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Unit of Molecular Prevention and Therapy of Human Diseases (CNRS-URA3012), Institut Pasteur, 28 rue Docteur Roux, F-75724 Paris Cedex 15, France
Correspondence
Hugues Bedouelle
hbedouel{at}pasteur.fr
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ABSTRACT |
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-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency. Published online ahead of print on 25 June 2007 as DOI 10.1099/vir.0.83028-0.
Present address: Unit of Molecular Retrovirology (CNRS-URA3015), Institut Pasteur, F-75724 Paris Cedex 15, France. 
Present address: Department of Biology, University of Cergy-Pontoise, F-95302 Cergy-Pontoise Cedex, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). They are transmitted by Aedes mosquitoes and infect between 50 million and 100 million persons each year. The disease generally takes a mild form, dengue fever, but severe forms, dengue haemorragic fever and dengue shock syndrome, have recently become epidemic (Gubler, 2002).
The immune response against an infection by DENV involves both a humoral component, in the form of neutralizing antibodies, and a cell-mediated component (Guzman & Kouri, 2002). The preferential reactivation of the memory B cells that correspond to a primary infection, and an antibody-dependent enhancement of infection, might constitute triggering mechanisms of the severe forms during a secondary infection by a different viral serotype (Halstead, 2003; Mongkolsapaya et al., 2003). A molecular understanding of the events that lead to antibody neutralization, enhancement or escape is critical to the development of efficient and secure vaccines and therapeutics.
DENV1–DENV4 are enveloped RNA viruses, like all flaviviruses. The structures of the whole virus and of the envelope glycoprotein E (gpE) have been solved by electron cryomicroscopy and X-ray crystallography (Modis et al., 2003, 2005; Zhang et al., 2003). Ninety dimers of gpE cover the surface of the virus. Each monomer comprises three ectodomains, ED1–ED3, and a transmembrane segment. ED2 includes the dimerization interface, a glycosylation site and the peptide of fusion with the cellular membrane. ED3 is continuous and comprises residues 296–400 of gpE. Its fold is compact, immunoglobulin-like and stabilized by a disulfide bond between residues Cys302 and Cys333.
Several types of cellular receptor have been identified for DENV: highly sulfated heparan sulfates (hsHS) (Chen et al., 1997), the lectins DC-SIGN and L-SIGN (Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003) and the laminin-binding protein (Thepparit & Smith, 2004; Tio et al., 2005). The structure of the complex between DENV and the carbohydrate-recognition domain of DC-SIGN has been determined by electron cryomicroscopy. This structure shows that DC-SIGN interacts with the glycosylation site of the ED2 domain, that the interaction involves two adjacent dimers of gpE and that ED3 remains available for interactions with other cellular receptors (Pokidysheva et al., 2006). Numerous data indicate that ED3 plays an important role in the infectious process (Hung et al., 2004; Se-Thoe et al., 2000; Thullier et al., 2001). In particular, many antibodies that are specific for ED3 are neutralizing (Crill & Roehrig, 2001; Roehrig, 2003). Of note, their epitopes are generally sensitive to elimination of the disulfide bond (Roehrig et al., 2004).
Monoclonal antibody mAb4E11 was raised against DENV1. Immunofluorescence experiments on cells of the mosquito Aedes albopictus, infected with various flaviviruses, and Western blot experiments on their total extracts have shown that mAb4E11 recognizes exclusively the viruses of the dengue group, and not other flaviviruses (Megret et al., 1992). Plaque-reduction assays, performed on monkey Vero cells, have shown that mAb4E11 neutralizes the four serotypes of DENV with varying efficacies (Thullier et al., 1999). ELISAs using truncated recombinant gpE proteins as antigens have shown that the epitope of mAb4E11 is included within the ED3 domain (Megret et al., 1992; Thullier et al., 1999). Selection of peptides from a random library by the method of phage display has shown that residues 306–314 of gpE from the DENV1 serotype (gpE.DEN1) constitute a mimotope of the mAb4E11 epitope (Thullier et al., 2001). Its paratope has been partially characterized by alanine-scanning mutagenesis of its third hypervariable loops (Bedouelle et al., 2006; Renard et al., 2003). This mutagenesis study of the Fab fragment of mAb4E11 has shown that the two residues that come from its diversity segment, H-Trp96 and H-Glu97 (Kabat's numbering), together provide >85 % of the free energy of interaction with the ED3.DEN1 antigen.
The objective of our study was to map the energetic epitope of mAb4E11 as completely and precisely as possible, by alanine-scanning mutagenesis of the ED3 domain. A comparison of the mapped epitope with the available sequence, structure and functional data has enabled us to propose structural mechanisms for the cross-recognition of the four DENV serotypes by mAb4E11, the exclusion of the other flaviviruses and its neutralization potency.
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METHODS |
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) and HB2151 (Carter et al., 1985), and plasmids pPE1 (Bedouelle et al., 2006), pET20b+ (Novagen) and those coding for the ED3 domain (Catteau et al., 2003), have been described previously. The viral strains used are listed in Table 1. pPE1 codes for a hybrid, Fab4E11-H6, between the Fab fragment of mAb4E11 and a hexahistidine tag. SB medium was described by Sambrook & Russell (2001). Ampicillin was added at 200 µg ml–1. Buffer A contained 20 mM Tris/HCl, 500 mM NaCl; buffer B contained 50 mM Tris/HCl, 150 mM NaCl. The pH of buffers A and B, 7.0 or 7.9, was chosen according to the protein and was distant by 
1 unit from its pI. PBS was purchased from Sigma-Aldrich.
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). The DNA segment of the E gene that encodes ED3 was amplified by PCR from plasmidic clones with oligonucleotide primers that introduced NcoI and XhoI restriction sites at its 5' and 3' ends, respectively. The PCR product was then inserted between the corresponding sites of pET20b+. For yellow fever virus (YFV), the NcoI site that pre-existed in the E gene was removed through a silent mutation before the PCR by changing codon GGC314 into GGT. For West Nile virus (WNV) and Japanese encephalitis virus (JEV), codons 400–406 or 399–405, respectively, of the E gene were absent from the parental plasmid clone. They were optimized for E. coli and reintroduced through one of the PCR primers. Mutations were constructed by site-directed mutagenesis with synthetic oligonucleotides and using previously published methods (Kunkel et al., 1987; Urban et al., 1997). All genetic constructs were verified by DNA sequencing.
Production and purification of proteins.
The Fab4E11-H6 fragment was produced in E. coli strain HB2151 and purified as described previously (Bedouelle et al., 2006). The ED3-H6 domains were produced in E. coli strain BL21(DE3) from recombinant plasmids (Table 1). The bacteria were grown at 24 °C in SB medium with ampicillin until A600 reached 1.5–2.0 and then induced for 2.5 h with 1.0 mM IPTG to obtain expression of the recombinant gene. The following steps were performed at 4 °C in buffer A. The bacteria were harvested, resuspended in 1 mg polymyxin B sulfate (Sigma-Aldrich) ml–1, 5 mM imidazole (25 ml for 1 l culture) with stirring for 1 h, then centrifuged at 15 000 g for 30 min. The supernatant (periplasmic fluid) was sonicated, filtered through a 0.22 µm pore filter and loaded onto a column of NiNTA resin [Qiagen; 1 ml (l culture)–1]. The bound protein was washed with 20 mM imidazole (20 vols resin), then eluted by a step gradient of imidazole. The protein fractions were analysed by SDS-PAGE under reducing conditions. The fractions that were homogeneous at >95 % were pooled, dialysed against buffer B and stored at –80 °C.
Protein characterization.
The concentration of the ED3-H6 domain was measured by spectrometry. The absorption coefficients were calculated as described by Pace et al. (1995). The concentration of the purified Fab4E11-H6 fragment was measured with a BioRad Protein Assay kit, using BSA (Roche) as a standard. The mass of ED3.DEN1-H6 was measured with an API 365 triple quadrupole mass spectrometer (Sciex) as follows. A purified preparation was dialysed extensively against 50 mM ammonium carbonate (pH 9.5) and then lyophilized. The sample was dissolved in a mixture of water, methanol and formic acid, and the measurement of its mass was performed exactly as described previously (Renard et al., 2002).
Fluorescence experiments were performed with 1 µM protein at 20 °C in buffer B. The wavelength of the excitation light was 278 nm. The wavelength at which the intensity of the emitted light was maximal (
max) was obtained from the fluorescence spectra exactly as described previously (Monsellier & Bedouelle, 2005).
Ellman's test was performed as described by Creighton (1989). Each reaction contained >3 µM protein. The amino acid Cys was used as a standard to establish a calibration curve, and BSA, which contains one free residue of Cys, was used as a positive control. The formation of nitrobenzoate from 5,5'-dithionitrobenzoic acid (Sigma-Aldrich) was measured from A412. The lower limit of detection of the spectrophotometer was equal to an A412 of 0.005 and corresponded to 0.44 µM free Cys.
Indirect ELISA.
ELISA experiments were performed in PBS and microtitre plates as described by Harlow & Lane (1988), except that the wells of the plates were washed three times with 0.05 % (v/v) Tween in PBS and three times with PBS alone between each step. The wells were coated with 1 µg ED3-H6 domain ml–1 and blocked with 3 % BSA (w/v). Measurements were performed at 25 °C in 1 % BSA. The immobilized domains were incubated with 0.4 nM Fab4E11-H6 for 1 h, except for the blank wells. The captured molecules of Fab4E11-H6 were revealed successively with an antibody specific for the Fab fragments of mouse IgG and conjugated with biotin, a conjugate between streptavidin and alkaline phosphatase, and p-nitrophenyl phosphate as a substrate (all from Sigma-Aldrich). A405 was measured and corrected by subtraction of the blank.
Determination of the equilibrium constants by competition ELISA.
The dissociation constants at equilibrium in solution, KD, between the Fab4E11-H6 fragment and the ED3-H6 antigens or their mutant derivatives were measured by a competition ELISA (Friguet et al., 1985). The assay was performed at 25 °C in PBS containing 1 % BSA. Fab4E11-H6 at a constant concentration (0.2 nM) and the ED3-H6 derivative at 12 different concentrations were first incubated together in solution for 20 h, to reach equilibrium. The concentration of free Fab4E11-H6 was then measured by an indirect ELISA in a microtitre plate whose wells had been coated with a 0.3 µg ml–1 solution of ED3.DEN1-H6. The bound molecules of Fab4E11-H6 were revealed with a goat antibody specific for the Fab fragment of mouse IgG and conjugated with either alkaline phosphatase or biotin. In the latter case, the bound antibody was revealed with a conjugate between streptavidin and alkaline phosphatase (all from Sigma-Aldrich). The raw ELISA data were processed as follows. Let us consider the equilibrium of association and dissociation between an antigen L and an antibody P to give the complex L : P. If [L]0 is the total concentration of L in the reaction, [P]0 the total concentration of P, [P] the concentration of the free molecules of P and KD the dissociation constant of the complex L : P, then the laws of mass action and conservation imply that [P] is the solution of the following equation (Rondard et al., 1997):
[P]2+(KD+[L]0–[P]0)[P]–KD[P]0=0 (equation 1)
The indirect ELISA for measuring [P] was performed in conditions such that:
A=A
+(A0–A)[P]/[P]0 (equation 2)
where A was the absorbance at 405 nm. We fitted equation 2 to the experimental values of A, with the solution of equation 1 as the expression of [P], 2x10–10 M for [P]0, 12 different and fixed concentrations for [L]0, and KD, A0 and A as floating parameters.
The free energy of dissociation between L and P is then equal to 
G=–RTln(KD), and the variation of free energy on mutation of L is equal to G=G(wt)–G(mut), where wt and mut refer to wild-type and mutant molecules of L. The standard error (SEM) of G was calculated from the SEM values of G by using the formula:
[SEM(G)]2=[SEM{G(wt)}]2+[SEM{G(mut)}]2 (equation 3)
Structural analyses.
The crystallographic structures of the gpE homodimer (PDB accession no. 1oan) and homotrimer (PDB accession no. 1ok8) from DENV2 (Modis et al., 2003, 2004), and that of the gpE homodimer from WNV (PDB accession no. 1ztx) (Kanai et al., 2006), were analysed with the WHAT IF program (Vriend, 1990). Computations were performed with the KALEIDAGRAPH software (Synergy Inc.).
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). These plasmids coded for hybrids ED3-H6 between ED3 and a hexahistidine tag under control of the phage T7 promoter and pelB signal sequence. The ED3-H6 domains and their mutant derivatives (see below) were purified from periplasmic extracts by affinity chromatography on a nickel-ion column. Analysis by SDS-PAGE under reducing conditions showed that their apparent and theoretical molecular masses (between 12 017 and 12 891 Da) were in agreement.
Structural integrity of the parental ED3 domains
The SignalP program (Bendtsen et al., 2004) predicted that the products of the recombinant genes carried by plasmids pLB11, pVP4, pVP5 and pVP6 would be cleaved between the signal peptide and the first residue of the ED3 domain (Table 1). An analysis of the purified ED3.DEN1-H6 protein by mass spectrometry gave molecular masses equal to 12 472.69±0.63 and 12 603.69±0.50 Da. These experimental masses were equal to the theoretical masses for ED3.DEN1-H6 domains whose disulfide bond was formed correctly and whose N-terminal residue was either Gly296 or Met-Gly296 (12 472.20 and 12 603.40 Da, respectively). They did not correspond to the theoretical mass of an ED3.DEN1-H6 domain with reduced Cys residues. We confirmed the absence of free Cys residues in our purified preparations of the ED3-H6 domains from DENV1, WNV, JEV and YFV by Ellman's test. The proportion of reactive Cys residues was below the lower limit of detection under our experimental conditions and corresponded to >94 % of the ED3-H6 molecules with a disulfide bond in all cases (see Methods).
Fluorescence of Trp and Tyr residues varies with the folding state of proteins. The ED3.DEN1-H6 domain comprises a unique and conserved residue of Trp, in position 391, and three residues of Tyr. The fluorescence intensity of ED3.DEN1-H6 decreases by 2.5-fold between 0 and 8 M urea upon excitation at 278 nm and its max shifts from 340 to 350 nm. These variations of the fluorescence intensity and max are consistent with the unfolding of ED3.DEN1-H6 by urea, the partial burial of Trp391 in the interior of the folded domain, its full exposure to the solvent in the unfolded domain and the solvent-accessible surface area of its side chain (20.7 %) in the crystal structure of gpE.DEN2 (Modis et al., 2003; Monsellier & Bedouelle, 2005). Thus, the native ED3.DEN1-H6 domain had the expected molecular mass, disulfide bond and partial burial of Trp391 in the interior of a folded structure, according to the above biochemical and biophysical criteria.
Recognition between Fab4E11 and four flaviviruses
We used a recombinant Fab fragment of mAb4E11 in our recognition experiments to avoid the problems associated with the bivalency of a whole antibody. We measured the constant of dissociation, KD, at 25 °C in solution between the Fab4E11-H6 fragment and the wild-type ED3.DEN1-H6(wt) domain by a competition ELISA in solution, in which the concentration of the antigen varied (Fig. 1; see Methods). The value of KD was equal to 0.12±0.01 nM and was fully consistent with the value that we obtained previously with another recombinant form of the antigen (Bedouelle et al., 2006).
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). The wells of a microtitre plate were coated with a high concentration of ED3-H6 (1 µg ml–1). At the end of a reaction period that was prolonged intentionally (3 h at 20 °C and then 20 h at 4 °C), the ELISA signal was equal to 3.4±0.4 (mean±SEM) for ED3.DEN1-H6, 0.025±0.004 for ED3.JE-H6 and lower for ED3.WN-H6 and ED3.YF-H6. Therefore, the signals for JEV, WNV and YFV were <1 % of the signal for DENV1. These results showed that Fab4E11-H6 did not recognize the ED3 domains of the three non-cognate flaviviruses.
Energetic contributions of gpE residues 306–314 to the epitope
A synthetic nonapeptide, corresponding to residues 306–314 of gpE.DEN1, constitutes a mimotope of the mAb4E11 epitope (see Introduction). To analyse the energetic contributions of these nine residues to the binding of mAb4E11 in the environment of the ED3.DEN1-H6 domain, we changed them individually into Ala by site-directed mutagenesis, except for Ala313, which was changed into Gly. We produced and purified the mutant ED3.DEN1-H6 domains with yields similar to that for the wild type, except for the F306A mutant, which we were unable to produce. We determined the KD values for the interaction between the mutant ED3.DEN1-H6 domains and Fab4E11-H6 as for the wild type (Table 2). The values of KD and the corresponding free energies of interaction, G, showed that residues 307–312 of gpE.DEN1 belonged to the energetic epitope of Fab4E11 [0.5<G<5.0 kcal mol–1 (1 kcal mol–1=4.184 kJ mol–1)], contrary to residues 313 and 314. Three residues, Lys307, Leu308 and Lys310, constituted hot spots of binding energy (G>1.5 kcal mol–1). The sum of the G values for the mutations of residues 307–312 was equal to 13.5 kcal mol–1 and was therefore close to the free energy of interaction between the wild-type ED3.DEN1-H6 domain and Fab4E11-H6, G=13.6 kcal mol–1 (Table 2). This comparison showed that residues 307–312 constituted a major part of the mAb4E11 epitope.
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). Second, we have shown in a previous study that the diversity segment of mAb4E11 comprises two residues, H-Trp96 and H-Glu97; these two residues are major contributors to the interaction with ED3.DEN1 (G6 kcal mol–1 for each mutation into Ala), and they are highly exposed to the solvent in a three-dimensional model of the Fab4E11 fragment (Bedouelle et al., 2006). Thus, the epitope of mAb4E11 should contain both positively charged residues to interact with the negatively charged H-Glu97, and a hydrophobic pocket to bind H-Trp96.
Our alanine-scanning mutagenesis of residues 306–314 of the ED3.DEN1-H6 domain showed that the positively charged residues Lys307 and Lys310 and the hydrophobic residue Leu308 of gpE.DEN1 were hot spots of binding energy (G=3.9, 5.0 and 2.1 kcal mol–1, respectively). We therefore analysed the structural environment of residue 308 in the crystal structure of gpE.DEN2 (Modis et al., 2003). We observed that it belongs to a major hydrophobic cluster at the surface of gpE.DEN2, including Ile308 and Ile312 on one -strand and Leu387, Leu389 and Trp391 on an adjacent and parallel -strand (Fig. 2).
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). We found that three hydrophobic residues, Leu387, Leu389 and Trp391, contributed significantly to the energy of interaction with Fab4E11-H6 (G>0.5 kcal mol–1) and that Leu389 constituted a hot spot of binding energy (G=1.8±0.2 kcal mol–1). These results showed that the epitope of mAb4E11 was discontinuous.
Epitope transplantation
The proportion of identical amino acid residues between gpE of DENV and those of other flaviviruses is about 40 %. As expected at this level of identity, the structures of the ED3 domains from DENV2, DENV3, DENV4, JEV and WNV, solved by nuclear magnetic resonance or X-ray crystallography, are similar (Kanai et al., 2006; Modis et al., 2003, 2005; Nybakken et al., 2005; Volk et al., 2004, 2007; Wu et al., 2003). Among the nine residues that belonged to the energetic epitope of mAb4E11 in ED3.DEN1-H6, six are different in YFV and seven in JEV and WNV (Table 3). To confirm that these nine residues belong to the epitope of mAb4E11, to test whether they constitute its whole epitope and to check whether they are responsible for its specificity of recognition, we introduced them at the homologous positions of the ED3.JE-H6, ED3.WN-H6 and ED3.YF-H6 domains by site-directed mutagenesis. We measured the KD values between the Fab4E11-H6 fragment and the chimeric domains by competition ELISA in solution (Table 3). These values (4.0, 5.9 and 204 nM, respectively) showed that it was possible to introduce the discontinuous epitope of mAb4E11 into different viral backgrounds and yet maintain its recognition by Fab4E11-H6 with a low-nanomolar affinity. This showed the quasi-completeness of the mapped epitope.
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). We found that the signal of interaction between the Fab4E11-H6 fragment and the purified mutant domain ED3.DEN1-H6(L308F, V312P, L389H) was <1 % of the signal for the wild-type domain ED3.DEN1-H6(wt) in an indirect ELISA (see Methods). This result showed that the introduction of bulky cyclic residues at these three positions of the mAb4E11 epitope abolished the interaction between its Fab fragment and ED3.DEN1-H6.
Structural integrity of the ED3.DEN1-H6 derivatives
We could not produce the F306A mutant of the ED3.DEN1-H6 domain in E. coli. The side chain of residue Phe306 is fully buried in the crystal structure of the gpE.DEN2 protein and interacts with the side chain of Cys302. Therefore, mutation F306A could prevent the correct folding of ED3.DEN1-H6 and hence its production. More generally, some mutations might induce a misfolding of ED3.DEN1-H6 and thus affect the interaction with Fab4E11-H6 indirectly. To test this assumption, we performed Ellman's test on all mutant ED3.DEN1-H6 domains with G>0.5 kcal mol–1 and recorded their fluorescence spectra. We found results in Ellman's test and the fluorescence spectra that were identical for the wild-type and mutant ED3.DEN1-H6 domains, except for the spectrum of the W391A mutant, which did not contain any Trp residue. Therefore, the mutations that we introduced, including W391A, did not grossly modify the folding of ED3.DEN1-H6, and the measured variations of the KD and G values resulted from local effects of the side-chain change.
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G>0.5 kcal mol–1): residues 307–312, 387, 389 and 391. Residues Lys307, Leu308, Lys310 and Leu389 constituted hot spots of binding energy (G>1.5 kcal mol–1). In the crystal structure of gpE.DEN2, the polypeptide backbone of residues 306–314 associates with and is antiparallel to that of residues 320–326, at the edge of a first -sheet. Similarly, the backbone of residues 387–393 is antiparallel to that of residues 374–380 at the edge of a second -sheet. Consequently, the epitope of mAb4E11 straddles the edges of two -sheets that associate through tertiary interactions (Fig. 2a). It is a discontinuous and conformational epitope. The structure shows that the complete epitope is formed only if ED3.DEN1 is folded properly, and we verified that none of the mutations that we constructed, except F306A, affected this folding.
Residues Lys307 (restricted to the aliphatic portion of its side chain), Leu308 and Val312 are engaged in tertiary interactions with residues Leu387, Leu389 and Trp391 (Fig. 2b). These six residues form a hydrophobic pocket at the surface of the ED3 domain. Therefore, the epitope of mAb4E11 includes two basic residues, Lys307 and Lys310, and a hydrophobic pocket that could interact with the diversity residues, H-Trp96-Glu97, of mAb4E11. These two residues constitute the main hot spots of binding energy with the ED3.DEN1 antigen (Bedouelle et al., 2006).
Completeness of the epitope
The variations of the free energy of interation between ED3.DEN1 and Fab4E11 for the nine mutations that had significant effects (G>0.5 kcal mol–1) had a sum equal to 17.1 kcal mol–1. This sum comprised 13.5 kcal mol–1 coming from residues 307–312 and 3.6 kcal mol–1 from Leu387, Leu389 and Trp391. It was higher than the free energy of interaction, G=13.6 kcal mol–1, between ED3.DEN1 and Fab4E11. This comparison suggested that the nine identified residues constituted the entirety of the epitope and indicated that the effects of some mutations were not fully independent. We showed the total absence of recognition between Fab4E11 and the ED3 domains from JEV, WNV and YFV in indirect ELISA experiments. We introduced the nine residues of the mAb4E11 epitope from ED3.DEN1 into the equivalent positions of ED3.JE, ED3.WN and ED3.YF. As a result of these transplantations, the KD values for the interaction between Fab4E11 and either the chimeric ED3.JE/DEN1 domain or the chimeric ED3.WN/DEN1 domain were in the low-nanomolar range. These experiments confirmed the quasi-completeness of the mAb4E11 epitope, as characterized here. The values of KD were higher for the chimeric domains than for the wild-type ED3.DEN1 domain, probably because the epitope residues were in different structural contexts. Consistently, the polypeptide backbones of ED3.DEN2 and ED3.WN are not exactly superimposable in the corresponding crystal structures (Kanai et al., 2006; Modis et al., 2003).
The interaction between mAb4E11 and the synthetic peptide that corresponds to residues 306–314 of gpE.DEN1 has a KD value equal to 3 µM and an associated G value equal to 7.5 kcal mol–1 (Thullier et al., 2001). Therefore, the sum of the G values for residues 307–312 of the ED3.DEN1 domain was higher than the value of G for the linear mimotope by 6 kcal mol–1. This comparison indicated that the synthetic peptide mimics residues 307–312 of ED3.DEN1 only partially and that the structural context of these residues is important for their recognition by mAb4E11. Consistently, residues 309–311 form a bulge outside -strand 306–314 and the surface of gpE (Fig. 2).
Specificity for the DENV group
We analysed the sequences and structures of ED3 domains from representative flaviviruses in the region of the epitope to understand the specificity of mAb4E11 towards DENV and its discrimination against the other flaviviruses (Figs 2 and 3). Residues 307 and 309 of DENV1 are variable among DENV; therefore, they cannot be involved in the cross-reactivity of mAb4E11 towards the four serotypes of DENV. Residues 310, 387 and 391 of DENV1 are conserved among the flaviviruses and take the values Lys/Arg, Leu/Ile and Trp, respectively. Therefore, they cannot be involved in the specificity of mAb4E11 for the flaviviral group of DENV. In contrast, the triad of aliphatic residues Leu/Ile308, Val/Ile/Met312 and Leu/Ile389, which participate in the formation of a hydrophobic pocket at the surface of the DENV virion, is replaced by bulky cyclic residues, Trp/Phe, Pro and His/Tyr, respectively, in the other flaviviruses. These bulky residues could prevent the binding of a hydrophobic residue, e.g. the diversity residue H-Trp96 of mAb4E11. Consistently, we found that the triple mutation (L308F, V312P, L389H) of the ED3.DEN1 domain abolished its recognition by the Fab4E11 fragment. Thus, the identities of the residues in positions 308, 312 and 389 could explain both the cross-reactivity of mAb4E11 towards the four serotypes of DENV and the rejection of the other flaviviruses.
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). Studies of natural and engineered variants of DENV2 have shown that residue 390 of gpE controls the replicative efficiency of the virus in cultured cells and its virulence in the mouse (Pryor et al., 2001; Sanchez & Ruiz, 1996). Antibody mAb4E11, which interacts with the neighbouring residues in positions 389 and 391, could prevent essential interactions between the virus and some cellular components by steric hindrance and thus be neutralizing. Interestingly, the epitope of mAb4E11 remains exposed to the solvent in the trimeric post-fusion structure of gpE (Modis et al., 2004).
The infectivity of DENV2 may depend on the binding of gpE to hsHS decorating the surface of some cell types (Chen et al., 1997). Moreover, the ED3.DEN2 and ED3.DEN4 domains bind hsHS in vitro and four conserved Lys residues have been implicated in this interaction, including Lys310 (Hung et al., 2004; Pattnaik et al., 2007). The binding of mAb4E11 to gpE could therefore sterically hinder the interaction between hsHS molecules and gpE.
Comparison with neutralizing epitopes in other flaviviruses
Several neutralizing epitopes have been mapped in the ED3 domain of flaviviruses through point mutations, synthetic peptides and structural studies of complexes between antibodies and antigens (Table 4). Here, we used alanine-scanning mutagenesis because it is the most sure and most well-documented method to map functional epitopes (Greenspan & Di Cera, 1999; Rondard & Bedouelle, 1998). We used isolated ED3 domains as antigens because their conformation is independent of concentration, which varied between 10 pM and 10 µM in our experiments, whereas the oligomerization state and conformation of the corresponding gpE ectodomains vary between flaviviruses and with concentration, which would preclude reliable measurements and comparisons of KD values (Kanai et al., 2006; Modis et al., 2004). Moreover, we checked that the mutations did not affect the structure of the ED3.DEN1 domain by using two sensitive conformational probes that were located in proximity to the epitope, i.e. the formation of a disulfide bond between Cys302 and Cys333 and the fluorescence of Trp391. The available data on the neutralizing epitopes of other flaviviruses show that some antibodies interact with the same regions as mAb4E11 (italicized residues in Table 4). However, none of these antibodies makes functional interactions with the residues that are homologous to Leu387, Leu389 or Trp391 in DENV1, and none makes functional interactions simultaneously with the two polypeptide segments that together form the epitope of mAb4E11. Therefore, the mechanism of recognition between mAb4E11 and gpE seems fully novel in the flaviviruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bedouelle, H., Belkadi, L., England, P., Guijarro, J. I., Lisova, O., Urvoas, A., Delepierre, M. & Thullier, P. (2006). Diversity and junction residues as hotspots of binding energy in an antibody neutralizing the dengue virus. FEBS J 273, 34–46.[CrossRef][Medline]
Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783–795.[CrossRef][Medline]
Bhardwaj, S., Holbrook, M., Shope, R. E., Barrett, A. D. & Watowich, S. J. (2001). Biophysical characterization and vector-specific antagonist activity of domain III of the tick-borne flavivirus envelope protein. J Virol 75, 4002–4007.
Buchen-Osmond, C. (2003). The universal virus database ICTVdB. Comput Sci Eng 5, 16–25.
Carter, P., Bedouelle, H. & Winter, G. (1985). Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13, 4431–4443.
Catteau, A., Kalinina, O., Wagner, M. C., Deubel, V., Courageot, M. P. & Despres, P. (2003). Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM. J Gen Virol 84, 2781–2793.[CrossRef]
Cecilia, D. & Gould, E. A. (1991). Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants. Virology 181, 70–77.[CrossRef][Medline]
Chambers, T. J., Halevy, M., Nestorowicz, A., Rice, C. M. & Lustig, S. (1998). West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness. J Gen Virol 79, 2375–2380.[Abstract]
Chen, Y., Maguire, T., Hileman, R. E., Fromm, J. R., Esko, J. D., Linhardt, R. J. & Marks, R. M. (1997). Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med 3, 866–871.[CrossRef][Medline]
Creighton, T. E. (1989). Disulphide bonds between cysteine residues. In Protein Structure: a Practical Approach, pp. 155–168. Edited by T. E. Creighton. Oxford: IRL Press.
Crill, W. D. & Roehrig, J. T. (2001). Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol 75, 7769–7773.
Friguet, B., Chaffotte, A. F., Djavadi-Ohaniance, L. & Goldberg, M. E. (1985). Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 77, 305–319.[CrossRef][Medline]
Gao, G. F., Hussain, M. H., Reid, H. W. & Gould, E. A. (1994). Identification of naturally occurring monoclonal antibody escape variants of louping ill virus. J Gen Virol 75, 609–614.
Goncalvez, A. P., Purcell, R. H. & Lai, C. J. (2004). Epitope determinants of a chimpanzee Fab antibody that efficiently cross-neutralizes dengue type 1 and type 2 viruses map to inside and in close proximity to fusion loop of the dengue type 2 virus envelope glycoprotein. J Virol 78, 12919–12928.
Greenspan, N. S. & Di Cera, E. (1999). Defining epitopes: it's not as easy as it seems. Nat Biotechnol 17, 936–937.[CrossRef][Medline]
Gubler, D. J. (2002). Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol 10, 100–103.[CrossRef][Medline]
Guzman, M. G. & Kouri, G. (2002). Dengue: an update. Lancet Infect Dis 2, 33–42.[CrossRef][Medline]
Halstead, S. B. (2003). Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 60, 421–467.[CrossRef][Medline]
Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Heinz, F. X., Collett, M. S., Purcell, R. H., Gould, E. A., Howard, C. R., Houghton, M., Moormann, R. J. M., Rice, C. M. & Thiel, H. J. (2000). Family Flaviviridae. In Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses, pp. 859–878. Edited by M. V. E. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego, CA: Academic Press.
Hiramatsu, K., Tadano, M., Men, R. & Lai, C. J. (1996). Mutational analysis of a neutralization epitope on the dengue type 2 virus (DEN2) envelope protein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit reduced mouse neurovirulence. Virology 224, 437–445.[CrossRef][Medline]
Holzmann, H., Heinz, F. X., Mandl, C. W., Guirakhoo, F. & Kunz, C. (1990). A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model. J Virol 64, 5156–5159.
Hung, J. J., Hsieh, M. T., Young, M. J., Kao, C. L., King, C. C. & Chang, W. (2004). An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells. J Virol 78, 378–388.
Jennings, A. D., Gibson, C. A., Miller, B. R., Mathews, J. H., Mitchell, C. J., Roehrig, J. T., Wood, D. J., Taffs, F., Sil, B. K. & other authors (1994). Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis. J Infect Dis 169, 512–518.[Medline]
Jiang, W. R., Lowe, A., Higgs, S., Reid, H. & Gould, E. A. (1993). Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence. J Gen Virol 74, 931–935.
Jiang, W., Bonnert, T. P., Venugopal, K. & Gould, E. A. (1994). A single chain antibody fragment expressed in bacteria neutralizes tick-borne flaviviruses. Virology 200, 21–28.[CrossRef][Medline]
Kanai, R., Kar, K., Anthony, K., Gould, L. H., Ledizet, M., Fikrig, E., Marasco, W. A., Koski, R. A. & Modis, Y. (2006). Crystal structure of West Nile virus envelope glycoprotein reveals viral surface epitopes. J Virol 80, 11000–11008.
Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154, 367–382.[Medline]
Leitmeyer, K. C., Vaughn, D. W., Watts, D. M., Salas, R., Villalobos, I., de Chacon, Ramos, C. & Rico-Hesse, R. (1999). Dengue virus structural differences that correlate with pathogenesis. J Virol 73, 4738–4747.
Li, L., Barrett, A. D. & Beasley, D. W. (2005). Differential expression of domain III neutralizing epitopes on the envelope proteins of West Nile virus strains. Virology 335, 99–105.[CrossRef][Medline]
Lin, C. W. & Wu, S. C. (2003). A functional epitope determinant on domain III of the Japanese encephalitis virus envelope protein interacted with neutralizing-antibody combining sites. J Virol 77, 2600–2606.
Lin, B., Parrish, C. R., Murray, J. M. & Wright, P. J. (1994). Localization of a neutralizing epitope on the envelope protein of dengue virus type 2. Virology 202, 885–890.[CrossRef][Medline]
Lok, S. M., Ng, M. L. & Aaskov, J. (2001). Amino acid and phenotypic changes in dengue 2 virus associated with escape from neutralisation by IgM antibody. J Med Virol 65, 315–323.[CrossRef][Medline]
Mandl, C. W., Guirakhoo, F., Holzmann, H., Heinz, F. X. & Kunz, C. (1989). Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. J Virol 63, 564–571.
Megret, F., Hugnot, J. P., Falconar, A., Gentry, M. K., Morens, D. M., Murray, J. M., Schlesinger, J. J., Wright, P. J., Young, P. & other authors (1992). Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein. Virology 187, 480–491.[CrossRef][Medline]
Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. (2003). A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A 100, 6986–6991.
Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. (2004). Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313–319.[CrossRef][Medline]
Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. (2005). Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein. J Virol 79, 1223–1231.
Mongkolsapaya, J., Dejnirattisai, W., Xu, X. N., Vasanawathana, S., Tangthawornchaikul, N., Chairunsri, A., Sawasdivorn, S., Duangchinda, T., Dong, T. & other authors (2003). Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med 9, 921–927.[CrossRef][Medline]
Monsellier, E. & Bedouelle, H. (2005). Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength. Protein Eng Des Sel 18, 445–456.
Navarro-Sanchez, E., Altmeyer, R., Amara, A., Schwartz, O., Fieschi, F., Virelizier, J. L., Arenzana-Seisdedos, F. & Despres, P. (2003). Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses. EMBO Rep 4, 723–728.[CrossRef][Medline]
Niedrig, M., Klockmann, U., Lang, W., Roeder, J., Burk, S., Modrow, S. & Pauli, G. (1994). Monoclonal antibodies directed against tick-borne encephalitis virus with neutralizing activity in vivo. Acta Virol 38, 141–149.[Medline]
Nybakken, G. E., Oliphant, T., Johnson, S., Burke, S., Diamond, M. S. & Fremont, D. H. (2005). Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 437, 764–769.[CrossRef][Medline]
Oliphant, T., Engle, M., Nybakken, G. E., Doane, C., Johnson, S., Huang, L., Gorlatov, S., Mehlhop, E., Marri, A. & other authors (2005). Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 11, 522–530.[CrossRef][Medline]
Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4, 2411–2423.[Medline]
Pattnaik, P., Babu, J. P., Verma, S. K., Tak, V. & Rao, P. V. (2007). Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate. J Chromatogr B Analyt Technol Biomed Life Sci 846, 184–194.[CrossRef][Medline]
Pokidysheva, E., Zhang, Y., Battisti, A. J., Bator-Kelly, C. M., Chipman, P. R., Xiao, C., Gregorio, G. G., Hendrickson, W. A., Kuhn, R. J. & Rossmann, M. G. (2006). Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN. Cell 124, 485–493.[CrossRef][Medline]
Pryor, M. J., Carr, J. M., Hocking, H., Davidson, A. D., Li, P. & Wright, P. J. (2001). Replication of dengue virus type 2 in human monocyte-derived macrophages: comparisons of isolates and recombinant viruses with substitutions at amino acid 390 in the envelope glycoprotein. Am J Trop Med Hyg 65, 427–434.[Abstract]
Renard, M., Belkadi, L., Hugo, N., England, P., Altschuh, D. & Bedouelle, H. (2002). Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies. J Mol Biol 318, 429–442.[CrossRef][Medline]
Renard, M., Belkadi, L. & Bedouelle, H. (2003). Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors. J Mol Biol 326, 167–175.[CrossRef][Medline]
Roehrig, J. T. (2003). Antigenic structure of flavivirus proteins. Adv Virus Res 59, 141–175.[CrossRef][Medline]
Roehrig, J. T., Bolin, R. A. & Kelly, R. G. (1998). Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology 246, 317–328.[CrossRef][Medline]
Roehrig, J. T., Volpe, K. E., Squires, J., Hunt, A. R., Davis, B. S. & Chang, G. J. (2004). Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein. J Virol 78, 2648–2652.
Rondard, P. & Bedouelle, H. (1998). A mutational approach shows similar mechanisms of recognition for the isolated and integrated versions of a protein epitope. J Biol Chem 273, 34753–34759.
Rondard, P., Goldberg, M. E. & Bedouelle, H. (1997). Mutational analysis of an antigenic peptide shows recognition in a loop conformation. Biochemistry 36, 8962–8968.[CrossRef][Medline]
Ryman, K. D., Ledger, T. N., Campbell, G. A., Watowich, S. J. & Barrett, A. D. (1998). Mutation in a 17D–204 vaccine substrain-specific envelope protein epitope alters the pathogenesis of yellow fever virus in mice. Virology 244, 59–65.[CrossRef][Medline]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Press.
Sanchez, I. J. & Ruiz, B. H. (1996). A single nucleotide change in the E protein gene of dengue virus 2 Mexican strain affects neurovirulence in mice. J Gen Virol 77, 2541–2545.
Schlesinger, J. J., Chapman, S., Nestorowicz, A., Rice, C. M., Ginocchio, T. E. & Chambers, T. J. (1996). Replication of yellow fever virus in the mouse central nervous system: comparison of neuroadapted and non-neuroadapted virus and partial sequence analysis of the neuroadapted strain. J Gen Virol 77, 1277–1285.
Se-Thoe, S. Y., Ling, A. E. & Ng, M. M. (2000). Alteration of virus entry mode: a neutralisation mechanism for dengue-2 virus. J Med Virol 62, 364–376.[CrossRef][Medline]
Seif, S. A., Morita, K. & Igarashi, A. (1996). A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice. Virus Res 43, 91–96.[CrossRef][Medline]
Serafin, I. L. & Aaskov, J. G. (2001). Identification of epitopes on the envelope (E) protein of dengue 2 and dengue 3 viruses using monoclonal antibodies. Arch Virol 146, 2469–2479.[CrossRef][Medline]
Tassaneetrithep, B., Burgess, T. H., Granelli-Piperno, A., Trumpfherer, C., Finke, J., Sun, W., Eller, M. A., Pattanapanyasat, K., Sarasombath, S. & other authors (2003). DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med 197, 823–829.
Thepparit, C. & Smith, D. R. (2004). Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor. J Virol 78, 12647–12656.
Thullier, P., Lafaye, P., Megret, F., Deubel, V., Jouan, A. & Mazie, J. C. (1999). A recombinant Fab neutralizes dengue virus in vitro. J Biotechnol 69, 183–190.[CrossRef][Medline]
Thullier, P., Demangel, C., Bedouelle, H., Megret, F., Jouan, A., Deubel, V., Mazie, J. C. & Lafaye, P. (2001). Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism. J Gen Virol 82, 1885–1892.
Tio, P. H., Jong, W. W. & Cardosa, M. J. (2005). Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3. Virol J 2, 25[CrossRef][Medline]
Urban, A., Neukirchen, S. & Jaeger, K. E. (1997). A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR. Nucleic Acids Res 25, 2227–2228.
Volk, D. E., Beasley, D. W., Kallick, D. A., Holbrook, M. R., Barrett, A. D. & Gorenstein, D. G. (2004). Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus. J Biol Chem 279, 38755–38761.
Volk, D. E., Lee, Y. C., Li, X., Thiviyanathan, V., Gromowski, G. D., Li, L., Lamb, A. R., Beasley, D. W., Barrett, A. D. & Gorenstein, D. G. (2007). Solution structure of the envelope protein domain III of dengue-4 virus. Virology 364, 147–154.[CrossRef][Medline]
Vriend, G. (1990). WHAT IF: a molecular modeling and drug design program. J Mol Graph 8, 52–56.[CrossRef][Medline]
Wu, S. C. & Lin, C. W. (2001). Neutralizing peptide ligands selected from phage-displayed libraries mimic the conformational epitope on domain III of the Japanese encephalitis virus envelope protein. Virus Res 76, 59–69.[CrossRef][Medline]
Wu, S. C., Lian, W. C., Hsu, L. C. & Liau, M. Y. (1997). Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res 51, 173–181.[CrossRef][Medline]
Wu, K. P., Wu, C. W., Tsao, Y. P., Kuo, T. W., Lou, Y. C., Lin, C. W., Wu, S. C. & Cheng, J. W. (2003). Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein. J Biol Chem 278, 46007–46013.
Zhang, W., Chipman, P. R., Corver, J., Johnson, P. R., Zhang, Y., Mukhopadhyay, S., Baker, T. S., Strauss, J. H., Rossmann, M. G. & Kuhn, R. J. (2003). Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 10, 907–912.[CrossRef][Medline]
Received 25 March 2007;
accepted 11 June 2007.
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trace of the ED3.DEN2 domain. Residues 307–312 and 387–391 are represented in black. (b) Epitope in the structure of gpE.DEN2. The nine residues of the epitope are labelled. The interface between residues 307–312 and 387–391 forms a hydrophobic pocket. Note that several residues of the epitope differ between DENV1 and DENV2 (Fig. 3