Article

Structures of the Zika Virus Envelope Protein and Its

Complex with a Flavivirus Broadly Protective
Antibody
Graphical Abstract Authors
Lianpan Dai, Jian Song, Xishan Lu, ...,
Cheng-Feng Qin, Jianxun Qi,
George F. Gao

Correspondence
qincf@bmi.ac.cn (C.-F.Q.),
jxqi@im.ac.cn (J.Q.),
gaof@im.ac.cn (G.F.G.)

In Brief
Zika virus (ZIKV) is a mosquito-borne
flavivirus. Dai et al. report the structures
of ZIKV envelope (E) protein and its
complex with a flavivirus broadly
protective antibody, which reveals
antibody recognition of a conserved
fusion loop. Antibody binds to ZIKV-E
with high affinity and neutralizes currently
circulating ZIKV strains in mice.

Highlights Accession Numbers

d Report structures of ZIKV E protein and its complex with a 5JHM
broadly neutralizing antibody 5JHL

d ZIKV-E contains a unique, positively charged patch, which

may influence host attachment

d The ZIKV-E-antibody structure reveals antibody recognition

of a conserved fusion loop

d Antibody binds ZIKV-E and neutralizes circulating ZIKV

strains in vitro and in mice

Dai et al., 2016, Cell Host & Microbe 19, 696704

May 11, 2016 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.chom.2016.04.013
 Cell Host & Microbe

Article

Structures of the Zika Virus

Envelope Protein and Its Complex
with a Flavivirus Broadly Protective Antibody
Lianpan Dai,1,11 Jian Song,2,3,11 Xishan Lu,4,11 Yong-Qiang Deng,5,11 Abednego Moki Musyoki,2,3 Huijun Cheng,1
Yanfang Zhang,4 Yuan Yuan,2,3 Hao Song,2,3 Joel Haywood,2,3 Haixia Xiao,4 Jinghua Yan,2,3,6,7,8 Yi Shi,1,2,3,6,7
Cheng-Feng Qin,5,* Jianxun Qi,2,* and George F. Gao1,2,3,4,6,7,9,10,*
1Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China
2CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100071, China
3Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China
4Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
5State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100101, China
6Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third Peoples Hospital, Shenzhen 518112, China
7Center for Influenza Research and Early-warning (CASCIRE), Chinese Academy of Sciences, Beijing 100101, China
8CAS Key Laboratory of Microbial Physiology and Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101,

China
9Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China
10National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC),

Beijing 102206, China

11Co-first author

*Correspondence: qincf@bmi.ac.cn (C.-F.Q.), jxqi@im.ac.cn (J.Q.), gaof@im.ac.cn (G.F.G.)

http://dx.doi.org/10.1016/j.chom.2016.04.013

SUMMARY
Zika virus (ZIKV), a mosquito-borne flavivirus, is a
current global public health concern. The flavivirus
envelope (E) glycoprotein is responsible for virus en-
try and represents a major target of neutralizing anti-
bodies for other flaviviruses. Here, we report the
structures of ZIKV E protein at 2.0 A and in complex
with a flavivirus broadly neutralizing murine antibody
2A10G6 at 3.0 A. ZIKV-E resembles all the known fla-
vivirus E structures but contains a unique, positively
charged patch adjacent to the fusion loop region of
the juxtaposed monomer, which may influence host
attachment. The ZIKV-E-2A10G6 complex structure
reveals antibody recognition of a highly conserved
fusion loop. 2A10G6 binds to ZIKV-E with high affinity
in vitro and neutralizes currently circulating ZIKV
strains in vitro and in mice. The E protein fusion
loop epitope represents a potential candidate for
therapeutic antibodies against ZIKV.
INTRODUCTION
Zika virus (ZIKV), a Flaviviridae family member, is a single-
stranded, positive-sense RNA virus with a 10.7 Kb genome en-
coding a single polyprotein that is cleaved into three structural
proteins (C, prM/M, and E) and seven non-structural proteins
(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral and
host proteases (Lindenbach and Rice, 2003). ZIKV was first iso-
lated from a sentinel rhesus monkey in the Zika Forest of Uganda
in 1947 and can be transmitted to different species by mosqui-
toes of the genus Aedes (Dick et al., 1952). For a long time,
ZIKV has been commonly circulating in the tropical regions in Af-
rican and Asian countries (Faye et al., 2014) and is classified into
African and Asian genotypes by phylogenetic analysis (Haddow
et al., 2012). In 2007, a large epidemic of Asian genotype ZIKV
was reported in Yap Island and Guam, Micronesia, and then be-
tween 2013 and 2014, a further outbreak was reported in French
Polynesia (Cao-Lormeau and Musso, 2014). The 2015 outbreak
of ZIKV epidemic has caused more than 30,000 cases of infection
in Brazil and rapidly spread to South and Central America, the
Caribbean (Faria et al., 2016; Petersen et al., 2016), and other
countries including China (Li et al., 2016). Recently, growing evi-
dence has indicated that ZIKV infections are linked to fetal and
newborn microcephaly and serious neurological complications,
such as Guillain-Barre syndrome (GBS) (Mlakar et al., 2016; Pe-
tersen et al., 2016). Moreover, it is noted that ZIKV can also
possibly be transmitted by sexual behavior, with high viral loads
detected in semen from infected patients (Mansuy et al., 2016).
To date, no clinically approved vaccines or therapeutics are avail-
able to control and prevent ZIKV infections; consequently, great
efforts are being made to improve our understanding of ZIKV
pathogenesis and vulnerability.
The recently published cryoelectron microscopy (cryo-EM)
structures of ZIKV reveal that ZIKV displays a similar structure
to other known flaviviruses (Kostyuchenko et al., 2016; Sirohi
et al., 2016). For flavivirus, mature virus particles contain 180
copies of the E protein and membrane (M) protein on the
envelope and display an icosahedral arrangement in which 90
E dimers completely cover the viral surface (Kuhn et al., 2002;
Zhang et al., 2013a). Upon entry into host cells via endocytosis,

696 Cell Host & Microbe 19, 696704, May 11, 2016 2016 Elsevier Inc.

the acidic endosomal environment triggers an irreversible
conformational change in the E protein and a transition from a
dimer to trimer formation that leads to the membrane fusion event
(Modis et al., 2004). In the ER, newly assembled virus progeny
form immature virions and exhibit a spiky surface anchored
with 60 trimeric protrusions of the E and precursor-membrane
(prM) heterodimers (Plevka et al., 2011). During virus maturation,
a low pH environment in the trans-Golgi network (TGN) induces
the reorganization of the E-prM heterodimers into E homodimers
(Yu et al., 2008). This structural rearrangement exposes the
cleavage site of prM for digestion by the host protease, furin. Af-
ter prM is cleaved, the protein dissociates from the particles upon
release into the pH neutral extracellular space. During the matu-
ration process, not all of E-prM heterodimer can be cleaved by
the furin, and then uncleaved E-prM heterodimer will revert
back to a spiky immature trimeric structure (Yu et al., 2008).
Flavivirus E protein is responsible for virus entry and represents
a major target for neutralizing antibodies. Thus far, a number of
potent flavivirus type-specific neutralizing monoclonal anti-
bodies (mAb) have been found to target E protein domain III (Bel-
tramello et al., 2010; Oliphant et al., 2005), while less potent
cross-neutralizing mAbs have been shown to be specific for the
fusion loop region or E-dimer region (Dejnirattisai et al., 2015;
Rouvinski et al., 2015; Smith et al., 2013). Although less potent,
antibodies targeting the highly conserved fusion loop epitope
(FLE) in domain II comprise a significant fraction of the humoral
response against flaviviruses (Beltramello et al., 2010; Costin
et al., 2013; Dejnirattisai et al., 2015; Lai et al., 2008; Oliphant
et al., 2005, 2007; Roberson et al., 2007; Smith et al., 2013;
Tsai et al., 2013). Moreover, FLE mAbs from several previous re-
ports have shown protective efficacy against flavivirus in vivo
(Beltramello et al., 2010; Deng et al., 2011; Lai et al., 2013; Sultana
et al., 2009; Vogt et al., 2011). Notably, most FLE mAbs are sen-
sitive to the substitution of W101 in the fusion loop, with several of
these also being sensitive to the substitution of G106, L107, and
F108 (Dejnirattisai et al., 2015; Tsai et al., 2013). Nevertheless, the
molecular basis of the mAb-E interaction is still elusive.
Figure 1. Overall Structure of the ZIKV-E
Protein
(A) Schematic diagram of domain organization for
ZIKV-E. Domain I (red), domain II (yellow), and
domain III (blue). A 48-residue stem segment links
the stably folded ZIKV-E ectodomain with the
C-terminal transmembrane anchor.
(B) Dimer structure of ZIKV-E. ZIKV-E has
three distinct domains: b-barrel-shaped domain I,
finger-like domain II, and immunoglobulin-like
domain III. Domain II is responsible for the dimer-
ization of ZIKV-E. The fusion loop is buried by the
domains I and II of the other ZIKV-E monomer.
Secondary structural elements are labeled. See
also Figures S1 and S2 and Table S1.
Here, we have determined the crystal
structures of free ZIKV-E ectodomain pro-
tein in its dimeric form and its complex
with the flavivirus broadly protective mu-
rine antibody 2A10G6. This antibody has
previously been shown by our group to
exhibit in vitro neutralization activity against dengue virus sero-
type 1-4 (DENV1-4), yellow fever virus (YFV) and West Nile virus
(WNV) and to confer in vivo animal protection against DENV1-4
and WNV (Deng et al., 2011). The crystal structure reveals
that ZIKV-E resembles all the known flavivirus E structures, which
have three distinct domains: a central b-barrel-shaped domain I,
an elongated finger-like domain II, and a C-terminal immunoglo-
bulin-like domain III. 2A10G6 binds to the ZIKV-E protein with a
high binding affinity and neutralizes the ZIKV infection in vitro.
Significantly, 2A10G6 completely protects mice against the circu-
lating ZIKV strain in vivo, indicating a therapeutic potential.
Furthermore, the complex structure of ZIKV-E/2A10G6 revealed
that 2A10G6 binds to the tip of ZIKV-E domain II at a perpendic-
ular angle, embedding the fusion loop of ZIKV-E in a hydrophobic
hole formed by both the heavy chain and light chain. The hydro-
phobic residues W101, L107, and F108 form the majority of inter-
actions between 2A10G6 and ZIKV-E and are highly conserved in
the majority of flaviviruses, providing an explanation for the
broadly neutralizing capacity of 2A10G6 (Deng et al., 2011).
RESULTS
Structure of Prefusion-Form ZIKV-E Protein
Here, we expressed a large fragment (residues 1409) of ZIKV-E
protein (Figure 1A), lacking the stem region and C-terminal trans-
membrane anchor, in E. coli cells. Initially, we obtained insoluble
inclusion bodies, and then we applied an in vitro refolding
method to obtain soluble E protein (termed as sE). Square-like
crystals were grown using the sitting drop vapor diffusion
crystallization method, and the best crystal diffracted to a high
resolution of 2.0 A. The ZIKV sE structure was determined by
molecular replacement method, using the dengue virus E protein
structure (PDB: 1OKE) as a template. After several rounds of
refinement, the final Rfree and Rwork values of 0.23 and 0.20,
respectively, were obtained (Table S1).
The overall structure of ZIKV sE resembles previously re-
ported flavivirus E protein structures (Lescar et al., 2001;
Cell Host & Microbe 19, 696704, May 11, 2016 697

Luca et al., 2012; Modis et al., 2003; Rey et al., 1995) and has
three distinct domains: a central b-barrel (domain I), an elon-
gated finger-like structure (domain II), and a C-terminal immu-
noglobulin-like module (domain III) (Figure 1). The central
domain I contains around 130 residues in three segments, res-
idues 151, 132192, and 280295 (Figure 1A). The central
domain I is folded into an eight-stranded b-barrel with an addi-
tional N-terminal A0 strand (Figure 1B). The 150 loop (residues
147161, between strands E0 and F0), which contains the
potential N154 glycosylation site, likely represents a highly flex-
ible loop in this domain due to a lack of electron density in this
region. The finger-like domain II is formed by two segments,
residues 52131 (between strands D0 and E0) and residues
193279 (between strands H0 and I0) (Figure 1). The C-terminal
domain III (residues 296403) displays an IgG-like fold where
the AABE sheet and disordered D strand are contacted by
the cd loop of the adjacent sE monomer.
Domain II is responsible for the dimerization of sE monomer,
leading to an extended, but interrupted, dimer interface, and
thus there are holes in the dimer at either side of domain II (Fig-
ure 1B). The central dimer interface is mainly constituted by the
aB helix and j strand elements of each sE monomer, whereas
the distal dimer interface is mainly created by the hydrophobic
interaction between the cd loop and the crevice formed by
domains I and III of the adjacent sE monomer (Figure 1B). The hy-
drophobic cd loop represents the fusion loop (residues 98109),
which is responsible for the membrane fusion between host cell
and virus membranes during virus entry, and is highly conserved
in flaviviruses.
In addition, comparison of an electrostatic surface potential
map of ZIKV-E with other structure-known flavivirus E,
including tick-borne encephalitis virus (TBEV), DENV2,
DENV3, Japanese encephalitis virus (JEV), and WNV, revealed
698 Cell Host & Microbe 19, 696704, May 11, 2016
Figure 2. Binding of ZIKV-E to a Broadly
Neutralizing Antibody 2A10G6
(A) Gel filtration profile of ZIKV-E and 2A10G6 Fab.
The ZIKV-E and 2A10G6 Fab proteins elute as
single monomer peaks in the gel filtration curves,
respectively. The ZIKV-E/2A10G6 Fab complex
displays a shifted complex peak with an extra
2A10G6 Fab peak. All the samples were assessed
by SDS-PAGE.
(B) BIAcore diagram of 2A10G6 Fab bound to
ZIKV-E protein. The 2A10G6 Fab binds to the
ZIKV-E protein with a high affinity and slow ki-
netics. The KD value was calculated by the
BIAcore 3000 analysis software (BIAevaluation
Version 4.1).
that a strong positive charge is found at
the fusion loop region of E-dimer due to
the presence of a conserved R99
residue at the protein surface. Notably,
however, ZIKV displays a uniquely
positively charged patch region adja-
cent to the fusion loop region in domain
I due to the existence of R138, R164,
and K166 unique to the ZIKV, making
the two positively charged regions combine together (Figures
S1 and S2).
Binding, Neutralization, and Protection of 2A10G6
to ZIKV
Previously, we have reported a flavivirus broadly protective
mouse monoclonal antibody 2A10G6 (Deng et al., 2011).
Phage-display biopanning experiments revealed that the
2A10G6 antibody bound to an epitope within the highly
conserved flavivirus fusion loop peptide, the 98DRXW101 motif
(Deng et al., 2011). Further, in vitro neutralizing and in vivo animal
protection experiments illustrated that 2A10G6 was able to
potently neutralize the DENV1-4, WNV, and YFV and confer pro-
tection in a mouse model from lethal challenges of DENV1-4 and
WNV (Deng et al., 2011). As ZIKV E protein also displays a
conserved 98DRXW101 motif, we hypothesized that 2A10G6
should also bind to ZIKV E protein.
The sE protein and 2A10G6 Fab eluted as single monomer
peaks with high purity in gel filtration assays (Figure 2A).
Following incubation of the ZIKV sE and 2A10G6 Fab together
at a molar ratio of 1:1.5, we found that the 2A10G6 Fab/sE
complex eluted as a peak preceding the sE and 2A10G6 Fab
monomer peaks, followed by a small 2A10G6 Fab peak (Fig-
ure 2A). To further investigate the binding affinity between sE
and 2A10G6 Fab, we performed surface plasmon resonance
(SPR) experiments and found that 2A10G6 Fab binds to the
ZIKV sE protein with a high affinity of 2.7 nM (Figure 2B).
To investigate the neutralizing potential of 2A10G6 to ZIKV
infection, the current circulating ZIKV Asian linage strain SZ01
isolated from the serum of an imported case in China was
used (Deng et al., 2016). The in vitro neutralizing activity of
mAb 2A10G6 for ZIKV was assessed using a plaque reduction
assay; indeed, the antibody exhibited neutralizing activity, with

a 50% plaque reduction neutralization titer (PRNT50) of 249 mg/ml
(Figure 3A). Though not as high as those for dengue virus sero-
type 1-4 (DENV1-4) and yellow fever virus (YFV), a single dose
of 500 mg 2A10G6 mAb provided complete protection in vivo
against ZIKV infection in the A129 mouse model in a 60% mortal-
ity setting of the control group (Figure 3B).
Complex Structure of E Protein with 2A10G6 Antibody
In order to elucidate the molecular basis of this interaction, the
ZIKV sE and 2A10G6 Fab were incubated at a molar ratio of
1:1, and a crystallization screen was performed with the mixed
protein complex. An X-ray diffraction dataset was collected,
and the complex structure was determined by molecular
replacement at a resolution of 3.0 A.
The solved complex structure reveals that 2A10G6 binds to
the tip of the finger-like domain II at a perpendicular angle via
the fusion loop and bc loop. In particular, the fusion loop enters
into a hole formed by both heavy and light chains, while the bc
loop contacts only the heavy chain (Figure 4A). Seven residues
(W101, G102, G104, C105, G106, L107, and F108) in the fusion
loop and two residues (T76 and Q77) in the bc loop of ZIKV sE
contribute to the binding of 2A10G6 (Figure 4B and Table 1).
Notably, the projecting residue W101 deeply inserts the hole in
the binding interface of 2A10G6 (Figure 4B). For 2A10G6, 13 res-
idues (T38, H40, G55, I56, D57, N60, G62, T63, N64, R104, Y107,
A108, and L109) from the heavy chain and six residues (Q91,
Y93, N94, S95, Y96, and P97) from the light chain participate
in the interaction with ZIKV sE (Figure 4C). The heavy-chain res-
idues (H40, R104, A108, and L109) and the light-chain residues
(Q91, Y93, and P97) constitute the inner hydrophobic ring of
the hole that can tightly envelope the aromatic residue W101 in
the fusion loop of sE, while the other residues form the outer
ring of the hole.
Detailed structural analysis demonstrates that four second-
ary elements of 2A10G6 surround the tip of the finger-like
domain of sE, including three elements (HFR2 strand, HCDR2
and HCDR3 loops) from the heavy chain and one element
(LCDR3 loop) from the light chain (Figure 5A). T38 and H40 in
the HFR2 strand contribute 23 contacts, including one
hydrogen bond between H40 and the main chain of G106 in
the fusion loop of sE (Figure 5B and Table 1). G55, I56, D57,
N60, G62, T63, and N64 in the HCDR2 loop contribute 45 con-
tacts, including two hydrogen bonds between N60 and the res-
idues (T76 and Q77) in the bc loop of sE and one hydrogen
Figure 3. Neutralization Activity and Pro-
tection of 2A10G6 against ZIKV
(A) Neutralization activity of 2A10G6 to ZIKV. ZIKV
was mixed with 5-fold serial dilutions of 2A10G6.
Then neutralization activity was evaluated by pla-
que reduction assays in duplicates using BHK21
cells. Data are represented as mean SEM. The
figure represents two independent experiments.
(B) Therapeutic activity of 2A10G6 against ZIKV in
A129 mice. 5-week-old A129 mice were inoculated
with 105 PFU of ZIKV. At 24 hr post virus infection,
mice were passively transferred a single inocula-
tion of 500 mg antibody 2A10G6, or PBS as control.
The survival of the mice was monitored until
21 days post infection. The survival curves were
constructed using data with five mice for each arm.
bond between N64 and the main chain of F108 (Figure 5B
and Table 1). R104, Y107, A108, and L109 in the HCDR3
loop contribute 62 contacts, including one hydrogen bond be-
tween R104 and the main chain of W101 in the fusion loop of sE
(Figure 5B and Table 1). Q91, Y93, N94, S95, Y96, and P97 in
the LCDR3 loop contribute 63 contacts, which are mainly
comprised of non-polar interactions with the aromatic residues
W101 and F108 in fusion loop of sE (Figure 5B and Table 1).
Reciprocally, the hydrophobic residues W101, L107, and
F108 in the fusion loop of sE contribute the majority (145 of
197 contacts) of the interaction with 2A10G6.
Comparison of Fusion Loop Targeting Neutralizing
Antibodies to Flavivirus E
During the maturation of flaviviruses, E protein domain II forms
the binding partner of precursor membrane glycoprotein (prM)
(Figure 6A). As immature flavivirus particles are transported
through the cellular secretory pathway, cellular furin proteases
cleave prM resulting in the release of the pr peptide and forma-
tion of mature virus particles, concurrent with the change from
an acidic to neutral pH environment. Interestingly, 2A10G6 binds
to the tip of domain II at a perpendicular angle (Figure 6B), which
should not interfere with the prM binding site on the side of
domain II (Figure 6A). This indicates that 2A10G6 is able to
bind to immature virus particles or partially immature virus parti-
cles but is unable to bind to fully mature virus particles, which
present in a compact E-dimer arrangement on the envelope
where the fusion loop is buried in a crevice formed by domain I
and III from the other monomer.
Similar FLE antibodies have been found in a significant fraction
of the humoral response to flaviviruses in humans, and W101,
G106, L107, and F108 have been reported as the key residues
for the binding of these FLE antibodies (Beltramello et al.,
2010; Costin et al., 2013; Dejnirattisai et al., 2015; Lai et al.,
2008; Oliphant et al., 2005, 2007; Roberson et al., 2007; Smith
et al., 2013; Tsai et al., 2013). Previously, only the complex struc-
ture of WNV sE protein and the FLE antibody E53 had been
solved, illustrating that E53 binds to the tip of domain II at a tilted
angle on the opposite side of prM binding site (Figures 6C and
S3) (Cherrier et al., 2009), away from the canonical FLE antibody
binding-dependent aromatic residues W101 and F108 in the
fusion loop. Our 2A10G6/ZIKV sE complex structure reveals
the binding mode of classical, W101, and F108 amino acid sub-
stitution-sensitive FLE antibodies.
Cell Host & Microbe 19, 696704, May 11, 2016 699

In contrast to FLE antibodies, a previous study on broadly
neutralizing human antibodies against dengue viruses revealed
recognition determinants to be at a serotype-invariant site at
the E-dimer interface, including the exposed main chain of the
fusion loop and two conserved glycan chains, which also create
the binding site for the prM (Figures 6D and S3) (Rouvinski et al.,
2015). These E-dimer-dependent (EDE) neutralizing antibodies
are able to bind to mature or partially immature virus particles
but are unable to bind to the immature virions.
DISCUSSION
The association of ZIKV infection with microcephaly in fetuses
and neurological diseases such as GBS has caused international
concern (Petersen et al., 2016). Here, we found that our previ-
ously reported potently neutralizing antibody 2A10G6, which tar-
gets the fusion loop region of some flavivirus E proteins, can also
protect against ZIKV infection in vivo and can potentially be used
as a therapeutic candidate for ZIKV infection. A complex crystal
structure of 2A10G6 and ZIKV-E revealed that 2A10G6 forms the
majority of contacts with the hydrophobic residues of the fusion
loop, including W101, L107, and F108. These three residues are
highly conserved in all of the isolated ZIKV strains and in most of
the human-infected flaviviruses, including DENV 1-4, WNV, and
YFV (Figure S2) (Deng et al., 2011). Structural models also show
that 2A10G6 likely recognizes the DENV-E and WNV-E via a
similar binding mode (Figure S4).
Previously, two classes of dengue virus broadly neutralizing
antibodies have been revealed: the FLE-specific neutralizing
antibodies and the EDE-specific neutralizing antibodies (Dejnir-
700 Cell Host & Microbe 19, 696704, May 11, 2016
Figure 4. Complex Structure of 2A10G6 Fab
Bound to ZIKV-E
(A) Overall complex structure. The 2A10G6 Fab
binds to the domain II tip of ZIKV-E monomer at a
perpendicular angle with the fusion loop and bc
loop involved in the interaction.
(B and C) Cartoon representations of interacting
residues in the ZIKV-E (B) and 2A10G6 Fab (C).
The fusion loop of ZIKV-E deeply inserts into a hole
formed by both the heavy chain and light chain of
2A10G6, while the bc loop contacts only the heavy
chain. See also Figure S4.
attisai et al., 2015). The FLE-specific
antibodies usually have a lower neutral-
izing capacity and higher antibody-de-
pendent enhancement (ADE) effect than
the EDE-specific antibodies (Dejnirattisai
et al., 2015). However, the FLE-specific
antibodies are generally cross-reactive
and are able to neutralize different flavivi-
ruses. Structural studies of EDE-specific
neutralizing antibodies have revealed
that the recognition determinants are
found at a serotype-invariant site at the
E-dimer interface, including the exposed
main chain of the fusion loop and the
two conserved glycan chains (N67- and
N153-linked glycans) (Rouvinski et al., 2015). These two glyco-
sylation sites are not highly conserved in other flaviviruses.
Moreover, ZIKV does not possess the N67-linked glycosylation
site, and the N154-linked glycosylation site (equivalent to the
N153-linked glycosylation site in DENV) is absent in some of
the isolated ZIKV strains (Table S2). Furthermore, several resi-
dues in b strand, 150 loop, ij loop, and A strand, which are critical
for DENV EDE mAb binding, are not conserved in ZIKV and other
flaviviruses (Figure S2). Importantly, as ZIKV sE structure dis-
plays a unique positively charged patch at the binding regions
of EDE antibodies (Figure S1), the EDE-specific antibodies may
not be effective against ZIKV infection. However, many other
known flavivirus FLE-specific antibodies, which target the highly
conserved fusion loop, may be able to neutralize ZIKV, as
confirmed by our neutralizing profile of 2A10G6. Interestingly,
during the preparation of our manuscript, the cryo-EM structure
of ZIKV was published, illustrating an intact 150 loop and N154
glycosylation site that may function as a host attachment site
for the virus. Herein, we describe a unique, positively charged
patch that surrounds the 150 loop of ZIKV, which may also affect
virus attachment and hence influence transmission and disease.
We propose two possible explanations for the protection
mechanisms of 2A10G6. First, during flavivirus infection, the
hidden epitopes for neutralizing antibodies such as the FLE-spe-
cific mAbs may become exposed either through the incomplete
cleavage of prM that has been shown to create a mosaic of
both mature and immature viruses or through virus conforma-
tional transition at physiological temperatures (above 34 C), as
has been described in DENV (Fibriansah et al., 2013; Plevka
et al., 2011; Zhang et al., 2013b). Blocking the key residues
 EXPERIMENTAL PROCEDURES
Table 1. Interaction between 2A10G6 and sE
a
Antibody Contacts sE Total Contacts Protein Preparation, Expression, and Purification
H Chain T38 6 G106 134 The coding sequence for ectodomain residues 1409 of envelope protein (sE)
H40 14, 3 W101, G106 (1)b from ZIKV BeH818995 strain (GeneBank: KU365777) were codon optimized
and cloned into pET21a vector with a C-terminal His6-tag. The sE proteins
G55 1 L107 were expressed in Escherichia coli strain BL21(DE3) as inclusion bodies and
I56 3 L107 then refolded in vitro using the method as previously described (Liu et al.,
D57 3, 1 L107, Q77 2011), with some modifications. Briefly, aliquots of inclusion body were diluted
dropwise into a stirring refolding buffer (100 mM Tris [pH 8.0], 400 mM L-Arg
N60 7, 5 T76 (1), Q77 (1)
HCl, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione,
G62 3, 5 T76, L107 and 5% glycerol) for overnight refolding. Subsequently, the refolded protein
T63 2 L107 was concentrated using an Amicon 400 concentrator with 30 kDa cutoff
membrane and then adjusted to 20 mM Tris (pH 8.0), 50 mM NaCl, and 5%
N64 1, 14 L107, F108 (1)
glycerol. sE proteins were further purified in an AKTA Pure System by gel filtra-
R104 23, 3, 10, 8 W101 (1), G104, tion on a HiLoad 16/60 Superdex 200 PG column (GE Healthcare).
C105, G106 The coding sequences for heavy chain and light chain of murine mAb
Y107 4 W101 2A10G6 were codon optimized and cloned into pFastBac-Dual for expression
as a Fab. A previously described gp67 signal peptide sequence was added to
A108 10 W101
the N terminus of both chains for protein secretion, and His6-tag was fused at
L109 8 W101 the C terminus of heavy chain for affinity purification. 2A10G6 Fab was ex-
L Chain Q91 4 W101 63 pressed by Bac-to-Bac baculovirus expression system in Tricoplusia ni
Y93 19, 6, 9 W101, G102, F108 (High 5) cells (Invitrogen) as previously described (Shi et al., 2013; Zhang
et al., 2010). Soluble Fab was harvested from the culture supernatants
N94 3 F108 by metal affinity chromatography using a HisTrap HP 5 ml column (GE Health-
S95 9 F108 care) and subsequently purified by gel filtration on a Hiload 16/60 Superdex
Y96 3 F108 200 PG column (GE Healthcare) in buffer consisting of 20 mM Tris (pH 8.0)
and 150 mM NaCl.
P97 1, 9 W101, F108
For functional analysis, mAb 2A10G6 was used, and its preparation has
a
Numbers represent the number of atom-to-atom contacts between the been described previously (Deng et al., 2011). Briefly, the hybridoma
antibody residues and the sE residues, which were analyzed by the Con- producing mAb 2A10G6 (IgG1) was maintained by limiting dilution and
tact program in CCP4 suite (the distance cutoff is 4.5 A). subsequently purified from mouse ascites using Protein G affinity columns
b
Numbers in the parentheses represent the number of hydrogen bonds (GE Healthcare).
between the antibody residues and the sE residues, which were analyzed
by the Contact program in CCP4 suite (the distance cutoff is 3.5 A). Analytical Gel Filtration and Purification of Fab/E Complex
The 2A10G6 Fab was mixed with purified ZIKV sE protein for 30 min incubation
at 4 C. The samples from individual Fab, E protein, and their mixtures were
(such as W101) for virus fusion may neutralize the virus infection then loaded onto calibrated Superdex 200 GL column (GE Healthcare),
in vivo. Furthermore, recent work on HIV has also demonstrated respectively. The chromatographs were recorded and overlaid. The pooled
proteins were analyzed on 12% SDS-PAGE gel and stained with Coomassie
that the fusion peptide may represent a viable target for broadly
blue.
neutralizing mAbs in another viral system (Chen et al., 2014; Frey The 2A10G6 Fab/ZIKV-E complex was separated from excess Fab by si-
et al., 2008; Lai et al., 2014). This hypothesis supports the ze-exclusion chromatography, and the buffer was exchanged to 20 mM Tris
previously proposed concept that both immature and mature (pH 8.0), 50 mM NaCl, and 5% glycerol. The Fab/ZIKV-E complex was further
flavivirus virions contribute to infection and pathogenesis concentrated by 30 kDa cutoff membrane (Amicon) for subsequent
(Nelson et al., 2008). Second, the protection efficacy of crystallization.
2A10G6 in ZIKV may not be due merely to its weakly neutralizing
Neutralization Assay
activity, but rather may be through another effector functional
Neutralizing activity of mAb 2A10G6 was measured using a standard plaque
mechanism. As recently demonstrated, Fcg receptor and com- reduction neutralization with BHK21 cells as previously described (Deng
plement-dependent effector can also provide in vivo protection et al., 2011). Briefly, 5-fold serial dilutions of mAb 2A10G6 were added to
against other flaviviruses (Vogt et al., 2011). The similar mecha- approximately 200 PFU of ZIKV SZ01 strain (Deng et al., 2016) and incubated
nism has also been extensively elucidated in antibody therapy for 1 hr at 37 C. Then, the mixture was added to BHK21 cell monolayers in a
against other viruses including Ebola and influenze (Audet 12-well plate in duplicate and incubated for 1 hr at 37 C. The mixture was
et al., 2014; Qiu et al., 2012; Wilson et al., 2000; Zeitlin et al., removed, and 1 ml of 1.0% (w/v) LMP agarose (Promega) in DMEM plus 4%
(v/v) FBS was layered onto the infected cells. After further incubation at
2011) (DiLillo et al., 2016).
37 C for 4 days, the wells were stained with 1% (w/v) crystal violet dissolved
In summary, we have shown 2A10G6 to be able to neutralize in 4% (v/v) formaldehyde to visualize the plaques. PRNT50 values were deter-
and provide protection against ZIKV via recognition of the ZIKV mined using non-linear regression analysis.
E protein fusion loop region. As we have previously shown
2A10G6 to be able to efficiently neutralize many other flavivi- Animal Protection Experiment
ruses (Deng et al., 2016), including DENV1-4, YFV, and WNV, ZIKV SZ01 strain was isolated as previously described (Deng et al., 2016) and
was propagated in C6/36 cells. For antibody protection against ZIKV
which exhibit this highly conserved fusion loop, it is likely that
challenge, groups of 5-week-old A129 male mice (n = 5) were injected intraper-
the binding modes of 2A10G6 are the same as that of ZIKV in
itoneally (i.p.) with 105 PFU of ZIKV. 24 hr post infection, mice were passively
these flaviviruses. This antibody, especially after being human- transferred a single inoculation of 500 mg antibody 2A10G6, or PBS as
ized, may represent an auspicious therapeutic for the treatment control via i.p. injection. The animals were monitored daily for death. All exper-
of flavivivirus infection. iments were carried out according to ethical guidelines and approved by the

Cell Host & Microbe 19, 696704, May 11, 2016 701

Experimental Animal Ethic and Welfare Committee of Beijing Institute of Micro-
biology and Epidemiology.
Crystallization, Data Collection, and Structure Determination
Crystallization trials were performed in sitting drops of 600 ml. All crystals were
obtained by mixing equal volumes of protein and reservoir solution, using a
Mosquito LCP robot (TTP LabTech). Diffraction-quality crystals of free sE
were obtained at 4 C in a condition consisting of 0.2 magnesium acetate
tetrahydrate, 0.1 sodium cacodylate (pH 6.5), and 20% w/v PEG8000 at a pro-
tein concentration of 10 mg/ml. Diffraction-quality crystals of 2A10G6 Fab/sE
702 Cell Host & Microbe 19, 696704, May 11, 2016
Figure 5. Analysis of the Detailed Interac-
tion between the 2A10G6 and ZIKV-E
(A) Four polypeptide elements, three from the
heavy chain (light pink) and one from the light chain
(cyan), surround the domain II tip of ZIKV-E.
(B) The heavy chain forms five hydrogen bonds
(green dashed lines) with the ZIKV-E residues. The
hydrophobic residues W101, L107, and F108 in
the fusion loop contribute most of the contacts
between 2A10G6 and ZIKV-E.
complex were finally obtained at 18 C in a con-
dition consisting of 0.2 M sodium chloride,
0.1 Na/K phosphate (pH 6.5), and 25% PEG1000.
Diffraction data were collected at Shanghai Syn-
chrotron Radiation Facility (SSRF) BL17U (wave-
length, 0.97930 A). For data collection, all crystals
were cryo-protected by briefly soaking in reservoir
solution supplemented with 20% (v/v) glycerol
before flash-cooling in liquid nitrogen. All the data-
sets were processed with HKL2000 software (Ot-
winowski and Minor, 1997). The structures of sE
and 2A10G6 were both determined by the molec-
ular replacement method using Phaser (Read,
2001) with previously reported dengue virus 2 E
protein structure (PDB: 1OKE) as the search
model. The E-2A10G6 complex structure was
further determined by the molecular replacement
with the solved monomer structure of sE and the
Fab structure (PDB: 4GMS) as the search models,
respectively. The atomic models were completed
with Coot (Emsley and Cowtan, 2004) and refined
with phenix.refine in Phenix (Aad et al., 2010), and
the stereochemical qualities of the final models
were assessed with PROCHECK (Laskowski et al., 1993). Data collection,
processing, and refinement statistics are summarized in Table S1.
Surface Plasmon Resonance Assay
The SPR analysis was carried out using a BIAcore 3000 machine with CM5
chips (GE Healthcare) at room temperature (25 C). The buffers for all proteins
used were exchanged to a BIAcore buffer consisting of 10 mM HEPES
(pH 7.5), 150 mM NaCl, and 0.005% (v/v) Tween-20 via gel filtration. The
ZIKV sE protein was immobilized on the chip at about 1,000 response units
(RU). Subsequently, gradient concentrations of 2A10G6 Fab (0, 1.9, 3.9, 7.8,
Figure 6. Comparison with Other Fusion
Loop-Targeting Neutralizing Antibodies
(A) Structure of prM-E heterodimer of dengue virus
serotype 2 (DENV2) (PDB: 3C5X). The pr peptide is
shown in green, with domain coloring described
previously in Figure 1.
(B) Structure of ZIKV-E/2A10G6 complex with
coloring described previously in Figure 5.
(C) Structure of fusion loop epitope (FLE)-specific
E53 in complex with the West Nile virus E protein
(PDB: 3I50). The heavy chain is in lemon, and the
light chain is in wheat.
(D) Structure of E-dimer epitope (EDE)-specific
antibody A11 in complex with the DENV2 E protein
(PDB: 4UT7). The heavy chain is in light gray, and
the light chain is in light orange. See also Figures
S2 and S3 and Table S2.
15.6, 31.3, 62.5, 125, 250, and 500 nM) were used to flow over the chip surface.
After each cycle, the sensor surface was regenerated via a short treatment
using 10 mM NaOH. The binding kinetics were analyzed with the software
BIAevaluation Version 4.1 using 1:1 Langmuir binding model.
ACCESSION NUMBERS
The accession numbers for the crystal structures of ZIKV-E and
ZIKV-E/2A10G6 Fab complex are PDB: 5JHM and 5JHL, respectively.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and two tables and can be found with this article online at
http://dx.doi.org/10.1016/j.chom.2016.04.013.
AUTHOR CONTRIBUTIONS
G.F.G., C.-F.Q., J.Q., and Y.S. designed and supervised the study. L.D., J.S.,
X.L., Y.-Q.D., H.C., A.M.M., Y.Z., Y.Y., and H.S. conducted the experiments.
J.Q. collected the data sets and solved the structures. Y.S., L.D., and G.F.G.
analyzed the data and wrote the paper. J.H., H.X., and J.Y. participated in
the manuscript editing and discussion.
ACKNOWLEDGMENTS
This work was supported by the Task-Force of Zika Virus Research from the
Chinese Academy of Sciences (CAS), the Emergency Task-Force Project
(Grant No. 81641001) of the National Natural Science Foundation of China
(NSFC), Zika Special Project of the National Infectious Disease Control S&T
Grand Project, and Strategic Priority Research Program of the Chinese Acad-
emy of Sciences (XDB08020100). We thank the staff of BL17U beamline at
Shanghai Synchrotron Radiation Facility for assistance during data collection.
Y.S. is supported by the Excellent Young Scientist Program of the Chinese
Academy of Sciences and the Youth Innovation Promotion Association CAS
(Grant No. 2015078). C.-F.Q. is supported by the Excellent Young Scientist
Program from the NSFC (No. 81522025) and the Newton Advanced Fellowship
from the Academy of Medical Sciences, UK and NSFC. G.F.G. is a leading
principal investigator of the NSFC Innovative Research Group (Grant No.
81321063). We thank Shihua Li, Gary Wong, Kefang Liu, and Helin Zhang for
their technical support.
Received: March 30, 2016
Revised: April 18, 2016
Accepted: April 22, 2016
Published: May 2, 2016
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