of the RNA substrates, was found to be adja-cent to the single SAM-binding site (Fig. 1). These resultssuggest that SAM, located in the same binding pocket forSAH, donates methyl groups to both the N-7 and 2�-O posi-tions during flavivirus RNA cap methylations. However, sincethe two reactions have different methyl acceptors that arelocated at different positions, the 5� terminus of RNA mustbe repositioned during the two methylations. In line with theflavivirus MTases, the N-7 and 2�-O MTases of vesicular sto-matitis virus were recently suggested to share a single SAM-binding site (22).
We hypothesize that the flavivirus MTases bind the RNAsubstrates in different positions to carry out two distinct methyltransfer reactions. During flavivirus cap methylation, the gua-nine N-7 would first be positioned in proximity to the methylgroup of SAM, to generate m7GpppA-RNA (Fig. 8). Structure
superposition of the WNV and DENV-2 MTases onto theEcm1 N-7 MTase-substrate complex (10) did not show anysteric hindrance between the guanosine cap analogue and theWNV and DENV-2 MTases (data not shown), providing struc-tural feasibility. Our mutagenesis results indicate that K61,D146, and E218, but not K182, within the conserved K-D-K-Etetrad likely contribute to the binding of the cap structure, tomodulate the N-7 methylation (Fig. 4C).
Once the guanine N-7 has been methylated, the 5� terminusof m7GpppA-RNA is repositioned. The m7Gppp moietymoves into a GTP-binding pocket (Fig. 8), as previously de-scribed for the DENV-2 MTase (9). Residues involved in bind-ing of the m7Gppp moiety are superimposable between theDENV-2 and WNV MTase structures: the guanine base stacksagainst the aromatic ring of F24, the ribose 2�-OH forms hy-drogen bonds with the side chains of K13 and N17, the ribose3�-OH interacts with K13, and the �–phosphate forms hydro-gen bonds with R28 and S150. The binding of the m7Gpppmoiety precisely repositions the ribose 2�-OH of the first tran-scribed adenosine in close proximity to SAM. Our mutagenesisexperiments showed that any alteration to the K61-D146-K182-E218 tetrad completely abolishes 2�-O methylation (Fig. 4B),suggesting that the tetrad forms the active site of 2�-O MTasethrough a SN2 methyl transfer mechanism. The latter conclu-sion is further supported by the observation that the optimalpH for 2�-O methylation is at pH 10 (Fig. 3A). Structuralalignment with the VP39 2�-O MTase suggests that K182 withinthe WNV tetrad directly participates in the deprotonation ofribose 2�-OH (Fig. 4A). Since the pKa of Lys is at pH 10, a highpH would facilitate the deprotonation of K182, which would inturn favor the deprotonation of the 2�-OH, leading to efficient
FIG. 8. Model for the sequential guanine N-7 and ribose 2�-OHmethylations of the RNA cap by flavivirus MTase. The SAM- andm7G-binding sites are shaded and depicted by an oval and a rect-angle, respectively. The 5�-terminal nucleotides of the WNV genome,GpppAGUA-RNA, are shown.
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formation of the SN2-like transition state so as to accomplishthe methyl transfer (17–19). The requirement of high pH formethylation has also been reported for DIM-5 (a histone H3lysine 9 MTase) (44).
What is the biological relevance of the optimal pH of 10 for2�-O methylation? It is not unreasonable to speculate that,during flavivirus infection, the replication complexes associ-ated with the endoplasmic reticulum could lower the local pKaof the Lys residues within the K-D-K-E motif, allowing 2�-Omethylation to occur at a physiological pH. The difference inoptimal pH and concentrations of NaCl and MgCl2 for N-7 and2-O methylation (Fig. 3) also supports the existence of distinctmechanisms for the two MTase activities. Finally, the 5� captranslocation model (Fig. 8) is supported by our recent foot-printing results: the 5� termini of RNA substrates interact withNS5 and may undergo conformational change during the se-quential methylation reactions (7a).
The processivity of the two methylation events is confrontedwith a single SAM-binding site on the flavivirus MTase. Oncethe guanine N-7 is methylated, the SAH needs to be replacedwith a fresh SAM before the ribose 2�-O methylation canoccur. Alternatively, the m7GpppA-RNA needs to disassociatefrom the SAH-bound MTase and to reassociate with an SAM-bound enzyme for the 2�-O methylation. The latter scenario isreminiscent of the reovirus �2 protein-mediated RNA capping,in which the N-7 and 2�-O methylations are sequentially exe-cuted by two separate MTase domains (35). In support of (butnot distinguishing between) the above two possibilities, wefound that an RNA substrate with preexisting N-7 methylation,m7GpppA-RNA, could readily be methylated to m7GpppAm-RNA (Fig. 2D). The results demonstrate that flavivirus 2�-Omethylation can be uncoupled from the N-7 methylation invitro. However, our current results do not prove that the 2�-Omethylation is dependent on the prior N-7 methylation.
A panel of mutant WNV was prepared for examination ofthe requirement of MTase activities for flavivirus life cycle.Upon transfection into susceptible cells, genome-length RNAscontaining Ala substitutions within the K-D-K-E motif showeddecreases in levels of specific infectivity, viral protein synthesis,RNA replication, and virion production compared to the wild-type RNA (Fig. 5). In addition, the mutant viruses displayedsmaller plaque morphology and slower growth kinetics inmammalian Vero and mosquito C6/36 cells (Fig. 6). Only mu-tant viruses containing the K61A, K182A, and E218A substitu-tions, which abolished the 2�-O but not the N-7 MTase activity,could be recovered from the RNA-transfected cells (Fig. 6).These results demonstrate that 2�-O MTase activity is impor-tant, but not essential, for the WNV reproduction. Consistentwith the cell culture results, we showed that the K61A andK182A viruses were attenuated in vivo and can be used toprotect mice from later challenge with wild-type WNV (Table2). These findings underscore the feasibility of using MTase asa target for flavivirus vaccine development.
In contrast to mutations that affected 2�-O MTase, cellstransfected with an RNA containing the D146A substitution,which abolished both the N-7 and 2�-O MTase activities (Fig.4), did not yield any virus in our specific infectivity assay (day3 to 4 p.t.) (Fig. 5). However, continuous culturing of thetransfected cells produced viruses with a wild-type phenotype;sequencing of the recovered virus showed a reversion of the
engineered mutation to the wild-type D146. The results suggestthat the N-7 MTase activity is essential for the WNV repro-duction. The conclusion was further supported by the stepwisereversion of A3E3D at position 146 of the MTase after adouble-nucleotide mutant D146A2 RNA was transfected intocells (Fig. 7A). Mutant D146E virus was viable but exhibitedsmall plaques. Remarkably, when the D146E mutation wasengineered into recombinant protein, the D146E MTase wasactive in the N-7 methylation, but not the 2�-O methylation(Fig. 7C and D). Why is D146 more critical than other tetradresidues? It could be, at least in part, explained by the crystalstructure of the MTase, in which D146 is the closest to SAMamong the four K-D-K-E motif residues (Fig. 4A). D146 notonly provides stabilization within the motif but also forms ahydrogen bond with the amine nitrogen N-2 of the bound SAH(Fig. 1B). Modeling suggests that an E146, but not other testedmutants, can also form hydrogen bond with the bound SAH(data not shown). Therefore, D146 may participate in bindingof both SAM and RNA substrates in addition to its role incatalysis. Nevertheless, the essential role of the N-7 MTase inthe viral life cycle, together with its specific methylation activityon viral RNA cap (7a), suggests that the flavivirus MTase is anovel target for antiviral therapy.
Why is the N-7 MTase activity essential in the flavivirus lifecycle? We previously showed the critical role of N-7 MTasemay function at the level of viral translation (34). Alternatively,flavivirus RNA cap formation could be coupled to viral RNAreplication or other steps in the viral life cycle. In support ofthis possibility, RNA templates containing a 5� cap were re-ported to enhance de novo RNA synthesis catalyzed by recom-binant RdRp of WNV (1). The RdRp domain of DENV-2 wasshown to recognize 5� RNA elements so as to promote RNAsynthesis (12). In vesicular stomatitis virus, cap formation waspreviously found to be required for nonabortive viral mRNAtranscription (40); amino acid substitutions to the conservedK-D-K-E tetrad of the MTase were also shown to attenuateviral replication in cell culture (21). Further studies are re-quired to define the intricate modulations between flavivirusRNA capping and RNA synthesis.
ACKNOWLEDGMENTS
We are grateful to Kiong Ho for providing recombinant VP39 pro-tein and for helpful discussions and to Corey Bennett and Aaloki Shahfor technical assistance. We thank the Molecular Genetics Core, theCell Culture Core, Mass Spectrometry and Proteomics Core, and theMacromolecular Crystallography Facility at the Wadsworth Center forDNA sequencing for maintenance of BHK and Vero cells, for verifi-cation of mutant MTases, and for crystal screening, respectively.
The work was partially supported by contract AI25490 and grantsAI061193 and AI065562 from NIH. The BSL-3 animal facility at theWadsworth Center was used, which is funded in part by the NortheastBiodefense Center’s animal core (NIH/NIAID U54 AI05 7158). D.R.is supported by a postdoctoral fellowship from the National Sciencesand Engineering Research Council of Canada. X-ray diffraction datafor this study were measured at beamline X4A of the national syn-chrotron light source, which is supported by the Department of En-ergy, by grants from the NIH, and by the New York Structural BiologyCenter.
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