Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein.
ABSTRACT Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced widely enough (at least 32 A) that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.
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ABSTRACT: Dengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Secondary infection by a different serotype is a risk factor for severe dengue. Monoclonal antibody mAb4E11 neutralizes the four serotypes of DENV with varying efficacies by recognizing an epitope located within domain-III (ED3) of the viral envelope (E) protein. To better understand the cross-reactivities between mAb4E11 and the four serotypes of DENV, we constructed mutations in both Fab4E11 fragment and ED3, and we searched for indirect interactions in the crystal structures of the four complexes. According to the serotype, 7 to 12 interactions are mediated by one water molecule, 1 to 10 by two water molecules, and several of these interactions are conserved between serotypes. Most interfacial water molecules make hydrogen bonds with both antibody and antigen. Some residues or atomic groups are engaged in both direct and water-mediated interactions. The doubly-indirect interactions are more numerous in the complex of lowest affinity. The third complementarity determining region of the light chain (L-CDR3) of mAb4E11 does not contact ED3. The structures and double-mutant thermodynamic cycles showed that the effects of (hyper)-mutations in L-CDR3 on affinity were caused by conformational changes and indirect interactions with ED3 through other CDRs. Exchanges of residues between ED3 serotypes showed that their effects on affinity were context dependent. Thus, conformational changes, structural context, and indirect interactions should be included when studying cross-reactivity between antibodies and different serotypes of viral antigens for a better design of diagnostics, vaccine, and therapeutic tools against DENV and other Flaviviruses. Copyright © 2014 John Wiley & Sons, Ltd.Journal of Molecular Recognition 04/2014; 27(4):205-14. · 3.01 Impact Factor
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ABSTRACT: Several dengue vaccine candidates have been evaluated in early clinical phase, and some are scheduled for efficacy testing in population-based studies. Given the advancements in dengue vaccine development, there is an increased interest in identifying immunological correlates of protection for these vaccines in order to facilitate their evaluation, further refinement, production and registration. To this end, the WHO Initiative for Vaccine Research (IVR) convened a consultation on primary and secondary immunological correlates of protection induced by dengue vaccines. The meeting was held on the 17th and 18th of November, 2005 at WHO headquarters in Geneva. The consultation was a first dedicated review of the available data in support of establishing correlates. It is concluded that it is not yet possible to define one specific set of correlates, the consultation concluded in recommendations that should help to gather the missing evidence in conjunction with future vaccine trials.Vaccine 06/2007; 25(21):4130-9. · 3.49 Impact Factor
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ABSTRACT: Dengue is among the most prevalent and important arbovirus diseases of humans. To effectively control this rapidly spreading disease, control of the vector mosquito and a safe and efficacious vaccine are critical. Despite considerable efforts, the development of a successful vaccine has remained elusive. Multiple factors have complicated the creation of a successful vaccine, not the least of which are the complex, immune-mediated responses against four antigenically distinct serotypes necessitating a tetravalent vaccine providing long-lasting protective immunity. Despite the multiple impediments, there are currently many promising vaccine candidates in preclinical and clinical development. Here, the recent advances in dengue virus vaccine development are reviewed and the challenges associated with the use of these vaccines as a public health tool are briefly discussed.Expert Review of Vaccines 08/2013; 12(8):933-53. · 4.22 Impact Factor
JOURNAL OF VIROLOGY, Jan. 2005, p. 1223–1231
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 2
Variable Surface Epitopes in the Crystal Structure of Dengue Virus
Type 3 Envelope Glycoprotein†
Yorgo Modis,1Steven Ogata,2David Clements,2and Stephen C. Harrison1*
Howard Hughes Medical Institute, Children’s Hospital and Harvard Medical School, Boston, Massachusetts,1
and Hawaii Biotech, Inc., Aiea, Hawaii2
Received 2 July 2004/Accepted 11 August 2004
Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral
attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes
enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein
E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and
tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are
all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against
epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize
serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably
inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced
widely enough (at least 32 Å) that they can interact with multiple carbohydrate recognition domains on
oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.
Dengue virus, a member of the flavivirus family, imposes one
of the largest social and economic burdens of any mosquito-
borne viral pathogen (12). There is no specific treatment for
infection, and control of dengue virus by vaccination has
proved elusive (5). The presence of multiple dengue virus
serotypes in circulation leads to an increase in pathogenesis
(10). The principal reason for this enhancement is thought to
be as follows (see reference 14 for a review). Structural differ-
ences at the viral surface in different serotypes mean that
neutralizing antibodies raised against one serotype are likely to
bind other serotypes with deceased affinity. As the affinity of an
antibody decreases, so does its ability to neutralize the virus.
Lower-affinity antibodies will fail to neutralize, but they may
still be present on virus particles in sufficient numbers to facil-
itate infection of cells bearing immunoglobulin G (IgG) Fc
receptors (15, 31). Alternatively, some high-affinity antibodies
that are neutralizing at high titers can enhance infection at
subneutralizing concentrations by facilitating cell uptake of
virus of the cognate serotype (3, 16, 38).
Flaviviruses other than dengue virus are important human
pathogens, including yellow fever, West Nile, tick-borne en-
cephalitis (TBE), and Japanese encephalitis viruses (5). Three
structural proteins (C, M, and E) and a lipid bilayer package
the positive-strand RNA genome (30). The core nucleocapsid
protein, C, assembles with RNA on the cytosolic face of the
endoplasmic reticulum membrane. The assembling core buds
through the endoplasmic reticulum membrane, thereby acquir-
ing an envelope that contains the major envelope glycoprotein,
E, and the so-called precursor membrane protein, PrM. The
particle passes through the secretory pathway, where a furin-
like protease cleaves PrM to M in a late trans-Golgi compart-
ment. The cleavage, which removes most of the ectodomain of
PrM, releases a constraint on E and primes the particle for
low-pH-triggered membrane fusion. Uncleaved, immature
particles are not fusion competent (5, 30). In addition to form-
ing the outer protein shell (24), E also binds a receptor on the
cell surface. Receptor binding directs the virion to the endo-
cytic pathway. E responds to the reduced pH of an endosome
by conformational rearrangement (36). The conformational
change induces fusion of the viral and host-cell membranes,
allowing the viral genome to enter the cytoplasm.
It is still unclear what receptor dengue virus recognizes on
the cell surface. Moreover, the virus may recognize different
receptors in human (host) and mosquito (vector) cells (18). It
has been proposed that patches of positively charged residues
on the surface of domain III bind to negatively charged hepa-
ran sulfate on the cell surface (6, 18). Many cell types express
heparan sulfate, however, and a more specific protein receptor
is thought to be required to target dengue virus to permissive
cell types, immature dendritic cells in particular (51). Glycans
on the virus surface have recently been implicated in receptor
binding by the finding that DC-SIGN, a mannose-specific, oli-
gomeric C-type lectin on the cell surface, is essential for pro-
ductive infection of dendritic cells (39, 48). These glycans must
belong to E, as it is the only glycoprotein on the surface of the
mature virion. DC-SIGN is specific to immature dendritic cells,
which have been proposed to be the primary target cells upon
introduction of dengue virus into the bloodstream (51).
The structures of the E protein ectodomains from dengue
virus type 2 (DEN-2) and from TBE virus have been deter-
mined by X-ray crystallography (35, 44). We now report the
structure of a soluble fragment (residues 1 to 393) of the E
protein from DEN-3. This fragment contains all but about 50
residues of the E-protein ectodomain (Fig. 1). It closely re-
sembles its homologs from DEN-2 and TBE viruses in its
dimeric structure and in the details of its protein fold. The
* Corresponding author. Mailing address: Children’s Hospital, End-
ers 673, 320 Longwood Ave., Boston, MA 02115. Phone: (617) 432-
5606. Fax: (617) 432-5600. E-mail: Harrison@crystal.harvard.edu.
† Supplemental material for this article may be found at http://jvi
clustering of serotype-specific neutralization escape mutants in
domain III, which has been implicated in receptor binding (6,
18, 44), suggests that a likely mechanism of neutralization for
antibodies against these epitopes is inhibition of cellular at-
tachment. The structure also shows that neighboring glycans
on the viral surface are spaced in such a way that oligomeric
lectins such as DC-SIGN could bind tightly through multiple
MATERIALS AND METHODS
Expression and purification of DEN-3 sE. Soluble E protein (sE) from DEN-3
(42) was expressed and purified at Hawaii Biotech, Inc. (Aiea, Hawaii). The
protein was expressed in Drosophila melanogaster Schneider 2 cells (obtained
from American Type Culture Collection) with a pMtt vector (GlaxoSmithKline)
containing the DEN-3 (strain CH53489) PrM and E genes (nucleotides 437 to
2113), as previously described (20). The resulting PrM-E preprotein is processed
during secretion to yield sE, which was purified from the cell culture medium by
immunoaffinity chromatography, as previously described (8). Purified sE was
concentrated to 12 g liter?1in Amicon Ultra centrifugal filters (Millipore).
Crystallization and data collection. Crystals were grown at 20°C by hanging-
drop vapor diffusion by mixing equal volumes of protein solution and the fol-
lowing reservoir solution: 15% polyethylene glycol 4000, 0.1 M Tris-HCl (pH
8.5), and 0.2 M lithium sulfate. Crystals grew as rods in space group P212121with
the following cell dimensions: a ? 52.9 Å, b ? 68.63 Å, and c ? 270.2 Å. The
asymmetric unit contains two molecules of sE. Crystals were transferred to a
cryoprotective solution of 15% polyethylene glycol 4000, 0.1 M Tris-HCl (pH
8.5), 0.2 M lithium sulfate, and 30% glycerol before being frozen in liquid
nitrogen. Data were collected at 100 K on beamline F1 of the Cornell High
Energy Synchrotron Source (CHESS) at Cornell University, Ithaca, N.Y. The
data were processed with HKL2000 (43). Data collection statistics are presented
in Table 1.
Structure determination. The crystal structure of DEN-3 E was determined by
molecular replacement with a monomer of the DEN-2 E structure (35) (Protein
Data Bank code 1OAN) as the search model. Two monomers were placed
sequentially with AMoRe (40). The atomic coordinates of the three domains in
each monomer were then refined as rigid bodies with the Crystallography &
NMR System (CNS) (4). Amino acids in the model were mutated from the
DEN-2 to the DEN-3 sequence, and the model was rebuilt with O (23) based on
2Fo—Fcand Fo—FcFourier maps. Coordinates were refined against data be-
tween a 20- and 3.7-Å resolution by simulated annealing with torsion-angle
dynamics with CNS and then rebuilt with O in iterative cycles. Later cycles
included restrained refinement of B factors for individual atoms and energy
minimization against a maximum-likelihood target with CNS. In the final refine-
ment cycles, carried out with REFMAC5 (50), the rigid-body motion of the
protein molecules in the crystal was taken into account in terms of three tensors:
one for translation (T), one for libration (L), and one for correlations of libration
and translation (S) (46). This TLS refinement was alternated with cycles of
restrained positional and B-factor refinement against a maximum likelihood
target. Strict twofold noncrystallographic symmetry restraints were used through-
out refinement. The final model contains residues 1 to 392, 14 water molecules,
and two simple, fucosylated glycans per protein chain on residues 67 and 153,
respectively. The four glycans in the model each contain between one and six
ordered sugar rings. Refinement statistics are presented in Table 1. The stere-
ochemical quality of the atomic model was checked and validated with PRO-
Accession numbers. The atomic coordinates of the DEN-3 sE dimer have been
deposited in the Protein Data Bank under accession code 1uzg. Structure factors
were deposited under accession code r1uzgsf.
Molecular architecture of the DEN-3 sE dimer. The amino
acid sequences of DEN-2 strain PR159S1 and DEN-3 strain
FIG. 1. Structure of the sE dimer of dengue virus E sE in the mature virus particle. (A) The three domains of dengue virus sE. Domain I is
red, domain II is yellow, and domain III is blue. A 53-residue stem segment links the stably folded sE fragment with the C-terminal transmembrane
anchor. (B) The DEN-3 sE dimer viewed along its twofold symmetry axis. (C) The sE dimer viewed perpendicular to its twofold axis. The two
glycans on residues 67 and 153 of the two subunits (A and B) of the dimer are labeled.
1224MODIS ET AL. J. VIROL.
CH53489 E proteins are 68% identical. As expected at this
level of sequence identity, the soluble fragment of DEN-3 E
that we crystallized, DEN-3 sE, adopts the same three-dimen-
sional fold as DEN-2 sE (35). Each sE monomer consists of
three domains (Fig. 1A). Domain I, an eight-stranded ?-barrel,
organizes the structure. Two long insertions between pairs of
?-strands in domain I form the elongated domain II, which
bears the fusion loop at its tip. Domain II contains 12 ?-strands
and two ?-helices. Domain III is an IgC-like module, with 10
?-strands. In solution and in the crystals, two monomers of sE
assemble head to tail to form a dimer, with the long axis of
domain II roughly perpendicular to the dyad axis and the
fusion loop buried at the dimer interface (Fig. 1B).
Dengue viruses have two conserved N-linked glycosylation
sites at Asn-67 and Asn-153. The glycosylation site at Asn-153
is conserved in most flaviviruses, while the glycosylation site at
Asn-67 appears to be unique to dengue viruses and is absent in
other class 2 enveloped viruses. The loop bearing the glycan on
Asn-153 is two residues shorter in DEN-3 than in the other
dengue virus serotypes. Both glycans have been implicated in
cellular attachment and viral entry (13, 19, 39, 48). Ordered
sugars are visible at both glycosylation sites in the DEN-3 sE
structure, indicating that both are utilized in Drosophila cells,
as is also the case of DEN-2 sE expressed in Drosophila cells
(35). DEN-2 E harvested from virus grown in Aedes albopictus
C6/36 cells, however, has been reported to lack glycosylation at
Asn-153 (22). In our DEN-3 structure, the glycans on Asn-67
and Asn-153 form crystal contacts with either glycan or protein
residues from molecules related by the crystal symmetry, hence
contributing to crystallization (Fig. 2). These contacts explain
why relatively large portions of the glycans obey the crystal
symmetry. An ?1-6-linked fucose is visible on the first N-acetyl-
glucosamine of both the Asn-67 glycan (on the A chain only)
and the Asn-153 glycan. The sugar chain is disordered beyond
the first N-acetylglucosamine of the Asn-67 glycan, but up to
six sugar rings are visible in the electron density for the Asn-
153 glycan of the A chain. They were modeled as follows:
36)?133Man; that is, two N-acetylglucosamines with a fu-
cose on the first N-acetylglucosamine and three mannoses
linked to the second N-acetylglucosamine through the central
mannose (Fig. 1C). This structure (excluding the fucose) cor-
responds to the pentasaccharide mannose 3 (Man3), which is
the common core of all N-linked oligosaccharides. Analysis of
the carbohydrate composition of human immunodeficiency vi-
rus gp120 overexpressed in Drosophila Schneider 2 cells shows
that the glycans consist of simple and high-mannose oligosac-
charides (25) rather than the more complex carbohydrates
found on mammalian proteins (reviewed in reference 1). In-
deed, Drosophila Schneider 2 cells have a high level of endog-
enous mannosidase activity, which can result in trimming of the
full nine-mannose structure of high-mannose glycans to Man3
structures. Both DEN-2 and DEN-3 sEs are resistant to deg-
FIG. 2. Contacts involving the glycans of sE between molecules
related by the crystal symmetry. A closeup of the sE dimer (in red,
yellow, and blue for domains I, II, and III, respectively) viewed from
the side (inset) shows that both glycans form crystal contacts with an
adjacent molecule in the crystal (shown in lighter colors). Mannose
residues in the glycan on Asn-153 of chain A form hydrogen bonds
with the main chain and side-chain oxygen atoms of Ser-16 and the
side-chain amine of Lys-36. The N-acetylglucosamine linked to Asn-67
of chain B forms a hydrogen bond with a mannose in the glycan on
Asn-153 of chain B of a neighboring molecule in the crystal.
TABLE 1. Crystallographic data and refinement statistics
Data set (space group)P212121
Molecules per asymmetric unit
Cell edges a, b, c (A ˚)
Resolution range (A ˚)
Avg B factor (A ˚2)
Protein (chains A and B)
Rmsddbond length (A ˚)
Rmsd bond angle (°)
Rmsd bonded B factor (A ˚2)
Ramachandran plot (%)
52.9, 68.6, 270.2
aNumbers in parentheses are for the highest-resolution shell, 3.73 to 3.60 Å.
bRcryst, ?hkl?Fobs? ? ??Fcalc??/?hkl?Fobs?.
cRfree, Rcrystwith 5% of Fobssequestered before refinement.
dRmsd, root mean square difference.
VOL. 79, 2005STRUCTURE OF DENGUE VIRUS TYPE 3 ENVELOPE PROTEIN 1225
lycosylation by endoglycosidase H (which is specific for struc-
tures with five or more mannoses) but can be completely deg-
lycosylated with either peptide N-glycosidase F or
endoglycosidase D (data not shown), suggesting that the gly-
cans on dengue virus sE are efficiently trimmed to simple Man3
structures during expression in Schneider 2 cells. In support of
this notion, there is no evidence for glycan residues beyond the
Man3 core in any of the glycans in the DEN-2 or DEN-3 crystal
Comparison to the structure of the DEN-2 sE dimer. The
greatest difference between the DEN-2 (35) and DEN-3 sE
structures is in the relative orientation of domains I and II.
This difference is most apparent when domain I of a DEN-3 sE
monomer is superimposed on domain I of a DEN-2 monomer:
the orientations of domain II in each structure are then sepa-
rated by a 10° rotation at about a point near Gly-188 of DEN-3
(Fig. 3A and B). The altered domain orientation translates into
a difference in backbone atom positions of 13 Å at the fusion
loop at the tip of domain II and of up to 18 Å at the far end of
domain III of the other monomer in the dimer (Fig. 3B).
Variations in the relative orientations of domain I and domain
II of up to 5° have been observed as a result of a hinge motion
around Gly-188 in different crystal forms of DEN-2 (35) and
TBE sE (F. A. Rey and S. C. Harrison, unpublished work),
suggesting that the sE dimer is somewhat flexible in solution.
The 10° difference in the relative orientations of domains I and
II of DEN-2 and DEN-3 represents a significantly larger shift,
however. If it reflects a real structural difference between the
two serotypes rather than conformational flexibility of sE,
there could be slight differences in the surface features of
DEN-2 and DEN-3 virions.
The individual domains of sE in DEN-2 and DEN-3 have a
high degree of structural similarity, with root mean square
deviations for main chain atoms of 0.78 Å for domain I, 0.73 Å
for domain II, and 0.85 Å for domain III. Only three loops
adopt substantially different conformations. The loop (residues
153 to 158) that follows the second glycosylation site is two
residues shorter in DEN-3. The solvent-exposed loop (residues
167 to 170) between ?-strands in domain I also has a different
conformation in the two serotypes, largely because Pro-166 in
DEN-2 is replaced at the equivalent position in DEN-3 by
serine, a conformationally more flexible residue. The third
loop with substantial differences in main chain positions (up to
3 Å) spans residues 336 to 346 in DEN-3. Two glycine residues
at positions 340 and 342 in DEN-3 replace a leucine and a
lysine, respectively. The increased flexibility of glycine allows
the loop to reach a conformation in DEN-3 that would have
caused strain with the DEN-2 amino acid sequence.
Just over two-thirds of the residues in DEN-2 and DEN-3 sE
are identical. Of the residues that are not identical, about
one-third have similar length and polarity; the remaining 86
unconserved residues differ substantially in charge, polarity,
length, or conformational variability. These 86 residues are
distributed fairly evenly throughout the sE structure, although
there are none in the area around the fusion loop. They tend
to cluster instead in the loops of domain III and in a loop in
middle of domain II formed by residues 219 to 234 (Fig. 3C
and D). Of the 86 unconserved residues, 56 will be exposed on
the viral surface, based on TBE sE fitting into a structure of the
entire DEN-2 virion at 9.5 Å resolution, as derived by electron
cryomicroscopy (52). All but 5 of the remaining 30 uncon-
served residues are exposed on the sE protein surface but are
not expected to be accessible from outside the virion. Residues
59, 63, 143, 219, and 337 are unconserved despite being buried
in the protein core, but none of these residues have charged
side chains. The different binding affinities of neutralizing an-
tibodies to dengue virus serotypes are likely to be due to
variations in residues that are exposed on the viral surface. We
therefore conclude, based on the DEN-3 sE structure, that
only the 56 unconserved residues exposed on the viral surface
(or a subset thereof) are the most important contributors to
differential antibody binding.
Implications for immune recognition of different dengue
virus serotypes. Structural features on the surface of the virion
determine the specificity with which a given antibody will bind.
Differences in the surface landscape of the four dengue virus
serotypes mean that neutralizing antibodies raised against one
serotype are likely to bind other serotypes with deceased af-
finity. Antibodies with a sufficiently low binding affinity may fail
to neutralize the virus but may still be present in sufficient
numbers on virions that they can induce cell uptake through
interaction with IgG Fc receptors (15, 31). Dengue virus is able
to use these molecules as surrogate receptors. By the same
mechanism, some high-affinity antibodies that neutralize virus
of a certain serotype at high titers can increase cell uptake of
the same serotype at sufficiently low antibody concentrations
(3, 16, 38). The resulting enhancement of viral infection may
cause more acute pathologies such as hemorrhagic fever (15,
37). Antibody-dependent enhancement of dengue virus infec-
tion is a problem particularly upon reinfection with a second
serotype, because antibodies against the first serotype may
bind but not neutralize the second serotype. Indeed, the cir-
culation of multiple serotypes in Cuba and southeast Asia
where dengue fever is hyperendemic has coincided with the
emergence of more severe dengue pathologies, dengue hem-
orrhagic fever and dengue shock syndrome, but also provides
synergies between different serotypes. These synergies allow
multiple strains to coexist in human populations and to gen-
erate complex and persistent epidemic cycles (7, 10, 41). It is
therefore important to determine which surface features on
the dengue virion are responsible for immune recognition in
the different serotypes.
From the data presented above, we conclude that the dif-
ferential antibody binding to different dengue serotypes is
likely to be due to a variation in a subset of the 56 unconserved
residues that are exposed on the viral surface. These residues
cover the majority of the known epitopes responsible for es-
cape from neutralization by monoclonal antibodies. The es-
cape mutants that map to serotype-specific residues are not
evenly distributed throughout the structure. Instead, they lie
exclusively in domain III (Fig. 4). They include residue 291 (2),
residue 305 (29), and residues 381 to 383 (17, 18). Residue 388
is also unconserved and exposed on the viral surface. An as-
paragine at this position appears to cause increased incidences
of hemorrhagic fever, while charged residues reduce virulence
(28). Residue 388 may therefore participate, directly or indi-
rectly, in receptor binding. The localization of all these mu-
1226 MODIS ET AL.J. VIROL.
tants in domain III is significant, because domain III has been
proposed to bind a cell-surface receptor (see below). The clus-
tering of serotype-specific neutralization escape mutants in
domain III suggests that a likely mechanism of neutralization
for antibodies against these epitopes is inhibition of receptor
binding and cellular attachment.
While most well-characterized neutralizing antibodies rec-
ognize epitopes in domain III, some (both neutralizing and
nonneutralizing) have epitopes on one of the other two do-
mains of E (Fig. 4A and B; Table 2). Monoclonal antibodies
raised against epitopes in domain I are type specific (that is,
affected by residues not conserved across dengue serotypes)
but nonneutralizing (33). At the domain I-domain II boundary,
a Phe277Ser mutation causes escape from neutralization by
FIG. 3. Structure of the DEN-3 sE dimer superimposed on the DEN-2 sE dimer, using domain I from one monomer as the reference, viewed
along the twofold symmetry axes (A) and perpendicular to the twofold axes (B). The greatest difference between the two structures is a ?10°
rotation of domain II relative to domain I about a point near Gly-188 (indicated by a grey star). (C) The DEN-3 sE dimer with residues that are
not conserved in DEN-2 is shown in space-filling representation. Residues that are exposed on the viral surface are in magenta; residues that are
not exposed are in grey. (D) The same structure as in panel C but viewed perpendicular to the twofold axis.
VOL. 79, 2005STRUCTURE OF DENGUE VIRUS TYPE 3 ENVELOPE PROTEIN1227
FIG. 4. Antibody neutralization-escape mutants in dengue virus. (A) Three serotype-specific epitopes have been reported: residue 291 (2),
residues 301 to 307 (29), and residues 381 to 383 (17, 18). Side chains in all three epitopes are shown on the DEN-3 E structure in magenta in
space-filling representation. Changes at residue 388 (in pink), also unconserved and exposed, correlate with changes in virulence (28). Phe-277 (in
green) is conserved in the dengue virus serotypes, but its mutation to serine during passaging in cell culture leads to escape from neutralization
by IgM M10 (2, 27). (B) A side view of panel A is shown. (C) Stereoscopic view of a close-up of DEN-3 domain III, showing the four proposed
serotype-specific epitopes. The orientation is halfway between the orientations used in the models shown in panels A and B, and the close-up is
of the copy of domain III shown on the right in panels A and B. (D) The same view as in panel C is shown, but of DEN-2 (and with the homologous
IgM M10 and alters the pH threshold for fusion (27). Phe277,
conserved in all dengue serotypes (2), is accessible from the
viral surface (Fig. 4A). It is located in the kl-hairpin region of
domain II, which has a role in the conformational rearrange-
ment that drives membrane fusion (35, 36, 44). Other antibod-
ies to epitopes in domain II are also group reactive; that is,
they recognize residues that are strictly conserved in the dif-
ferent serotypes (33). These antibodies appear to neutralize
the virus by inhibiting membrane fusion (33, 45), rather than by
blocking attachment. Indeed, West Nile virus can attach and
enter endosomes in the presence of polyclonal antibodies, but
membrane fusion and viral entry are both inhibited (11).
Implications for receptor binding. Several lines of evidence
suggest that domain III binds a cellular receptor. Recombinant
DEN-2 domain III expressed alone interferes with infection by
blocking virion adsorption to both mammalian and mosquito
cells (18). Furthermore, a loop formed by residues 381 to 384
is responsible for the serotype-specific attachment of domain
III to mosquito (but not mammalian) cells (18). In tick-borne
viruses such as the TBE virus, this loop is four residues shorter
and forms a tight ?-turn (44). Many of the residues implicated
as determinants of host range, tropism, or virulence in various
flaviviruses are located in domain III (21, 44); see Table S1 in
the supplemental materials for examples in dengue virus). The
widespread occurrence of Ig modules in cell adhesion proteins
is also consonant with the notion that domain III participates
in attachment to a cellular receptor. From the structure of
DEN-2 sE in the postfusion conformation (36), none of the
neutralization-escape mutations in domain III are expected to
interfere with the conformational rearrangement that accom-
panies membrane fusion, and they do not alter the pH thresh-
old of fusion (Table 2). The most likely mechanism of neutral-
ization by antibodies recognizing epitopes on domain III is
therefore inhibition of cellular attachment. The efficiency of
the inhibition of viral attachment by these antibodies is rela-
tively low (55 to 60%), however, suggesting that other factors
may contribute to neutralization (45).
It is still unclear what molecules domain III recognizes on a
cell surface. Moreover, dengue virus may recognize different
receptors on human and mosquito cells (18). There are cur-
rently no candidate protein receptors, but it has been proposed
that patches of basic residues on the surface of domain III bind
to heparan sulfate on the cell surface, which carries a net
negative charge (6, 18, 49). The DEN-3 sE structure shows that
only the first of two putative heparan binding motifs (6) in
domain I of sE forms a cluster of positively charged residues on
the viral surface. The cluster consists of Arg-188, His-280,
Lys-282, Arg-284, Lys-286, Lys-289, and Lys-293, all of which
are conserved as basic residues across dengue virus serotypes
but not in TBE virus. Examination of the DEN-2 and DEN-3
sE structures reveals that another basic patch on the viral
surface is formed in the middle of domain II near the dimer
interface by residues Lys-58, Lys-64, Lys-128, and Lys-200. The
basic cluster is larger in DEN-2, as it also includes Lys-89,
Lys-122, and Lys-123. Most of the residues in this cluster are
not conserved in TBE virus. Thus, the positively charged
patches of lysines and arginines on domains I and II could in
principle contribute to heparan sulfate binding. Many cell
types express heparan sulfate, however, and a more specific
protein receptor is thought to be required to target dengue
virus to permissive cell types—immature dendritic cells in par-
Glycans on the dengue virus surface were recently impli-
cated in receptor binding when DC-SIGN, a mannose-specific,
C-type lectin on the cell surface of immature dendritic cells,
was found to be essential for productive infection (39, 48).
These glycans must belong to E, as it is the only glycoprotein
on the surface of the mature virion. Immature dendritic cells
have been proposed to be the primary target cells upon intro-
duction of dengue virus into the bloodstream (51). Dengue
virus infection of human dendritic cells is inhibited by anti-
DC-SIGN antibodies and by the soluble ectodomain of DC-
SIGN (39, 48). Flavivirus infection requires clathrin-mediated
uptake into the endocytic pathway and acidification in the
endosome (5, 39). The cytoplasmic tail of DC-SIGN contains
two sorting signals for the endocytic pathway (47); these sort-
ing signals could be responsible for targeting dengue virus to
the endosome. DC-SIGN is a type II integral membrane pro-
tein. Its extracellular domain has a stalk and a C-terminal
C-type carbohydrate recognition domain (CRD). The stalk
causes the protein to form tetramers through an ?-helical
coiled-coil interaction (34). The CRD selectively recognizes
TABLE 2. Dengue virus mutationsa
MutationSerotype MAbPhenotype Explanation
DEN-1 IgM M17 More virulent and hemagglutinating
at pH 5.8–7
At a hinge between domains I
and III; may rigidify the
hinge, requiring lower pH
for conformational switch
IgG G8D11 may interfere
with receptor binding (29)
IgM 6B2 may interfere with
receptor binding (32)
IgG 3H5 may interfere with
receptor binding (17),
probably in mosquito cells
and not in human cells (18)
E311GDEN-2 IgM 6B2None
E383G P384E/D/N G385K/S/A
DEN-2 IgG 3H5Reduced mouse neurovirulence.
aMutations in dengue virus that lead to escape from neutralization by monoclonal antibodies, resulting phenotypes, and possible explanations based on the structures
of sE of DEN-2 and -3. Residue numbers refer to DEN-2. For unconserved residues, the residue type in DEN-2 is listed in parentheses before the residue number.
Residues that are unconserved among dengue serotypes are in boldface type.
VOL. 79, 2005STRUCTURE OF DENGUE VIRUS TYPE 3 ENVELOPE PROTEIN1229
endogenous high-mannose oligosaccharides (9, 34), and it
binds glycopeptides bearing two such oligosaccharides with a 5-
to 25-fold-higher affinity (34). This oligovalency may be impor-
tant for recognition of dengue virus because dengue virus E
has two glycosylation sites at Asn-67 and Asn-153, unlike the
envelope proteins of other flaviviruses, which have only the
latter. Furthermore, the binding of a substrate is enhanced
substantially if the spacing between the glycans is sufficient for
each glycan to reach a CRD in the DC-SIGN tetramer (34).
The evolution of a second glycosylation site in dengue virus E
that is appropriately spaced from the first site may thus have
allowed E to bind DC-SIGN with sufficient affinity for it to
function as a receptor. Indeed, based on our structure of
DEN-3 sE and a fit of TBE E into the electron cryomicroscopy
structure of DEN-2 virions (52), the spacing between any two
neighboring glycans on the viral surface is at least 32 Å. This
distance should be sufficient to span between two CRDs in the
DC-SIGN tetramer, based on the crystal structure of the CRD-
mannose complex (9) and on the predicted structure of the
DC-SIGN tetramer (34). All four CRDs in a DC-SIGN tet-
ramer should thus be able to bind a glycan on the dengue
virion. The absence of glycosylation on Asn-153 in DEN-2 E
under certain conditions (22), however, suggests that certain
dengue virus strains or serotypes may be viable with only a
single glycan per E monomer, on Asn-67.
Conclusions. Comparison of the prefusion structures of E
from DEN-2 and DEN-3 shows that the differences are, as
anticipated, relatively subtle. Our analysis of the locations of
the known type- and group-specific epitopes poses the follow-
ing questions. Are antibodies that recognize epitopes con-
served among serotypes (e.g., those in domain II) also cross-
neutralizing at a reasonable titer, or are deviations from
conservation at the periphery of the antibody binding site suf-
ficient to prevent adequate interaction of an antibody with any
but the serotype against which it was raised? If there is some
degree of cross-neutralization, would increasing the titer of
such antibodies by a suitable immunization strategy lead to
useful cross-protection, or would the risk of antibody-depen-
dent enhancement outweigh potential benefits? Answers to
these and related questions will require careful analysis of the
spectrum of epitopes recognized by circulating antibodies in
patient sera, the changes in this spectrum as a function of time,
and the correlation of the results with the response to subse-
quent infection by a different dengue virus serotype. The E-
protein structures will allow such data to be interpreted di-
rectly in terms of well-defined molecular surfaces.
This work was supported by NIH grants CA-13202 and AI-57159 to
S.C.H. S.C.H. is a Howard Hughes Medical Institute Investigator.
We thank staff at the F1 Beamline of the Cornell High Energy
Synchrotron Source (CHESS) at Cornell University, Ithaca, N.Y., and
John Roehrig for comments on the manuscript.
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