Neutralizing antibodies can initiate genome releasefrom human enterovirus 71Pavel Plevkaa,1, Pei-Yin Limb, Rushika Pereraa,2, Jane Cardosab,c, Ampa Suksatua, Richard J. Kuhna,and Michael G. Rossmanna,3

aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; bSentinext Therapeutics, 10050 Penang, Malaysia; and cMAB Explorations,10050 Penang, Malaysia

Edited by Wah Chiu, Baylor College of Medicine, Houston, TX, and approved January 6, 2014 (received for review November 7, 2013)

Antibodies were prepared by immunizing mice with empty, imma-ture particles of human enterovirus 71 (EV71), a picornavirus thatcauses severe neurological disease in young children. The capsidstructure of these empty particles is different from that of themature virus and is similar to “A” particles encountered whenpicornaviruses recognize a potential host cell before genome release.The monoclonal antibody E18, generated by this immunization,induced a conformational change when incubated at temperaturesbetween 4 °C and 37 °C with mature virus, transforming infectiousvirions into A particles. The resultant loss of genome that wasobserved by cryo-EM and a fluorescent SYBR Green dye assayinactivated the virus, establishing the mechanism by which thevirus is inactivated and demonstrating that the E18 antibody haspotential as an anti-EV71 therapy. The antibody-mediated virusneutralization by the induction of genome release has not beenpreviously demonstrated. Furthermore, the present results indicatethat antibodieswith genome-release activity could also be producedfor other picornaviruses by immunization with immature particles.

Enterovirus 71 (EV71) is a picornavirus that causes hand, foot,and mouth disease (1). In infants and small children, the

infection may proceed to encephalitis that can be fatal or resultin permanent brain damage. EV71 virions are nonenvelopedwith a diameter of approximately 300 Å. The capsid has icosa-hedral, pseudo-T=3 symmetry with four viral proteins VP1, VP2,VP3, and VP4 in each icosahedral asymmetric unit (2, 3). Sub-units VP1, VP2, and VP3 have a jelly-roll fold common to manyviruses. VP4 is a small protein attached to the inner face of thecapsid. EV71 infections produce fully infectious RNA-filledparticles and empty immature particles that lack genome andcontain capsid protein VP0, the precursor of VP4 and VP2 (3).These empty particles have approximately 5% larger diameterthan the mature virions. Furthermore, the protomer formed byVP0, VP1, and VP3 is rotated by 5.4° relative to the protomerformed by VP1, VP2, VP3, and VP4 in the mature particle withrespect to the icosahedral symmetry axes. The empty particlesare presumably precursors of the mature infectious virions (3).Rhino and entero picornaviruses have a depression, called the

“canyon,” on the virion surface encircling the icosahedral five-fold axes (4). The canyon is frequently the site of binding ofpicornavirus receptors (5–8), although some receptor moleculesbind to other sites on picornavirus capsids (9, 10). Experimentalevidence indicates that binding of a receptor into the canyonresults in the expulsion of the “pocket factor” from the hydro-phobic cavity within VP1 (11–14). Ejection of the pocket factorleads to destabilization of virions. Such activated “A” particlesare characterized by expansion of the capsid, release of VP4, andexternalization of the VP1 N-termini (6). The organization of themajor capsid proteins in the A particle and in the immatureempty particles are similar (3). Transition of the virion to the Astate is a prerequisite for the release of the genome (15). Heatingof picornavirus particles to nonphysiological temperatures of 50 °Cto 60 °C can also induce transformation of virions to the A state invitro (6, 16, 17).

Here we present an analysis of the interactions of the mono-clonal antibodies E18 and E19 with EV71. By using cryo-EM, weshow that binding of E18 to EV71 causes the virus to change itsconformation to the A state and to eject much of its genome.This was further verified by fluorescence activation when SYBRGreen dyes interact with RNA. In contrast, although mAb E19does neutralize the virus, it has a quite different footprint on thevirus surface and does not cause ejection of the genome.

Results and DiscussionThe E18 and E19 antibodies were prepared by immunizing micewith empty, immature EV71 particles containing VP0 (18). BothE18 and E19 could neutralize the virus as intact antibodies or asFab fragments (Fig. 1). Both these mAbs can recognize confor-mational epitopes on the surface of heat-inactivated EV71 par-ticles by indirect ELISA. However, these antibodies could notrecognize linear epitopes by using immunoblot analysis (Fig. 2).The Fab fragments of these mAbs were incubated with EV71 forcryo-EM studies of the mAb–virion complexes. Visual inspectionof the cryo-EM micrographs showed that as many as 20% of theEV71 particles that had been incubated with E18 had lost muchor all of their RNA genome (Fig. 3 A, D, E, and F). In contrast

Significance

Enterovirus 71 (EV71) causes yearly outbreaks of hand, foot,and mouth disease in Southeast Asian countries includingChina and Malaysia. Some of the infected children developencephalitis that can be fatal or result in permanent braindamage. There are no anti-EV71 therapeutic agents available.Here it is shown that an antibody that had been generated byusing an immature EV71 virus as an antigen induced the re-lease of genome from EV71 virions, rendering the virus non-infectious. The induction of genome release is a mechanismby which antibodies can neutralize viruses. Furthermore, theapproach presented in the paper could be used to prepareantibodies with similar properties against related viruses thatinclude significant human pathogens.

Author contributions: P.P. and M.G.R. designed research; P.P., P.-Y.L., R.P., J.C., and A.S.performed research; P.P., P.-Y.L., R.P., J.C., R.J.K., and M.G.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Cryo-EM reconstructions were deposited with the EM Data Bank, www.emdatabank.org [accession numbers EMD-2397 (E18 full), EMD-2434 (E18 empty), andEMD-2436 (E19 Fab–EV71)]. The atomic coordinates have been deposited in the ProteinData Bank, www.pdb.org [PDB ID codes 4C0U (E18 full), 4C0Y (E18 empty), and 4C10(E19 Fab–EV71).1Present address: Central European Institute of Technology, Masaryk University, 625 00Brno, Czech Republic.

2Present address: Arthropod-Borne and Infectious Diseases Laboratory, Department ofMicrobiology, Immunology, and Pathology, Colorado State University, Fort Collins,CO 80523.

3To whom correspondence should be addressed. E-mail: mr@purdue.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1320624111/-/DCSupplemental.

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to the action of E18 Fab, only approximately 1% of the EV71particles incubated with E19 Fab had lost their genome (Fig. 3B and C). Thus, E18 but not E19 induced genome releasefrom virions.Separate reconstructions were made of the empty immature

EV71–E18 complexes and of the full EV71–E18 complexes. Theresolutions of these reconstructions were 10 Å and 20 Å, re-spectively, as judged by the resolution at which the Fourier shellcorrelation coefficient decreased to less than 0.5 (Fig. S1). Thecorrelation coefficient between the cryo-EM electron densitymaps of the capsid region of the heat-induced EV71 A particlesand that of the corresponding density of the EV71–E18 (full)complex was 0.83, whereas the correlation between the electrondensity of the native virus capsid and the EV71–E18 complexwas only 0.61 (Table 1). Thus, the E18 Fab had induced theconformational change of the EV71 virions to the A state (Fig. 4A and D), a conformational change similar to what was producedby heating EV71 virions to 56 °C (Fig. 4 D and G) (16). Con-sistent with these observations, ELISA tests showed that E18binds better to heat-induced EV71 A particles than to maturevirions (Fig. 5). The 3D cryo-EM reconstructions showed thatthe capsid structures of the RNA-containing EV71–E18 Fabcomplex and of the empty EV71–E18 Fab complex are nearlyidentical (correlation coefficient, 0.95; Fig. 4 A, B, D, and E andTable 1). Thus, the conformational change must be the consequenceof E18 binding to the virions, which then leads to genome release.

Although EV71 can be completely inhibited by E18, theelectron micrographs show that not all particles had releasedtheir genomes (Fig. 3F). However, the virus particles would beinactivated even if only a small part of the genome were releasedfrom the virion and degraded by RNAses. Such particles, eventhough noninfectious, would be evaluated as genome containingin our analysis. Indeed, the electron density corresponding to thegenome was lower in the EV71–E18 complex (minimum, −2.2,maximum, 1.1; average, −0.17) than in the EV71–E19 complex(minimum, −1.6; maximum, 2.0; average, −0.15), even though onlyfull-looking particles were used to calculate the (full) EV71–E18reconstruction.Alternatively, the neutralization of EV71 by E18 might be

achieved not only by inducing genome release but also by othermeans such as preventing receptor binding. Indeed, the E18footprint on the virion surface includes Lys-149 of VP2 that hasbeen implicated to have a role in attachment of EV71 to theP-selectin glycoprotein ligand-1 receptor (Fig. 6 and Fig. S2) (19).The E18 binding sites on the EV71 capsids are located

between VP4–VP2–VP3–VP1 protomers (Fig. 6A and Table 2).However, the protomer in both A particles (after receptorbinding) and empty, immature (before VP0 cleavage) particles isrotated by 5.4° relative to its position in the mature capsid withrespect to the icosahedral axes (3, 16). Because the E18 antibodywas generated by immunization with empty, immature particles,it is likely that, when E18 binds to mature EV71 particles, therewill occur an “induced fit” that requires local rearrangements ofthe capsid to an immature-like capsid conformation. Thus, pos-sibly the E18 antibody binds to the virus when the capsid tem-porarily and locally changes structure to be like an A particlebecause of the natural capsid dynamics. However, mature virionscontain VP2 and VP4 instead of VP0. Therefore, the capsidproteins reorganize to resemble A particles that are generatedwhen the virus recognizes a receptor and expels the pocketfactors (Fig. 4 A, G, H, and I). As the E18 Fabs bind across theinterface between protomers (Fig. 6A), binding of E18 to themature virion induces the protomers to rotate by 5.4° to the Aform. In contrast, E19 binds wholly within a single protomer(Fig. 6B), and therefore its binding does not require anyconformational change of the capsid. It has been shown thatpicornavirus genomes are released through channels at the ico-sahedral twofold axes that form upon transition of the virionsto the A state (3, 20). Therefore, binding of E18 to maturevirions results in a conformational change to A-like particles andthus facilitates the release of the genome. Hence, the release of

Fig. 1. Neutralization of EV71 by monoclonal antibodies E18 and E19.Whole IgG and Fab fragments of the monoclonal antibodies E18 (A) and E19(B) were used to inhibit EV71 at different concentrations (x axis) by usinga plaque reduction neutralization test. The red symbols represent wholeantibody and the blue symbols represent Fab fragments. Inhibition of viruswas represented as the percentage of plaques relative to plaques in thecontrol wells. We demonstrated neutralization of EV71 by Fab fragments aswell as by whole IgG, with whole IgG being more efficient than Fab fragments.

Fig. 2. Analysis of binding of E18 and E19 to EV71 viral proteins by immunoblot and indirect ELISA. (A) Mock- (M) and EV71-infected (V) cell lysates wereseparated by SDS/PAGE and transferred onto membrane. Membrane was probed with R525, E18, or E19. E18 and E19 did not bind denatured viral proteinsand, therefore, recognize conformational epitopes. (B) Indirect ELISA was performed by coating wells with recombinant viral proteins or heat-inactivatedEV71-infected cell lysates. Various concentrations of mAb were added in duplicates. The mAbs were detected by HRP assay (Materials and Methods). Theamount of bound mAbs are presented as average OD450 ± SDs.

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the genome upon E18 binding is achieved by a mechanism thatcould be similar to that of a receptor binding to a virus. Thepresent study of E18 binding to EV71 may indirectly providea description of a mechanism by which picornavirus receptorsthat bind outside of the canyon induce genome release.The effect of antibody binding on mature EV71 virions was

also evaluated with an assay that measured the interaction of thegenome with the RNA-binding fluorescent dyes SYBR Green Iand II (Fig. 3A and Fig. S3) (20, 21). The results demonstratedthat binding of E18 antibodies or Fab fragments to EV71 in-creased accessibility of the genome to the dyes at temperaturesbetween 4 °C and 37 °C, whereas E19 had limited effect on ge-nome accessibility (Fig. S3). However, these experiments do notdifferentiate between the genome being released from a virionand then interacting with the dyes outside the virion or the dyesentering the virion and interacting with the genome remaininginside the virion. However, these experiments do confirm thatbinding of E18 to the virus induces a conformational change thatallows communication between the inside of the capsid and theexternal environment.The Fab fragment of the E19 mAb binds primarily to the VP3

“knob” (4), a different site than that occupied by E18 (Fig. 6).Binding of E19 Fab to EV71 did not induce any detectablerearrangements of the capsid relative to the native state (Fig. 4C, F, and I). The correlation coefficient of the capsid of matureEV71 with the capsid of EV71 complexed with E19 was 0.81,whereas its correlation with the A particle capsid (heated maturevirus) was only 0.39 (Table 1). In other picornaviruses, binding ofreceptors to the knob region (Fig. 6) does not induce transition

of particles to the A state (9, 10), but require additional cor-eceptors for successful infection (10). The E19 footprint on theEV71 virion surface does not overlap with any putative receptorbinding sites (19, 22). However, it is possible that E19 mightneutralize the virus by preventing EV71 binding to an as yetunidentified cellular receptor. The geometry of binding of E18and E19 Fab fragments indicates that neither of the antibodiescould bind to the virus divalently as intact IgG.Virus capsids evolved to serve as efficient vessels for transport

of virus genetic material from one host to another. However,some of the functions that the capsids perform exert conflictingselection pressures on the design of the capsid. For instance,the capsids need to be stable to protect the viral genomes inthe extracellular environment, but they also need to release thegenome at the right time to initiate infection. Therefore, thecapsids are selected for optimal—not too high, not too low—stability. Infection of some picornavirus genera can be limited bysmall molecules that bind with high affinity into the VP1 pocketin place of the pocket factor (23, 24). These compounds inhibitinfection by overstabilizing the virions (14, 23). In contrast, thepresent results show that antibody binding can promote transi-tion of virions to the A state and deactivate EV71 by inducinguntimely genome release. It has been shown here that antibodiescapable of causing the release of genome can be generated byimmunization with empty particles containing VP0. As an ex-ample of this strategy, empty immature virus-like particles(VLPs) were purified on an E18 affinity column and were usedto immunize mice. The resultant sera were assayed to determinethe neutralizing antibody titres (Fig. S4). Mice immunized with

Fig. 3. Stability of EV71 virions and their complexes with E18 and E19. (A) Plot of time and temperature dependence of interaction of the fluorescent SYBRGreen I and II dyes with the EV71 genomic RNA. Native EV71 virions (blue line) as well as EV71 complexes with E18 (green line) and E19 (red line) wereincubated with the fluorescent dyes at 37 °C. The purple line represents a negative control without virus. Subsequently, the complexes were gradually heatedto 90 °C. The increase in fluorescence showed that the fluorescent dyes were binding to the genomic RNA. The numbers at 60, 120, and 360 min associatedwith each of the lines show the percentages of empty particles in cryo-EM images for each sample. Cryo-EM images of (B) EV71 virions and of (C) EV71complexed with E19 Fab and (D) EV71 complexed with E18 Fab that were incubated at 37 °C for 120 min. (E and F) Also shown are virions in the process ofgenome release observed in the E18–EV71 mixture. (Scale bars: 50 nm.)

Table 1. Correlation coefficients comparing electron density distribution in the capsid regionsof EV71 virions, A particles, and E18 and E19 antibody complexes

Structure EV71 mature EV71 “A” particle EV71 + E19 EV71 + E18 empty EV71 + E18 full

EV71 + E18 full 0.61 0.83 0.39 0.95 —

EV71 + E18 empty 0.42 0.57 0.48 —

EV71 + E19 0.81 0.39 —

EV71 “A” particle 0.52 —

EV71 mature —

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VLPs exhibited higher neutralizing antibody titers against EV71(geometric mean titer, 153) compared with control mice (geo-metric mean titer, 55). Pooled serum from VLP-immunized miceinhibited 60% of E18 binding indicating that serum from thesemice contains antibodies that recognize E18 epitope (Fig. S4).Thus, VLPs selected for having the E18 epitope can induceneutralizing antibodies in mice. Therefore, therapeutic anti-bodies with genome-release activity might also be obtainable forother picornaviruses using the approach described here.

Materials and MethodsPreparation of Fab Fragments of Monoclonal Antibodies. The Fab fragments ofthe antibodies were prepared with the use of the Pierce Fab Preparation Kit

according to the manufacturer’s instructions. Animal care and use wasconducted in accordance with the National Animal Welfare Standards andGuidelines of Malaysia under the Animals Act of 2006.

Immunoblot Analysis. Equal volumeofmock-andEV71-infectedcell lysateswereseparatedona 12% (wt/vol) SDS/PAGE, transferred tonitrocellulosemembrane,and probed with R525 (polyclonal antibody against EV71 VP1), E18, or E19.Bound antibody was detected by incubation with HRP-conjugated secondaryantibodies (Dako) followed by TMB membrane peroxidase substrate (KPL).

ELISA Analyses. An indirect ELISA was performed by coating Nunc-Immunoplate with recombinant viral proteins or heat-inactivated EV71-infectedrhabdomyosarcoma cell lysates as positive control. Nonspecific binding wasblocked using 5% skim milk, antibodies were added at various concentrations

Fig. 4. Cryo-EM reconstructions of EV71–Fab complexes showing also comparisons with native and expanded EV71 virions. (Top) Cryo-EM reconstructions of(A) genome containing EV71–E18, (B) empty EV71–E18, and (C) genome containing EV71–E19 complexes. The reconstructions are rainbow colored accordingto the distance of the surface from the particle center. (Middle) Center sections of the cryo-EM reconstructions of (D) genome containing EV71–E18, (E) emptyEV71–E18, and (F) genome containing EV71–E19 complexes. The sections are rainbow-colored according to the electron density height. (Bottom) Centersections of (G) heat-induced EV71 A particles, (H) empty particles after genome release, and (I) native EV71 capsid.

Fig. 5. Comparison of binding of E18 (A) and E19 (B) to heat-inactivated EV71 (i.e., A particles) and native EV71. The purified EV71 was stored on ice orincubated at 56 °C for 30 min. Serial dilutions of the samples were added to wells coated with polyclonal antibodies against VP1, and bound viral particleswere detected by the addition of E18 or E19 (Materials and Methods). The average OD values indicating amounts of bound mAbs ± SDs are shown.

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in duplicate, and bound antibodies were detected by using HRP-conjugatedanti-mouse IgG (Dako). SureBlue Reserve TMB microwell peroxidase substrate(KPL) was added for 5 min, 0.5 M HCl was added to stop the enzyme reaction,and wells were read at 450 nm.

A sandwich ELISA was performed whereby the wells were coated withR525 antibody against VP1, and PEG-precipitated EV71 that was untreated orheat-inactivated at 56 °C for 30 min was allowed to bind to the VP1 anti-body. The bound particles were detected by the monoclonal antibodies E18or E19, followed by HRP IgG (Dako) as described earlier.

A competitive ELISA was conducted to examine the presence of antibodiescontaining E18 epitope in mouse serum. Sera from four mice immunized withVLP that had high plaque reduction neutralization tests were pooled, andsera from four mice immunized with PBS solution were pooled for thecompetitive ELISA. Wells were coated with R525 followed by equal proteinconcentrations of mock- and EV71-infected RD cell lysates. Sera pooled frommice (at 1/250 dilution) were added into the wells, and HRP-conjugated E18was added immediately afterward. Reserve TMB microwell peroxidase sub-strate (KPL) was added for 5 min, 0.5 M HCl was added to stop the enzymereaction, and wells were read at 450 nm. Adjusted OD values were obtainedby subtracting OD of mock RD cell lysate from OD of EV71-infected RD cells.The relative percentage of binding of E18 was derived by dividing the ad-justed OD of samples by the adjusted OD of well containing only HRP-E18,multiplied by 100.

Plaque Reduction Neutralization Test. Different concentrations of Abs, Fabfragments, or heat-inactivated mouse serum were incubated in 1:1 volumeratios with infectious EV71 strain MY104 (300 pfu/mL) for 1 h at 37 °C. Thevirus–antibody (or Fab) mixture was inoculated in duplicates over Vero cellmonolayers in 24-well plates (Nunc/Thermo-Fisher). The monolayers wereprepared with 0.5 mL per well of Vero cells at 3 × 105 per milliliter in DMEMsupplemented with 5% FBS and antibiotics (all from Invitrogen) and left toadhere overnight before inoculation. Media was aspirated before in-oculation with 200 μL of the antibody (or Fab)–virus mixtures and incubatedin a CO2 incubator at 37 °C for 2 h before 1 mL of overlay was added con-taining DMEM supplemented with 2% FBS, antibiotics, and 1.5% carboxy-methyl cellulose. Plates were incubated at 37 °C with 5% CO2 for 4 d andstained with naphthalene black. Plaques were counted manually. The per-cent inhibition was determined relative to controls in which the mean

number of plaques in wells in which the virus had been incubated withmedia alone.

Virus Production and Purification. EV71 virions were produced and purified asdescribed previously (25).

VLP Production and Purification. Briefly, EV71 empty immature capsids wereproduced using a baculovirus expression system in which the complete P1coding sequence and the protease 3CD of EV71 were recombinantly inserteddownstream of the polyhedrin promoter and the recombinant baculoviruswas used to infect Sf9 cells at a multiplicity of infection of 0.1. The super-natant harvested on day 4 was clarified and concentrated by using tan-gential flow filtration (GE Healthcare Lifesciences), and the retentate wasrun through an affinity column prepared by coupling E18 to a HiTrap NHS-activated HP column (GE Healthcare Lifesciences). The particles bound wereeluted by using a glycine buffer at pH 3.0 and immediately neutralized to pH7.2with 1 M Tris·HCl. The particles were transferred to Dulbecco’s phosphate-buffered saline buffer (Invitrogen).

Immunization of Mice. Mice (n = 10 per group) were immunized with twodoses of DPBS or 10 μg of VLP in the presence of Imject Alum (Thermo Sci-entific) 3 wk apart. Serum were inactivated by incubation at 56 °C for 30min, and stored at −20 °C for further analysis.

CryoEM Data Collection and Reconstruction. Either the E18or E19 Fab fragmentswere incubatedwithEV71at 37 °C for 1, 2, or 6hat a ratioof three Fab fragmentsper icosahedral asymmetric unit of the virus. Small aliquots (3.5 μL) of this mix-ture were applied to holey carbon-coated grids, blotted with filter paper, andvitrified by plunging into liquid ethane. Electron micrographs were recorded onKodak SO-163 film by using a Philips CM200 FEG microscope. Micrographs weredigitizedwith a Nikon Cool-Pix scanner. The final averaged pixel size was 2.48 Å.The program e2boxer.py was used to box 8325 and 13346 particles for the E18and E19 complexes, respectively (26). The particles were corrected for the con-trast transfer function using the programs ctfit and e2projectmanager.py fromeman and eman2 packages (26, 27). The defocus ranged from 1.12 to 3.67 μm.The reconstruction was started by combining projections down twofold, three-fold and fivefold axes using the program starticos from the eman package (27).The EM reconstruction processes were performed using icosahedral averagingwith the same software. The resolution of the resultingmaps were estimated bycomparing structure factors of the virus shell computed from two independenthalf data sets (Fig. 3). For the final 3D reconstruction, data were included to theresolution (approximately 16Å, 9 Å, and 13 Å for the complexes with empty E18,full E18, and full E19, respectively) at which the correlation between the Fouriercoefficients of two independent data sets was better than 0.3.

Fitting of Fab Protein Data Bank Models into the Cryo-EM Density. The pro-gram EMfit was used to calibrate the exact magnification of the cryo-EMmapof EV71 reconstructions by comparing them with maps derived from thecrystallographically determined coordinates of EV71 mature and immatureparticles [Protein Data Bank (PDB) ID codes 3ZFE and 3VBO]. For the EV71–E19complex, the mature EV71 structure (PDB ID code 3ZFE) was used to model

Fig. 6. Antibody footprints on the EV71 surface. The figure shows 2D projections of the EV71 virion surface. Residues of capsid proteins VP1, VP2, and VP3 areoutlined in blue, green, and red, respectively. Residues involved in binding (A) E18 and (B) E19 are shown in bright colors. The footprints of E18 and E19 areoutlined by yellow lines in A and B, respectively. The border of one VP4–VP2–VP3–VP1 protomer is indicated by a dotted line. Positions of twofold, threefold, andfivefold icosahedral symmetry axes are shown as ovals, triangles, and pentagons, respectively. One icosahedral asymmetric unit is outlined by a triangle.

Table 2. EMfit statistics for fitting of Fab fragments intocryo-EM electron density maps.

Fab Sumf Clash −Den

E18 54.9 0.0 5.0E19 38.6 0.0 17.0

Clash, percentage of atoms in the model that have clashes with symmetryrelated protein molecules; −Den, percentage of atoms positioned in nega-tive density; Sumf, average value of density at atomic positions normalizedby setting the highest density in the map to 100.

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the capsid. For the EV71–E18 complex, the model of the capsid was based onthe immature EV71 particle (PDB ID code 3VBO). “Difference maps” werethen calculated by masking out the density of the capsid by setting to zeroall grid points within 3 Å from any EV71 capsid protein atom. Modeled Fabfragments (based on PDB model ID number 1QGC) were then fitted into thedifference map by using the program EMfit (28) (Table 2).

Buried Surface Area and Residues Forming the Protein–Protein Interface. Theresidues forming the virus–Fab interfaces were identified with the Web service

Proteins, Interfaces, Structures, and Assemblies at the European BioinformaticsInstitute (www.ebi.ac.uk/msd-srv/prot_int/pistart.html) (29) based on buriedsurface area between the fitted Fab fragments and capsid proteins.

ACKNOWLEDGMENTS. We thank Sheryl Kelly for help with the preparationof the manuscript. Cryo-EM studies were supported by a National Institutesof Health Grant R01 AI 11219 (to M.G.R.). The production and characteriza-tion of antibodies in mice was performed by Jane Cardosa for a separateresearch project funded by MAB Explorations (Penang, Malaysia).

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