Functional Analysis of Two Cavities in Flavivirus NS5Polymerase□S

Received for publication, December 19, 2010, and in revised form, February 15, 2011 Published, JBC Papers in Press, February 23, 2011, DOI 10.1074/jbc.M110.214189

Gang Zou‡, Yen-Liang Chen‡, Hongping Dong‡, Chin Chin Lim‡, Li Jian Yap§, Yin Hoe Yau§, Susana Geifman Shochat§,Julien Lescar§, and Pei-Yong Shi‡1

From the ‡Novartis Institute for Tropical Diseases, Singapore 138670 and the §School of Biological Sciences, NanyangTechnological University, Singapore 637551, Singapore

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analy-sis of flavivirus RdRpdomains uncovered two conserved cavities(A and B). Both cavities are located in the thumb subdomainsand represent potential targets for development of allostericinhibitors. In this study, we used dengue virus as a model toanalyze the function of the twoRdRp cavities. Amino acids fromboth cavities were subjected to mutagenesis analysis in the con-text of genome-length RNA and recombinant NS5 protein; res-idues critical for viral replication were subjected to revertantanalysis. For cavity A, we found that only one (Lys-756) of theseven selected amino acids is critical for viral replication. Ala-nine substitution of Lys-756 did not affect the RdRp activity,suggesting that this residue functions through a nonenzymaticmechanism. For cavity B, all four selected amino acids (Leu-328,Lys-330, Trp-859, and Ile-863) are critical for viral replication.Biochemical and revertant analyses showed that three of thefour mutated residues (Leu-328, Trp-859, and Ile-863) functionat the step of initiation of RNA synthesis, whereas the fourthresidue (Lys-330) functions by interacting with the viral NS3helicase domain. Collectively, our results have provided directevidence for the hypothesis that cavity B, but not cavity A, fromdengue virus NS5 polymerase could be a target for rational drugdesign.

The genus Flavivirus from the family Flaviviridae containsmore than 70 viruses, many of which are important humanpathogens causingmajor public health threats worldwide. Den-gue virus (DENV)2 is a mosquito-borne flavivirus responsiblefor 50–100 million human infections and �20,000 deaths eachyear (1). Besides DENV, West Nile virus (WNV), Japaneseencephalitis virus, yellow fever virus, and tick-borne encepha-litis virus also cause significant human diseases (1). No antiviraltherapy is currently available for treatment of flavivirus infec-tions. Therefore, development of antiviral therapy is urgentlyneeded for flaviviruses.

The flavivirus genome is a single strand, plus-sense RNA ofabout 11 kb in length. The genomic RNAcontains a 5�-untrans-lated region (UTR), a single open reading frame, and a 3�-UTR.The single open reading frame encodes a long polyprotein thatis processed by viral and host proteases into 10 mature viralproteins (2). Three structural proteins (capsid, pre-membrane,and envelope) are primarily involved in virus particle forma-tion, and sevennonstructural proteins (NS1,NS2A,NS2B,NS3,NS4A, NS4B, and NS5) are mainly responsible for viral replica-tion (2). Among these, NS3 and NS5 possess enzymatic activi-ties and have been targeted for antiviral development. NS3functions as a protease (with NS2B as a cofactor), helicase,5�-RNA triphosphatase, and nucleoside triphosphatase (3–5).NS5 is the largest and the most conserved viral protein. TheN-terminal part of NS5 is a methyltransferase that methylatesthe N-7 and 2�-O positions of the viral RNA cap structure(6–9); the C-terminal part of NS5 has an RNA-dependent RNApolymerase (RdRp) activity (10, 11). Because the RdRp activityis essential for viral replication, and human host cells are devoidof RdRp, it is an attractive target for antiviral development.Two approaches have been successfully used to develop

RdRp inhibitors in clinical trials for hepatitis C virus (HCV)(12), a virus closely related to flavivirus. The first approach isbased on a nucleoside analogue. Nucleoside analogues functionas RNA chain terminators to block viral RNA synthesis. Thesecond approach is based on an allosteric inhibitor. Allostericinhibitors bind to pockets on polymerase to block conforma-tional changes required for enzyme function. Both nucleosideanalogues and allosteric inhibitors (targeting one of the fourpockets in HCV RdRp) are currently in clinical trials (12). Thesuccess of HCV RdRp inhibitors suggests that the sameapproaches could be used to develop inhibitors of flavivirusRdRp.Flavivirus RdRp initiates the RNA synthesis via a de novo

mechanism, which differs from a primer-dependent mecha-nism used by other viruses, such as poliovirus and SARS-CoV(10, 13–15). Crystal structures of RdRp from DENV-3 andWNV have been reported (16, 17). Like all polymerases, thestructure of flavivirus RdRps resembles a right hand with char-acteristic fingers, palm, and thumb subdomains. Malet et al.(18) recently proposed two cavities (named cavities A and B)that were conserved in the crystal structures of DENV andWNVRdRp. Both cavities are located in the thumb subdomainand could be potentially targeted for development of smallmol-ecule inhibitors. However, the biological relevance of the cavi-ties has not been validated.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 To whom correspondence should be addressed: 10 Biopolis Rd., 05-01Chromos, Singapore 138670. Tel.: 65-6722-2909; Fax: 65-6722-2916;E-mail: pei_yong.shi@novartis.com.

2 The abbreviations used are: DENV, dengue virus; RdRp, RNA-dependentRNA polymerase; WNV, West Nile virus; HCV, hepatitis C virus; IFA, immu-nofluorescence assay; SPR, surface plasmon resonance; p.t., post-transfec-tion; BBT, 2�-[2-benzothiazoyl]-6�-hydroxybenzothiazole.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 16, pp. 14362–14372, April 22, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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In this study, we performed a biochemical and genetic anal-ysis of the two cavities conserved among flavivirus RdRp.Mutagenesis analysis was used to analyze the function of thecavities in viral replication. Residues critical for viral replicationwere subjected to revertant analysis. In addition, the criticalresidues were further examined for their roles in RdRp activity.Our results demonstrate that cavity B, but not cavityA, is essen-tial for DENV replication.Mutations in cavity B could abrogateviral replication by decreasing the initiation of RdRp activity;alternatively, mutations in cavity B could suppress viral replica-tion by interference with the interaction between the NS5 andNS3 helicase domains. The results have provided functionalevidence that cavity B could be a rational target for drugdiscovery.

EXPERIMENTAL PROCEDURES

Cells and Viruses—Baby hamster kidney cells (BHK-21) andAfrican greenmonkey kidney cells (Vero) were cultured inDul-becco’s modified Eagle’s medium (DMEM) with 10% fetalbovine serum (FBS), 100 units/ml penicillin, and 100 �g/mlstreptomycin in 5% CO2 at 37 °C. DENV-2 TSV01 strain(GenBankTM accession number AY037116) was first isolatedfrom a patient in Australia in 1993 and then was passaged onC6/36 cells five times (19). The DENV-2 TSV01 from cell cul-ture fluids was aliquoted and stored at �80 °C.Plasmid Construction—To construct a full-length DENV-2

TSV01 cDNA clone, the virus stock was subjected to viral RNAextraction by RNeasy kit (Qiagen), and four cDNA fragments(A–D) covering the complete genomewere amplified fromviralRNA by RT-PCR using SuperScript III one-step RT-PCR kits(Invitrogen). The supplemental Fig. 1A depicts the cloningscheme to construct the cDNA of full-length genomic RNAof DENV-2. Fragment A contained an NheI restriction site, aT7 promoter sequence, and cDNA representing nucleotides1–2,428 of the viral genome, followed by an extra XhoI restric-tion site. The RT-PCR product of fragment A was cloned into apredigested low copy number plasmid pACYC177 (New Eng-land Biolabs), resulting in pACYC TSV-A plasmid. Fragment Bspanned from the EcoRI site (nucleoside position 2,340) to theXhoI site (nucleoside position 5,426). Fragment C spannedfrom the XhoI (nucleoside position 5,426) site to the AgeI site(nucleoside position 7,613). FragmentD spanned from theAgeIsite (nucleoside position 7,613) to the 3� end of the genome,followed by a hepatitis delta virus ribozyme sequence and anextra ClaI site. The RT-PCR products of fragments B–D wereindividually ligated into pCR2.1-TOPOwithTOPOTAcloningkit according to the manufacturer’s instructions (Invitrogen),resulting in constructs TA TSV-B, -C, and -D, respectively(supplemental Fig. 1A). Each subclone was validated by DNAsequencing before it was used for subsequent assembly. Next,fragment B was inserted into subclone pACYC TSV-A at theEcoRI and XhoI site, resulting in construct pACYC TSV-E.Fragment C was inserted into subclone TA TSV-D at the XhoIand AgeI sites, yielding plasmid TA TSV-F. Finally, the frag-ments C and D from plasmid TA TSV-F was inserted into con-struct pACYC TSV-E at the XhoI and ClaI sites, resulting inthe full-length cDNA clone pACYC FLTSV. Escherichia coliHB101 (Promega) was used as the host for construction and

propagation of cDNA clones. All restriction enzymes were pur-chased from New England Biolabs.The genome-length cDNA clones with cavity mutations in

NS5 RdRp were constructed by using the subclone TA TSV-F.The mutations were introduced into the plasmid TA TSV-Fby the QuikChange II XL site-directed mutagenesis kit (Stra-tagene); the mutated DNA fragment was cut and inserted intothe pACYC FLTSV at the NruI (located at nucleotide 7,737of the viral genome) and ClaI sites. All constructs were verifiedby DNA sequencing.In Vitro Transcription, RNA Transfection, Specific Infectivity

Assay, and Immunofluorescence Assay (IFA)—The genome-length RNA of DENV-2 TSV01 was transcribed in vitro fromthe corresponding cDNA plasmids that were linearized withClaI. A T7 mMESSAGE mMACHINE kit (Ambion) was usedfor RNA synthesis as described previously (6). The genome-length RNA was electroporated into BHK-21 cells as describedpreviously (20). After transfection of the genome-length RNA,cells were cultured at 37 °C for the first 24 h and then trans-ferred to 30 °C. We and others previously found that culturingthe transfected cells at 30 °C increased the yield of DENV (21,22). Supernatants were collected every 24 h until day 5 post-transfection. The culture medium containing viruses were ali-quoted and stored at �80 °C. The specific infectivity assay wasperformed as described previously (6). The E protein expres-sion in the transfected cells was monitored by IFA usingmouseanti-E mAb 4G2 antibody as described previously (23).Plaque Assay—Plaque assay was performed as described pre-

viously with minor modifications (24). Briefly, BHK-21 cells(ATCC) were seeded with a cell density of 1 � 105 per well in a24-well plate (Nalge Nunc) 2 days in advance. A series of 1:10dilutionsweremade bymixing 15�l of virus samplewith 135�lof RPMI 1640 medium containing 2% FBS, 100 units/ml peni-cillin, and 100�g/ml streptomycin. One hundredmicroliters ofundiluted and 10-fold dilutions of viral supernatant wereseeded to individual wells of 24-well plates. The plates wereincubated at 30 °C with 5% CO2 for 1 h with shaking every 15min, and then the virus inocula were replaced with 0.6 ml ofRPMI 1640 medium plus 0.8% methylcellulose (Aquacide II,Calbiochem) and 2% FBS. After 4 days of incubation at 37 °Cwith 5% CO2, the cells were fixed with 3.7% formalin andstained with 1% crystal violet. For some mutant viruses, theincubation period was more than 4 days to visualize theplaques.Selection and Sequencing of Revertant Viruses—Five mutant

viruses defective in viral replication were subjected to revertantanalysis by continuously culturing on Vero cells for sevenrounds. For each round of culturing, Vero cells (1� 106 cells) ina T-25 flask were infected with 500 �l of culture supernatantderived from the previous passaging and cultured in DMEMwith 2% FBS in 5% CO2 at 37 °C. The cytopathic effect wasmonitored under a microscope. The incubation period last 5days unless an apparent cytopathic effect appeared earlier. ViralRNA extracted from culture supernatants of passages 2 and 7was subjected to RT-PCR using SuperScript III one-step RT-PCR kits (Invitrogen). The entire NS5 region or the completeviral genome was sequenced.

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RdRp Assay—Recombinant protein of full-length DENV-4NS5 was prepared using an E. coli expression system andpurified through affinity and gel filtration chromatography.Mutagenesis of the NS5 expression plasmid was performedusing QuikChange II XL site-directed mutagenesis kit. Thedetailed protocols for mutagenesis, protein expression, andpurification have been reported previously (25). Two types ofRdRp assays were performed, de novo RdRp assay and elonga-tion RdRp assays. The de novo RdRp reaction (25 �l) contained50 mM HEPES, pH 8.0, 10 mM KCl, 5 mM MgCl2, 2 mM MnCl2,10mMDTT, 0.5mMATP, 0.5mMUTP, 0.5mMCTP, 2.5�Ci of[�-33P]GTP (10 �Ci/�l, 3,000 Ci/mmol; PerkinElmer Life Sci-ence), 0.25 �g of NS5, 0.5 �g of RNA template. The RNA tem-plate was transcribed in vitro from a PCR product using a T7MEGAscript kit (Applied Biosystems). The PCR template con-tained a T7 promoter followed by a cDNA fragment represent-ing a DENV-2 subgenome with a deletion from nucleotide 169to 10,263 (GenBankTM accession number AF038403.1). The denovo RdRp reactions were incubated at 23 °C for 30 min; themixtures were passed through a MicroSpin G-25 column (GEHealthcare) to remove unincorporated NTPs; and the RNAelute was extracted with phenol/chloroform and precipitatedwith ethanol. The RNA pellet was dissolved in 10 �l of RNase-freewater,mixedwith an equal volumeof denaturingGel Load-ing Buffer II (Applied Biosystems), and loaded onto a 10%dena-turing polyacrylamide gelwith 7Murea.APhosphorImagerwasused to quantify the 33P-labeled RNA products. The elongationRdRp assay was performed using an RNA template and a 2�-[2-benzothiazoyl]-6�-hydroxybenzothiazole (BBT)-ATP as a sub-stratemimic (26). TheBBT-ATPwas chemically synthesized byattaching the BBT fluorophore group to the �-phosphate ofadenosine triphosphate. During the polymerization reaction,the adenosine monophosphate was incorporated into the RNAchain, releasing BBTppi as a by-product. BBTppi was thenhydrolyzed by calf intestinal alkaline phosphatase, producingtwo inorganic phosphates (Pi) and BBT, which yields fluores-cence. The details of assay conditions were recently reported(26).Surface Plasmon Resonance (SPR) Assay—SPR measure-

ments were performed on Biacore 3000 equipment (Biacore,GE Healthcare). All experiments were run with a constant flowrate of 30 �l/min HBS-EP buffer (10 mM HEPES, pH 7.4, 150mM NaCl, 3.4 mM EDTA, and 0.0005% P-20). Wild-type full-length DENV-4 NS5 and its mutants (single mutant, K330Aand G840R; double mutant K330A/G840R) were amine-cou-pled onto the surface of aCM5chip (Biacore,GEHealthcare). A3-fold serially diluted DENV-4 full-length NS3 (0.1–24 �M) orits helicase domain (1–244�M) was injected in duplicate acrossthe chip for 1 min, and dissociation was monitored for 2.5 min.Recombinant full-length NS3 and its helicase domain proteinswere prepared as reported previously (27). A short pulse (30 s)of 15 mM HCl was passed through the flow system to com-pletely remove any residual bound protein to prepare for thenext injection cycle. Raw sensorgrams obtained were aligned,solvent-corrected, and double-referenced using Scrubber 2software (BioLogic Software, Campbell, Australia). Processeddata were globally analyzed and fit to a simple 1:1 interaction

model using numeric integration to yield the individualaffinities.

RESULTS

Construction and Characterization of a Full-length cDNAClone of DENV-2 Strain TSV01—The low copy number plas-mid pACYC177 and E. coli HB101 were used to construct thecDNA clone of the DENV-2 TSV01 strain. The selection ofpACYC177 as the cloning vector was based on our previousexperience in WNV infectious clones (6). The supplementalFig. 1A shows the overall scheme of the cloning strategy (seedetails under “Experimental Procedures”). The assembled full-length cDNAclone contained aT7 promoter at the 5� end for invitro transcription and a 67-bp hepatitis delta virus ribozymesequence at the 3� end for generating the authentic 3� end viralsequence. The stability of the cDNA clone was tested by sixrounds of plasmid extraction and re-transformation into E. coliHB101. DNA sequencing of the 6th round plasmid did notshow any mutations throughout the cDNA insert (data notshown), demonstrating the stability of the clone.BHK-21 cells transfected with the in vitro transcribed

genome-lengthRNAexhibited an apparent cytopathic effect onday 5 post-transfection (p.t.). IFA of the transfected cellsshowed increasing numbers of cells expressing viral E proteinfromday 1 to 3;more than 80% of the cells were IFA-positive onday 3 p.t. (supplemental Fig. 1B). Plaque assay of the culturefluids showed an increase in viral titer from day 1 to 4, peakingat 1� 106 to 1� 107 pfu/ml onday 4 p.t. (supplemental Fig. 1C).Specific infectivity assay showed that transfection of BHK-21cells with 1 �g of RNA generated about 1.2 � 105 pfu of virus.The results demonstrate that the RNA transcribed from thecDNA clone was highly infectious.Structure and SequenceAnalyses of Cavities A andB ofDENV

RdRp—Structure analysis uncovered two cavities conserved inDENV and WNV RdRps (18). The two cavities (A and B) arelocated on two opposite sides of the thumb subdomain ofDENV and WNV RdRp. Fig. 1A shows the two cavities on thecrystal structure of DENV-3 RdRp. OnDENV-2 RdRp, cavity Ais formed by amino acids Lys-756, Gln-760, Ser-763, Asn-777,Cys-780, Ser-785, Thr-806, Glu-807, Asp-808, Met-809, Leu-810, and Tyr-882, among which Lys-756, Thr-806, Glu-807,Asp-808, Met-809, and Leu-810 are completely conservedamong various members of flaviviruses (Fig. 1B). Cavity B con-sists of amino acids Leu-327, Leu-328, Lys-330, Thr-858, Trp-859, Asn-862, Ile-863, and Ala-866, among which Leu-328,Trp-859, and Ile-863 are conserved among flaviviruses (Fig. 1B).Role of Cavity A in Viral Replication—To determine the bio-

logical function of cavity A, we selected seven amino acids formutagenesis analysis. As shown in Fig. 1A (left panel), Lys-756(red), Gln-760 (green), Cys-780 (blue), and Met-809 (tint) arelocated deep inside the cavity. Thr-806 (magenta), Glu-807(cyan), and Asp-808 (orange) form the left side wall of the cav-ity. Among the seven selected residues, five amino acids (Lys-756, Thr-806, Glu-807, Asp-808, and Met-809) are completelyconserved among flaviviruses; two residues (Gln-760 and Cys-780) have variations in yellow fever virus and/or tick-borneencephalitis virus RdRp (Fig. 1B). It should be noted that resi-

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FIGURE 1. Structure and sequence analyses of cavities A and B from DENV RdRp. A, cavities A and B in DENV-3 RdRp structure. The crystal structure ofDENV-3 RdRp (Protein Data Bank entry 2J7U) was used to illustrate cavity A (left panel) and cavity B (right panel). Amino acids constituting the cavities are labeledin yellow or other colors. Residues selected for mutagenesis analysis are colored as follows: for cavity A, Lys-756 (red), Gln-760 (green), Cys-780 (blue), Thr-806(magenta), Glu-807 (cyan), Asp-808 (orange), and Met-809 (tint); for cavity B, Leu-328 (red), Lys-330 (green), Trp-859 (blue), and Ile-863 (pink). The images of RdRpwere produced using PyMOL. B, amino acid sequence alignment of partial RdRp region from the four serotypes of DENV and other flaviviruses. The RdRp aminoacid sequences of DENV-1, DENV-2, DENV-3, DENV-4, WNV, yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) are derived from GenBankTM

accession numbers U88535, AY037116, M93130, AY947539, AF404756, X03700, and AF069066, respectively. Identical amino acids among all RdRps are shaded.The numbering of amino acid sequence is based on DENV-2. The residues involved in the formation of cavity A and B are indicated by F and � above thesequence, respectively. The mutated residues in this study are indicated by * below the sequence.

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dues Thr-806, Glu-807, Asp-808, and Met-809 also form theC-terminal part of the priming loop (16).Each of the selected residues wasmutated to Ala in the infec-

tious clone of DENV-2 TSV01. Equal amounts of wild-type(WT) and mutant genome-length RNAs were electroporatedinto BHK-21 cells. Viral protein synthesis, plaque morphology,and virus production were compared. For viral protein synthe-sis, IFAs (detecting viral E protein) showed that similar percent-ages of IFA-positive cells were detected for the WT andmutants T806A, E807A, and D808A RNAs at 72 h p.t.; mutantsQ760A, C780A, and M809A showed a slight decrease in thepercentage of IFA-positive cells, and mutant K756A remainedless than 1% IFA-positive cells at 72 h p.t. (Fig. 2A). For differentRNAmutants, the relative IFA-positive cells were similar whenthe transfected cells were analyzed at 24 and 48 h p.t. (data notshown). No infectious virus was recovered from the K756ARNA-transfected cells, whereas other mutant RNAs yieldedsimilar amounts of infectious viruses with no significant differ-ence in plaque morphology (Fig. 2, A and B). These results sug-gest that only K756A among the selected mutations in cavity Ais essential for viral replication.Role of Cavity B in Viral Replication—Four residues were

selected formutagenesis analysis of cavity B. As depicted in Fig.1A (right panel), Leu-328 (red) and Ile-863 (pink) are located atthe bottom of the cavity; Trp-859 (blue) is at the side of cavitywall; and Lys-330 (green) is on the surface of the cavity. The fourresidueswere individuallymutated toAla in the genome-lengthRNA of DENV-2 TSV01. Unlike cavity A mutants, genome-length RNAs containing the mutations in cavity B showed asignificant decrease in viral protein synthesis in the transfectedBHK-21 cells. At 72 h p.t., �5% of the cells transfected withI863ARNAexpressed viral protein; less than 1%of IFA-positivecells were detected for L328A and K330A; no IFA-positive cellswas detected in cells transfected withW859ARNA (Fig. 2C). Inagreement with the IFA results, only the I863A RNA-trans-fected cells yielded viruses (Fig. 2D) with tiny plaques (Fig. 2C);no plaque was detected from the L328A, K330A, and W859ARNA-transfected cells, up to 120 h p.t. (Fig. 2,C andD). Overall,the results indicate that cavity B plays a critical role in viralreplication.Effects of Mutations on RdRp Activity—To determine

whether the cavities play a direct role in RdRp activity, we pre-pared a panel of full-lengthDENV-4NS5 proteins and analyzedthe mutational effects on polymerase activity. The reason tochooseDENV-4NS5was that this proteinwasmore stable thanDENV-2 NS5 (data not shown). All five replication-defectiveNS5 mutations (L328A, K330A, K756A, W859A, and I863A)were engineered into recombinant full-length NS5 proteins.The proteins were analyzed in two types of RdRp assays. First, ade novo RdRp assay was performed using a viral subgenomicRNA as template. As shown in Fig. 3A, mutants L328A,W859A, and I863A exhibited only 15, 10, and 7% of the WTactivity, respectively, whereas mutants K330A and K756Aretained 80 and 82% of the WT activity, respectively.Because the de novo RdRp assay includes steps of initiation

and elongation, we next analyzed themutant NS5 in an elonga-tion RdRp assay (26). In contrast to the de novo synthesisresults, all mutants retained greater than 70% of the WT RNA

elongation activity (Fig. 3B). Fig. 3C summarizes the effects ofmutations on de novo and elongation RdRp activities. The com-parable elongation activities between theWT and mutant NS5suggest the following: (i) the engineered mutations do notchange the global conformation of the NS5 protein; and (ii) theobserved defect in de novo RNA synthesis for mutants L328A,W859A, and I863A is not due to variations in protein prepara-tions. These results allowed us to conclude that L328A,W859A, and I863A reduce viral replication on or beforede novoinitiation, whereas K330A and K756A decrease RNA replica-tion through a nonenzymatic mechanism.Revertant Analysis of Cavity A—Among the mutated amino

acids from cavities A and B, five of them resulted in replication-defective viruses (L328A, K330A, K756A, W859A, and I863A;Fig. 2). Only K756A belongs to cavity A; L328A, K330A,W859A, and I863A belong to cavity B (Fig. 1A). We performedrevertant analysis by continuous passaging of culture superna-tants on Vero cells for seven rounds (P1 to P7). The culturefluids from passages 2 (P2) and 7 (P7) were subjected to plaqueassay and sequencing analysis. For cavity A, both the P2 and P7supernatants of the K756A mutant produced plaque morphol-ogy similar to theWT; sequencing of the viruses revealed a truereversion to theWT sequence (data not shown). Together withthe result that the K756A RNA-transfected cells (P0) did notyield any infectious virus (Fig. 2, A and B), we conclude thatresidue Lys-756 in cavity A is essential for viral replication.Revertant Analysis of Cavity B—For cavity B, both P2 and P7

culture fluids derived from the L328A, K330A, and I863ARNAs produced plaques. Sequencing of the recovered virusesshowed various reversions. For L328Amutant, the P2 virus hada mixed population of Val (GTA) and Leu (CTA), whereas theP7 virus became a single population of Leu (CTA) (Fig. 4A).These results suggest that the L328Amutant underwent a step-wise reversion of Ala (GCA) 3 Val (GTA) 3 Leu (CTA)(mutant nucleotides are underlined), each step with a singlenucleotide change.For the K330Amutant, transfection of this mutant RNA into

BHK-21 cells did not yield any infectious virus (Fig. 2,C andD).However, the culture fluids from P2 and P7 yielded infectiousviruses with plaques smaller than the WT virus (Fig. 4B). Thesequencing result showed that both the P2 and P7 virusesretained the engineered mutation (Fig. 4B); complete genome-length sequencing of the P2 virus revealed a second site nucle-otide mutation G3A at position 10,087, which resulted in aGly3 Arg amino acid change at position 840 of NS5. To dem-onstrate whether the G840R substitution rescued the replica-tion defect of K330A RNA, we prepared two mutants genome-length RNAs containing the G840R alone or the K330A/G840Rdouble mutations. Transfection of BHK-21 cells with equalamounts of RNA showed that the G840R RNA generated fewerIFA-positive cells than theWTRNAdid, but the doublemutantK330A/G840RRNAproducedmore IFA-positive cells than theG840R RNA did (Fig. 4C). At 72 h p.t., equivalent amounts ofIFA-positive cells were observed between the WT and theK330A/G840R double mutant RNA-transfected cells. Theplaque size of G840R virus was smaller than K330A/G840Rviruses, and the plaques of both viruses are fainter and slightlysmaller than that of theWT virus (Fig. 4C). These results dem-

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FIGURE 2. Mutagenesis of cavities A and B using an infectious cDNA clone of DENV-2. A and C, analysis of viral E protein synthesis and plaque morphologyfor cavity A and B mutant viruses, respectively. BHK-21 cells were transfected with WT and mutant genome-length RNAs (10 �g), and analyzed for viral E proteinexpression by IFA at 72 h post-transfection (upper panel). Plaque morphologies of WT and mutant viruses were shown (lower panel). N.D., not detectable. B andD, production of cavity A and B mutant viruses after transfection, respectively. Viruses from culture supernatants were collected every 24 h. Viral titers weredetermined by plaque assay on BHK-21 cells. Error bars indicate the standard deviations from two independent experiments; dashed line, limit of sensitivity ofthe plaque assay.

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onstrate that the G840R adaptation is responsible for the res-toration of the replication of mutant K330A (see further mech-anistic analysis below).For the I863A mutant, the P0 culture fluids showed tiny

plaques (Fig. 2C). The P2 virus showed plaques of differentsizes, and the P7 virus showed only large plaques (Fig. 5A). Inagreement with the plaque morphology, sequencing analysisshowed that the P2 virus had a mixed population of Val (GTC)and Ala (GCC), whereas the P7 virus became a single popula-tion ofVal (GTC) (Fig. 5A). These results suggest that the I863Amutant underwent a reversion of Ala (GCC) 3 Val (GTC)through a single nucleotide change (mutant nucleotides areunderlined). Engineering the I863V substitution into genome-length RNA restored viral replication. After transfection intoBHK-21 cells, the I863V RNA and WT RNA generated equiv-alent amounts of IFA-positive cells from 24 to 72 h p.t. (com-pare Figs. 5Bwith 4C). The I863V RNA generated an infectiousvirus with plaque morphology similar to that of the WT virus(Fig. 5B). In addition, comparable viral titers were detected forWT and I863V mutant virus at various time points post-trans-fection; sequencing of the recovered virus showed that the engi-neered I863V mutation was retained without any other muta-

tions (data not shown). Next, we compared the RdRp activity ofrecombinant NS5 containing the I863A or I863V mutation. Inthe de novo synthesis assay, the I863V and I863ANS5 exhibited100 and 5% of the WT RdRp activity, respectively (Fig. 5C). Inthe elongation assay, the I863V and I863A NS5 exhibited 101and 73% of theWT RdRp activity, respectively (Fig. 5D). Theseresults strongly indicate that the reversion of I863A3 I863Vimproves viral replication through enhancement of RdRp activ-ity, especially at the step of initiation of RNA synthesis.For the W859A mutant, no infectious virus was recovered

(Fig. 2,C andD), even after seven rounds of passaging (data notshown), indicating that Trp-859 is essential for viral replication.To examine whether amino acids other than Trp at position859 could support viral replication, we substituted Trp-859with Phe, Lys, Asp, His, or Leu in the context of genome-lengthRNA. Equal amounts of WT and mutant RNAs were trans-fected intoBHK-21 cells.Only the cells transfectedwithW859FRNA showed a few number of IFA-positive cells (less than 1%)at 72 h p.t. (Fig. 6). None of theTrp-859 variant RNAs producedany infectious viruses, as judged by plaque assays (data notshown). These results demonstrate that Trp-859 in cavity B isessential for viral replication, even an aromatic amino acidsubstitution (W859F) could not sufficiently support viralreplication.

FIGURE 3. Effects of cavity mutations on de novo and elongation RdRpactivities. A, de novo RdRp activities of mutant NS5 proteins. B, elongationRdRp activities. The elongation assay measured the fluorescence generatedby using a substrate mimic BBT-ATP and RNA template, as reported previ-ously (26). Results are the average of two independent experiments per-formed in duplicate. C, summary of de novo and elongation RdRp activities.The relative de novo RdRp activities were calculated by comparing the prod-ucts generated using the mutant NS5 proteins with that produced using theWT protein (set as 100%). The relative RNA elongation activities were calcu-lated by comparing the slope from the mutant NS5 proteins with that of theWT protein (set as 100%).

FIGURE 4. Revertant analysis of cavity B mutant viruses, L328A andK330A. The mutant viruses were continuously cultured on Vero cells forseven rounds (P1 to P7). The plaque morphology and sequencing chromato-gram for P2 and P7 are presented for the L328A (A) and K330A (B) viruses. Theengineered mutant nucleotides are underlined. C, validation of the K330Aadaptive mutation. Revertant analysis of the P2 K330A virus revealed a sec-ond site mutation G840R in the RdRp domain (data not shown). For validationof G840R in restoring the replication of K330A mutation, two mutantgenome-length RNAs (G840R and K330A/G840R) were transfected intoBHK-21 cells. The transfected cells were monitored for viral E protein expres-sion at 24, 48, and 72 h post-transfection. Plaque morphologies were shownfor recovered viruses.

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K330A Mutation Affects Viral Replication by DecreasingNS3/NS5 Interaction—K330A mutation abolished viral repli-cation (Fig. 2,C andD) but did not affect RdRp activity (Fig. 3C),suggesting that the mutation affects viral replication through anonenzymatic mechanism. Revertant analysis showed that asecond sitemutation (G840R) in theRdRpdomain could rescuethe replication defect of the K330A mutation (Fig. 4C). Theseresults prompted us to test the hypothesis that K330Amutationaffects replication through decreasing the interactionNS5 withother replication component(s). Because the NS3/NS5 interac-tion has been reported forDENVand other flaviviruses (28, 29),wemeasured the binding affinity between the recombinant full-length NS3 and NS5 proteins using an SPR assay. As shown inFig. 7C, the K330Amutation increased theKD value of theNS3/NS5 interaction by 12.4-fold; the compensatory mutationG840R alone also increased the KD by 8.1-fold; remarkably, thedouble mutant K330A/G840R restored theKD value to 1.8-foldof the WT KD. The experimental curve of SPR fits closely to a1:1 binding model of NS3 and NS5. The slight discrepancybetween the experimental and theoretical curves (particularlyin the dissociation phase) is most likely due to NS3 aggregationat high concentrations. Such aggregation was observed when ahigh concentration ofNS3was fractionated through size-exclu-sion chromatography (data not shown).Because the NS3/NS5 interaction was previously mapped to

the helicase domain of NS3 (30, 31), we performed SPR assaysusing recombinant helicase domain (without protease domain)and various NS5 proteins. The results showed that theKD value

of helicase/NS5 was 4.4-fold higher than that of the full-lengthNS3/NS5 (Fig. 7C), indicating that the protease domain con-tributes to the NS3/NS5 interaction. Similar to the full-lengthNS3 results, mutation K330A increased the KD value of heli-case/NS5 interaction by 10.2-fold, and the double mutationK330A/G840R restored the KD value to 1.4-fold of theWT KD.However, the model fitting for the helicase domain is not asgood as the full-length NS3 (Fig. 7,A and B) due to the fact thatthe helicase domain is more prone to aggregation than the full-lengthNS3 at a high concentration (data not shown).Neverthe-less, these results indicate that the compensatory mutationG840R rescued the replication defect of K330A by restorationof the NS3/NS5 interaction.

DISCUSSION

Flavivirus RdRp is an attractive target for antiviral develop-ment. Both nucleoside analogues and non-nucleoside inhibi-tors of DENV RdRp have recently been reported (25, 32–34).The crystal structures ofWNV and DENV RdRps revealed twoconserved cavities that could be used for development of allo-steric inhibitors (18). Using both genetic and biochemicalapproaches, we probed the biological relevance of the two cav-ities. Fig. 8A summarizes the results about all critical residuesidentified in this study. For cavity A, we found that among thesevenmutated amino acids, onlyK756Amutationwas lethal forDENV replication; the other six mutated residues did not sig-nificantly affect viral replication (Fig. 2, A and B). It is worthnoting that four of the mutated residues (Thr-806, Glu-807,Asp-808, and Met-809) reside in the C-terminal region of thepriming loop (colored in yellow, Fig. 8B). For the K756A muta-tion, only true reversion could restore viral replication. In termsof mode-of-action, we found that the K756A mutation affectneither de novo RNA synthesis nor RNA elongation activity(Fig. 3). The latter result indicates that the K756A mutationreduces viral replication through a nonenzymatic mechanismand possibly by affecting interaction with other replicase com-ponent(s). However, structure analysis showed that residueLys-756 is located deep inside cavityA; the residue is behind the

FIGURE 5. Revertant analysis of cavity B mutant virus, I863A. A, plaquemorphology and sequencing chromatogram from the I863A P2 and P7viruses. The engineered mutant nucleotides are underlined. B, validation ofthe I863V revertant mutation. The I863V genome-length RNA was electropo-rated into BHK-21 cells. The transfected cells were monitored for E proteinexpression at 24, 48, and 72 h post-transfection. Plaque morphology of theI863V virus (P0) is shown. C, comparison of de novo RdRp activities among WT,I863V, and I863A NS5 proteins. The relative de novo RdRp activities (WT set as100%) are indicated below the denaturing gel. D, comparison of elongationRdRp activities among WT, I863V, and I863A NS5 proteins. The relative elon-gation activities were shown in parentheses (WT set as 100%). Results are theaverage of two independent experiments performed in duplicate.

FIGURE 6. Site-directed mutagenesis of Trp-859 in cavity B. BHK-21 cellswere transfected with WT and mutant genome-length RNAs (10 �g) and ana-lyzed for viral E protein synthesis by IFA at 72 h post-transfection. The engi-neered mutant nucleotides are underlined.

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C terminus of the priming loop (Fig. 8B). It is conceivable thatduring RNA synthesis, a local conformational change exposesresidue Lys-756 to the protein surface, allowing Lys-756 to

interact with other replicase component(s). Nevertheless, ourresults argue that cavity A is not an ideal target for designingsmall molecular inhibitors.

FIGURE 7. Compensatory mutation G840R rescued the replication defect of K330A through restoration of the NS3/NS5 interaction. A and B, SPRmeasurements of the interaction between the wild-type and mutants of NS5 and the full-length NS3 and its helicase domain, respectively. 3-Fold seriallydiluted DENV-4 full-length NS3 (0.1–24 �M) or its helicase domain (1–244 �M) were injected across immobilized DENV-4 NS5 (and its mutants) in duplicate (onlyone of the duplicates is shown in figure for clarity) for 1 min and allowed to dissociate in running buffer for another 2.5 min. The aligned and double-referencedsensorgrams were globally fit to a simple 1:1 interaction model (thin red lines). To verify the accuracy of our rate parameters, we also fit the same data to abivalent model (BIAEvaluation version 4.1, Biacore, GE Healthcare) and the results tallied with the 1:1 model. Hence, we chose to present the 1:1 model fit toavoid complication and to provide a better comparison between the full-length NS3 data and the NS3 helicase domain data. C, summary of the resultantaffinities for interaction between full-length DENV-4 NS3 and its helicase domain with wild-type NS5, its single mutant (K330A and G840R), and its doublemutant (K330A/G840R). The resultant affinity for interaction is reflected by KD (equilibrium dissociation constants), which was calculated using the ratio of kd/kafrom the SPR assay. RU, response units.

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In contrast to cavity A, all four mutated residues in cavity Bsignificantly reduced viral replication (Fig. 2,C andD). None ofthe mutations decreased RNA elongation activity by �27%;however, three mutations (L328A, W859A, and I863A)reduced de novo RNA synthesis by �85% (Fig. 3). Correlatedwith the de novo RNA synthesis results, revertant analysisshowed the following: (i) only true reversion could restore thereplication of L328ARNA (Fig. 4A); (ii) no revertant virus couldbe selected forW859ARNA; (iii) only amino acidVal, similar tothe wild-type Ile-863, was selected to restore the replication ofI863ARNA (Fig. 5). These results indicate that L328A,W859A,and I863A mutations reduced viral replication by decreasingthe initiation of RNA synthesis. Although these residues areaway from the active site and RNA tunnel (Fig. 8C), they mayparticipate in the formation of the RNA template-RdRp-NTPcomplex during the initiation of RNA synthesis. In support ofthis notion, the �1 loop on the surface of thumb subdomain ofHCV RdRp was previously shown to regulate the conforma-tions needed for de novo initiation and for elongation of RNAsynthesis; mutations that affect the �1 loop conformationselectively reduced de novo initiation (35). In this study, thethree mutations that selectively decreased RNA synthesis initi-ation (L328A, W859A, and I863A) are also located at the sameinterface between the�1 loop and the rest of thumb subdomainof DENV RdRp.

Unlike the three residues in cavity B discussed above, muta-tion K330A abolished viral replication but retained 80 and 81%of the wild-type activities of de novo and elongation RNA syn-thesis, respectively (Fig. 8A). These results indicate that theK330A mutation abrogated RNA replication through a nonen-zymatic mechanism. In support of this hypothesis, our SPRresults showed that the K330Amutation reduced the NS3/NS5interaction. Furthermore, revertant analysis showed that a sec-ond sitemutationG840R in the RdRp domain could restore thereplication of theK330Amutant. In agreementwith the geneticdata, our SPR results showed that, although the compensatorymutationG840R alone is also defective inNS3/NS5 interaction,the double mutant K330A/G840R restored the NS3/NS5 inter-action. On the crystal structure, Gly-840 (blue in Fig. 8C) is 24.1Å away from Lys-330 but is located on the surface of the thumbsubdomain. The switch from the wild-type Lys-330 � Gly-840to the revertant K330A/G840R maintained one positivelycharged residue, indicating that the positive charge on the sur-face of NS5 is important for the NS3/NS5 interaction. In sup-port of our results, Johansson et al. (30) previously suggestedthat a region between residues 320 and 368 ofDENVNS5 inter-acts with NS3 helicase domain.In summary, our results have demonstrated that cavity B is

essential for viral replication and could be targeted for develop-ment of the allosteric inhibitor of flavivirus NS5 polymerase.

FIGURE 8. A, summary of mutagenesis analysis of cavities A and B. Based on the IFA results, the replication levels of WT and mutants RNAs are categorized intostrong (����), medium (��), weak (�/�), and nonreplicative (�). B, crystal structure of DENV RdRp showing residues Thr-806, Glu-807, Asp-808, Met-809(all in yellow), and Lys-756 (in red) from cavity A. The priming loop is shown in yellow. The GDD active site of RdRp is shown in pink. C, crystal structure of DENVRdRp showing residues Leu-328, Lys-330, Trp-859, and Ile-863 (all in red) from cavity B. Amino acid Gly-840, a compensatory mutation for K330A, is labeled inblue. The illustrations for B and C were prepared using PyMOL.

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Pharmacological blockage of cavity B could potentially lead tosuppression of initiation of viral RNA synthesis and/or inhibi-tion of NS3/NS5 interaction. The SPR assay described in thisstudy is amendable for high throughput screening to identifyinhibitors of NS3/NS5 interaction.

Acknowledgments—We thank Ying Tan (Duke-National Universityof Singapore) and Sui Sum Yeong (Nanyang Technological Univer-sity) for technical support during the course of the study. We alsothank Shamala Devi for providing DENV-4 virus that was used forcloning the full-length ns5 gene.

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Functional Analysis of Dengue Virus RdRp Cavities

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Yau, Susana Geifman Shochat, Julien Lescar and Pei-Yong ShiGang Zou, Yen-Liang Chen, Hongping Dong, Chin Chin Lim, Li Jian Yap, Yin Hoe

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

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