Neutralizing antibodies can initiate genome release from human 71

Pavel 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- Here we present an analysis of the interactions of the mono- ture particles of human enterovirus 71 (EV71), a that clonal antibodies E18 and E19 with EV71. By using cryo-EM, we causes severe neurological disease in young children. The capsid show that binding of E18 to EV71 causes the to change its structure of these empty particles is different from that of the conformation to the A state and to eject much of its genome. mature virus and is similar to “A” particles encountered when This was further verified by fluorescence activation when SYBR recognize a potential host cell before genome release. Green dyes interact with RNA. In contrast, although mAb E19 The monoclonal antibody E18, generated by this immunization, does neutralize the virus, it has a quite different footprint on the induced a conformational change when incubated at temperatures virus surface and does not cause ejection of the genome. between 4 °C and 37 °C with mature virus, transforming infectious virions into A particles. The resultant loss of genome that was Results and Discussion observed by cryo-EM and a fluorescent SYBR Green dye assay The E18 and E19 antibodies were prepared by immunizing mice inactivated the virus, establishing the mechanism by which the with empty, immature EV71 particles containing VP0 (18). Both virus is inactivated and demonstrating that the E18 antibody has E18 and E19 could neutralize the virus as intact antibodies or as potential as an anti-EV71 therapy. The antibody-mediated virus Fab fragments (Fig. 1). Both these mAbs can recognize confor- neutralization by the induction of genome release has not been mational epitopes on the surface of heat-inactivated EV71 par- previously demonstrated. Furthermore, the present results indicate ticles by indirect ELISA. However, these antibodies could not that antibodies with genome-release activity could also be produced recognize linear epitopes by using immunoblot analysis (Fig. 2). for other picornaviruses by immunization with immature particles. The Fab fragments of these mAbs were incubated with EV71 for cryo-EM studies of the mAb–virion complexes. Visual inspection nterovirus 71 (EV71) is a picornavirus that causes hand, foot, of the cryo-EM micrographs showed that as many as 20% of the Eand mouth disease (1). In infants and small children, the EV71 particles that had been incubated with E18 had lost much infection may proceed to encephalitis that can be fatal or result or all of their RNA genome (Fig. 3 A, D, E, and F). In contrast in permanent brain damage. EV71 virions are nonenveloped with a diameter of approximately 300 Å. The capsid has icosa- Significance hedral, pseudo-T=3 symmetry with four viral proteins VP1, VP2, VP3, and VP4 in each icosahedral asymmetric unit (2, 3). Sub- Enterovirus 71 (EV71) causes yearly outbreaks of hand, foot, units VP1, VP2, and VP3 have a jelly-roll fold common to many and mouth disease in Southeast Asian countries including . VP4 is a small protein attached to the inner face of the and Malaysia. Some of the infected children develop capsid. EV71 infections produce fully infectious RNA-filled encephalitis that can be fatal or result in permanent brain particles and empty immature particles that lack genome and damage. There are no anti-EV71 therapeutic agents available. contain capsid protein VP0, the precursor of VP4 and VP2 (3). Here it is shown that an antibody that had been generated by These empty particles have approximately 5% larger diameter using an immature EV71 virus as an antigen induced the re- than the mature virions. Furthermore, the protomer formed by lease of genome from EV71 virions, rendering the virus non- VP0, VP1, and VP3 is rotated by 5.4° relative to the protomer infectious. The induction of genome release is a mechanism formed by VP1, VP2, VP3, and VP4 in the mature particle with by which antibodies can neutralize viruses. Furthermore, the respect to the icosahedral symmetry axes. The empty particles approach presented in the paper could be used to prepare are presumably precursors of the mature infectious virions (3). antibodies with similar properties against related viruses that Rhino and entero picornaviruses have a depression, called the include significant human pathogens. “canyon,” on the virion surface encircling the icosahedral five- fold axes (4). The canyon is frequently the site of binding of Author contributions: P.P. and M.G.R. designed research; P.P., P.-Y.L., R.P., J.C., and A.S. picornavirus receptors (5–8), although some receptor molecules performed research; P.P., P.-Y.L., R.P., J.C., R.J.K., and M.G.R. wrote the paper. bind to other sites on picornavirus capsids (9, 10). Experimental The authors declare no conflict of interest. evidence indicates that binding of a receptor into the canyon This article is a PNAS Direct Submission. results in the expulsion of the “pocket factor” from the hydro- 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), and phobic cavity within VP1 (11 14). Ejection of the pocket factor EMD-2436 (E19 Fab–EV71)]. The atomic coordinates have been deposited in the Protein leads to destabilization of virions. Such activated “A” particles Data Bank, www.pdb.org [PDB ID codes 4C0U (E18 full), 4C0Y (E18 empty), and 4C10 are characterized by expansion of the capsid, release of VP4, and (E19 Fab–EV71). externalization of the VP1 N-termini (6). The organization of the 1Present address: Central European Institute of Technology, Masaryk University, 625 00 major capsid proteins in the A particle and in the immature Brno, Czech Republic. 2 empty particles are similar (3). Transition of the virion to the A Present address: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, state is a prerequisite for the release of the genome (15). Heating CO 80523. of picornavirus particles to nonphysiological temperatures of 50 °C 3To whom correspondence should be addressed. E-mail: [email protected]. to 60 °C can also induce transformation of virions to the A state in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vitro (6, 16, 17). 1073/pnas.1320624111/-/DCSupplemental.

2134–2139 | PNAS | February 11, 2014 | vol. 111 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1320624111 Downloaded by guest on September 30, 2021 Although EV71 can be completely inhibited by E18, the electron micrographs show that not all particles had released their genomes (Fig. 3F). However, the virus particles would be inactivated even if only a small part of the genome were released from the virion and degraded by RNAses. Such particles, even though noninfectious, would be evaluated as genome containing in our analysis. Indeed, the electron density corresponding to the genome 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 only Fig. 1. Neutralization of EV71 by monoclonal antibodies E18 and E19. full-looking particles were used to calculate the (full) EV71–E18 Whole IgG and Fab fragments of the monoclonal antibodies E18 (A) and E19 reconstruction. (B) were used to inhibit EV71 at different concentrations (x axis) by using a plaque reduction neutralization test. The red symbols represent whole Alternatively, the neutralization of EV71 by E18 might be antibody and the blue symbols represent Fab fragments. Inhibition of virus achieved not only by inducing genome release but also by other was represented as the percentage of plaques relative to plaques in the means such as preventing receptor binding. Indeed, the E18 control wells. We demonstrated neutralization of EV71 by Fab fragments as footprint on the virion surface includes Lys-149 of VP2 that has well as by whole IgG, with whole IgG being more efficient than Fab fragments. been implicated to have a role in attachment of EV71 to the P-selectin glycoprotein ligand-1 receptor (Fig. 6 and Fig. S2)(19). TheE18bindingsitesontheEV71capsidsarelocated to the action of E18 Fab, only approximately 1% of the EV71 between VP4–VP2–VP3–VP1 protomers (Fig. 6A and Table 2). particles incubated with E19 Fab had lost their genome (Fig. 3 However, the protomer in both A particles (after receptor B C and ). Thus, E18 but not E19 induced genome release binding) and empty, immature (before VP0 cleavage) particles is from virions. rotated by 5.4° relative to its position in the mature capsid with Separate reconstructions were made of the empty immature respect to the icosahedral axes (3, 16). Because the E18 antibody EV71–E18 complexes and of the full EV71–E18 complexes. The was generated by immunization with empty, immature particles, resolutions of these reconstructions were 10 Å and 20 Å, re- it is likely that, when E18 binds to mature EV71 particles, there spectively, as judged by the resolution at which the Fourier shell will occur an “induced fit” that requires local rearrangements of correlation coefficient decreased to less than 0.5 (Fig. S1). The the capsid to an immature-like capsid conformation. Thus, pos- correlation coefficient between the cryo-EM electron density sibly the E18 antibody binds to the virus when the capsid tem- maps of the capsid region of the heat-induced EV71 A particles porarily and locally changes structure to be like an A particle and that of the corresponding density of the EV71–E18 (full) because of the natural capsid dynamics. However, mature virions complex was 0.83, whereas the correlation between the electron contain VP2 and VP4 instead of VP0. Therefore, the capsid density of the native virus capsid and the EV71–E18 complex proteins reorganize to resemble A particles that are generated was only 0.61 (Table 1). Thus, the E18 Fab had induced the when the virus recognizes a receptor and expels the pocket conformational change of the EV71 virions to the A state (Fig. 4 factors (Fig. 4 A, G, H,andI). As the E18 Fabs bind across the A and D), a conformational change similar to what was produced interface between protomers (Fig. 6A), binding of E18 to the by heating EV71 virions to 56 °C (Fig. 4 D and G) (16). Con- mature virion induces the protomers to rotate by 5.4° to the A sistent with these observations, ELISA tests showed that E18 form. In contrast, E19 binds wholly within a single protomer binds better to heat-induced EV71 A particles than to mature (Fig. 6B), and therefore its binding does not require any virions (Fig. 5). The 3D cryo-EM reconstructions showed that conformational change of the capsid. It has been shown that the capsid structures of the RNA-containing EV71–E18 Fab picornavirus genomes are released through channels at the ico- complex and of the empty EV71–E18 Fab complex are nearly sahedral twofold axes that form upon transition of the virions identical (correlation coefficient, 0.95; Fig. 4 A, B, D,andE and to the A state (3, 20). Therefore, binding of E18 to mature BIOPHYSICS AND Table 1). Thus, the conformational change must be the consequence virions results in a conformational change to A-like particles and of E18 binding to the virions, which then leads to genome release. thus facilitates the release of the genome. Hence, the release of COMPUTATIONAL BIOLOGY

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 were separated by SDS/PAGE and transferred onto membrane. Membrane was probed with R525, E18, or E19. E18 and E19 did not bind denatured viral proteins and, therefore, recognize conformational epitopes. (B) Indirect ELISA was performed by coating wells with recombinant viral proteins or heat-inactivated EV71-infected cell lysates. Various concentrations of mAb were added in duplicates. The mAbs were detected by HRP assay (Materials and Methods). The

amount of bound mAbs are presented as average OD450 ± SDs.

Plevka et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2135 Downloaded by guest on September 30, 2021 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 SYBR Green 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) were incubated with the fluorescent dyes at 37 °C. The purple line represents a negative control without virus. Subsequently, the complexes were gradually heated to 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 associated with 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) EV71 complexed 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 of genome release observed in the E18–EV71 mixture. (Scale bars: 50 nm.)

the genome upon E18 binding is achieved by a mechanism that of particles to the A state (9, 10), but require additional cor- could be similar to that of a receptor binding to a virus. The eceptors for successful infection (10). The E19 footprint on the present study of E18 binding to EV71 may indirectly provide EV71 virion surface does not overlap with any putative receptor a description of a mechanism by which picornavirus receptors binding sites (19, 22). However, it is possible that E19 might that bind outside of the canyon induce genome release. neutralize the virus by preventing EV71 binding to an as yet The effect of antibody binding on mature EV71 virions was unidentified cellular receptor. The geometry of binding of E18 also evaluated with an assay that measured the interaction of the and E19 Fab fragments indicates that neither of the antibodies genome with the RNA-binding fluorescent dyes SYBR Green I could bind to the virus divalently as intact IgG. and II (Fig. 3A and Fig. S3) (20, 21). The results demonstrated Virus capsids evolved to serve as efficient vessels for transport that binding of E18 antibodies or Fab fragments to EV71 in- of virus genetic material from one host to another. However, creased accessibility of the genome to the dyes at temperatures some of the functions that the capsids perform exert conflicting between 4 °C and 37 °C, whereas E19 had limited effect on ge- selection pressures on the design of the capsid. For instance, nome accessibility (Fig. S3). However, these experiments do not the capsids need to be stable to protect the viral genomes in differentiate between the genome being released from a virion the extracellular environment, but they also need to release the and then interacting with the dyes outside the virion or the dyes genome at the right time to initiate infection. Therefore, the entering the virion and interacting with the genome remaining capsids are selected for optimal—not too high, not too low— inside the virion. However, these experiments do confirm that stability. Infection of some picornavirus genera can be limited by binding of E18 to the virus induces a conformational change that small molecules that bind with high affinity into the VP1 pocket allows communication between the inside of the capsid and the in place of the pocket factor (23, 24). These compounds inhibit external environment. infection by overstabilizing the virions (14, 23). In contrast, the The Fab fragment of the E19 mAb binds primarily to the VP3 present results show that antibody binding can promote transi- “knob” (4), a different site than that occupied by E18 (Fig. 6). tion of virions to the A state and deactivate EV71 by inducing Binding of E19 Fab to EV71 did not induce any detectable untimely genome release. It has been shown here that antibodies rearrangements of the capsid relative to the native state (Fig. 4 capable of causing the release of genome can be generated by C, F, and I). The correlation coefficient of the capsid of mature immunization with empty particles containing VP0. As an ex- EV71 with the capsid of EV71 complexed with E19 was 0.81, ample of this strategy, empty immature virus-like particles whereas its correlation with the A particle capsid (heated mature (VLPs) were purified on an E18 affinity column and were used virus) was only 0.39 (Table 1). In other picornaviruses, binding of to immunize mice. The resultant sera were assayed to determine receptors to the knob region (Fig. 6) does not induce transition the neutralizing antibody titres (Fig. S4). Mice immunized with

Table 1. Correlation coefficients comparing electron density distribution in the capsid regions of 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 —

2136 | www.pnas.org/cgi/doi/10.1073/pnas.1320624111 Plevka et al. Downloaded by guest on September 30, 2021 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 according to the distance of the surface from the particle center. (Middle) Center sections of the cryo-EM reconstructions of (D) genome containing EV71–E18, (E) empty EV71–E18, and (F) genome containing EV71–E19 complexes. The sections are rainbow-colored according to the electron density height. (Bottom) Center sections of (G) heat-induced EV71 A particles, (H) empty particles after genome release, and (I) native EV71 capsid.

VLPs exhibited higher neutralizing antibody titers against EV71 according to the manufacturer’s instructions. Animal care and use was (geometric mean titer, 153) compared with control mice (geo- conducted in accordance with the National Animal Welfare Standards and metric mean titer, 55). Pooled serum from VLP-immunized mice Guidelines of Malaysia under the Animals Act of 2006. inhibited 60% of E18 binding indicating that serum from these mice contains antibodies that recognize E18 epitope (Fig. S4). Immunoblot Analysis. Equal volume of mock- and EV71-infected celllysateswere separated on a 12% (wt/vol) SDS/PAGE, transferred to nitrocellulose membrane, Thus, VLPs selected for having the E18 epitope can induce and probed with R525 (polyclonal antibody against EV71 VP1), E18, or E19. neutralizing antibodies in mice. Therefore, therapeutic anti- Bound antibody was detected by incubation with HRP-conjugated secondary BIOPHYSICS AND

bodies with genome-release activity might also be obtainable for antibodies (Dako) followed by TMB membrane peroxidase substrate (KPL). COMPUTATIONAL BIOLOGY other picornaviruses using the approach described here. ELISA Analyses. An indirect ELISA was performed by coating Nunc-Immuno Materials and Methods plate with recombinant viral proteins or heat-inactivated EV71-infected Preparation of Fab Fragments of Monoclonal Antibodies. The Fab fragments of rhabdomyosarcoma cell lysates as positive control. Nonspecific binding was the antibodies were prepared with the use of the Pierce Fab Preparation Kit blocked using 5% skim milk, antibodies were added at various concentrations

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 or incubated at 56 °C for 30 min. Serial dilutions of the samples were added to wells coated with polyclonal antibodies against VP1, and bound viral particles were detected by the addition of E18 or E19 (Materials and Methods). The average OD values indicating amounts of bound mAbs ± SDs are shown.

Plevka et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2137 Downloaded by guest on September 30, 2021 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 are outlined in blue, green, and red, respectively. Residues involved in binding (A)E18and(B) E19 are shown in bright colors. The footprints of E18 and E19 are outlined 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, and fivefold icosahedral symmetry axes are shown as ovals, triangles, and pentagons, respectively. One icosahedral asymmetric unit is outlined by a triangle.

in duplicate, and bound antibodies were detected by using HRP-conjugated number of plaques in wells in which the virus had been incubated with anti-mouse IgG (Dako). SureBlue Reserve TMB microwell peroxidase substrate media alone. (KPL) was added for 5 min, 0.5 M HCl was added to stop the enzyme reaction, and wells were read at 450 nm. Virus Production and Purification. EV71 virions were produced and purified as A sandwich ELISA was performed whereby the wells were coated with described previously (25). R525 antibody against VP1, and PEG-precipitated EV71 that was untreated or heat-inactivated at 56 °C for 30 min was allowed to bind to the VP1 anti- VLP Production and Purification. Briefly, EV71 empty immature capsids were body. The bound particles were detected by the monoclonal antibodies E18 produced using a baculovirus expression system in which the complete P1 or E19, followed by HRP IgG (Dako) as described earlier. coding sequence and the protease 3CD of EV71 were recombinantly inserted A competitive ELISA was conducted to examine the presence of antibodies downstream of the polyhedrin promoter and the recombinant baculovirus containing E18 epitope in mouse serum. Sera from four mice immunized with was used to infect Sf9 cells at a multiplicity of infection of 0.1. The super- VLP that had high plaque reduction neutralization tests were pooled, and natant harvested on day 4 was clarified and concentrated by using tan- sera from four mice immunized with PBS solution were pooled for the gential flow filtration (GE Healthcare Lifesciences), and the retentate was competitive ELISA. Wells were coated with R525 followed by equal protein run through an affinity column prepared by coupling E18 to a HiTrap NHS- concentrations of mock- and EV71-infected RD cell lysates. Sera pooled from activated HP column (GE Healthcare Lifesciences). The particles bound were mice (at 1/250 dilution) were added into the wells, and HRP-conjugated E18 eluted by using a glycine buffer at pH 3.0 and immediately neutralized to pH7.2 was added immediately afterward. Reserve TMB microwell peroxidase sub- with 1 M Tris·HCl. The particles were transferred to Dulbecco’s phosphate- strate (KPL) was added for 5 min, 0.5 M HCl was added to stop the enzyme buffered saline buffer (Invitrogen). reaction, and wells were read at 450 nm. Adjusted OD values were obtained by subtracting OD of mock RD cell lysate from OD of EV71-infected RD cells. Immunization of Mice. Mice (n = 10 per group) were immunized with two The relative percentage of binding of E18 was derived by dividing the ad- doses of DPBS or 10 μg of VLP in the presence of Imject Alum (Thermo Sci- justed OD of samples by the adjusted OD of well containing only HRP-E18, entific) 3 wk apart. Serum were inactivated by incubation at 56 °C for 30 multiplied by 100. min, and stored at −20 °C for further analysis.

Plaque Reduction Neutralization Test. Different concentrations of Abs, Fab CryoEM Data Collection and Reconstruction. Either the E18 or E19 Fab fragments fragments, or heat-inactivated mouse serum were incubated in 1:1 volume were incubated with EV71 at 37 °C for 1, 2, or 6 h at a ratio of three Fab fragments ratios with infectious EV71 strain MY104 (300 pfu/mL) for 1 h at 37 °C. The per icosahedral asymmetric unit of the virus. Small aliquots (3.5 μL) of this mix- virus–antibody (or Fab) mixture was inoculated in duplicates over Vero cell ture were applied to holey carbon-coated grids, blotted with filter paper, and monolayers in 24-well plates (Nunc/Thermo-Fisher). The monolayers were vitrified by plunging into liquid ethane. Electron micrographs were recorded on 5 prepared with 0.5 mL per well of Vero cells at 3 × 10 per milliliter in DMEM Kodak SO-163 film by using a Philips CM200 FEG microscope. Micrographs were supplemented with 5% FBS and antibiotics (all from Invitrogen) and left to digitized with a Nikon Cool-Pix scanner. The final averaged pixel size was 2.48 Å. adhere overnight before inoculation. Media was aspirated before in- The program e2boxer.py was used to box 8325 and 13346 particles for the E18 oculation with 200 μL of the antibody (or Fab)–virus mixtures and incubated and E19 complexes, respectively (26). The particles were corrected for the con- in a CO2 incubator at 37 °C for 2 h before 1 mL of overlay was added con- trast transfer function using the programs ctfit and e2projectmanager.py from taining DMEM supplemented with 2% FBS, antibiotics, and 1.5% carboxy- eman and eman2 packages (26, 27). The defocus ranged from 1.12 to 3.67 μm. methyl cellulose. Plates were incubated at 37 °C with 5% CO2 for 4 d and The reconstruction was started by combining projections down twofold, three- stained with naphthalene black. Plaques were counted manually. The per- fold and fivefold axes using the program starticos from the eman package (27). cent inhibition was determined relative to controls in which the mean The EM reconstruction processes were performed using icosahedral averaging with the same software. The resolution of the resulting maps were estimated by comparing structure factors of the virus shell computed from two independent Table 2. EMfit statistics for fitting of Fab fragments into half data sets (Fig. 3). For the final 3D reconstruction, data were included to the cryo-EM electron density maps. resolution (approximately 16 Å, 9 Å, and 13 Å for the complexes with empty E18, full E18, and full E19, respectively) at which the correlation between the Fourier Fab Sumf Clash −Den coefficients of two independent data sets was better than 0.3. E18 54.9 0.0 5.0 E19 38.6 0.0 17.0 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-EM map Clash, percentage of atoms in the model that have clashes with symmetry of EV71 reconstructions by comparing them with maps derived from the related protein molecules; −Den, percentage of atoms positioned in nega- crystallographically determined coordinates of EV71 mature and immature tive density; Sumf, average value of density at atomic positions normalized particles [Protein Data Bank (PDB) ID codes 3ZFE and 3VBO]. For the EV71–E19 by setting the highest density in the map to 100. complex, the mature EV71 structure (PDB ID code 3ZFE) was used to model

2138 | www.pnas.org/cgi/doi/10.1073/pnas.1320624111 Plevka et al. Downloaded by guest on September 30, 2021 the capsid. For the EV71–E18 complex, the model of the capsid was based on Proteins, Interfaces, Structures, and Assemblies at the European Bioinformatics the immature EV71 particle (PDB ID code 3VBO). “Difference maps” were Institute (www.ebi.ac.uk/msd-srv/prot_int/pistart.html) (29) based on buried then calculated by masking out the density of the capsid by setting to zero surface area between the fitted Fab fragments and capsid proteins. all grid points within 3 Å from any EV71 capsid protein atom. Modeled Fab fragments (based on PDB model ID number 1QGC) were then fitted into the ACKNOWLEDGMENTS. We thank Sheryl Kelly for help with the preparation difference map by using the program EMfit (28) (Table 2). of the manuscript. Cryo-EM studies were supported by a National Institutes of Health Grant R01 AI 11219 (to M.G.R.). The production and characteriza- Buried Surface Area and Residues Forming the Protein–Protein Interface. The tion of antibodies in mice was performed by Jane Cardosa for a separate residues forming the virus–Fab interfaces were identified with the Web service research project funded by MAB Explorations (Penang, Malaysia).

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