The EV71 strain 1095 structures of procapsid and mature virion 1 Javier O. Cifuente

a, Hyunwook Lee

a, Joshua D. Yoder

b, Kristin L. Shingler

a, Michael S. Carnegie

a, 2

Jennifer L. Yodera, Robert E. Ashley

a, Alexander M. Makhov

b, James F. Conway

b, Susan 3

Hafensteina* 4

5 aDepartment of Medicine, The Pennsylvania State University College of Medicine, 500 University 6

Drive, Hershey, Pennsylvania 17033, USA 7 bDepartment of Structural Biology, University of Pittsburgh School of Medicine, 3501 5th Ave, 8

Pittsburgh, Pennsylvania 15260, USA 9 Corresponding author: 10

Susan Hafenstein 11 Division of Infectious Diseases, MC H036 12 Department of Medicine, Penn State University College of Medicine 13 500 University Drive, Hershey PA 17033 14 Email: shafenstein@hmc.psu.edu 15

16 Author contributions: JOC, HL, and JDY performed research; MSC and KLS contributed processing 17 support; JDY and MSC collected crystal data; JDY processed crystal data; JLY contributed 18

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.03519-12 JVI Accepts, published online ahead of print on 1 May 2013

reagents/virus; REA performed microscopy; AMM and JFC collected cryo-EM data; JOC processed 19 and analyzed cryo-EM data; JOC, HL, and SH wrote the paper. 20 21 Word Count Abstract: 206 22 Word Count Manuscript: 4077 23 24 ABSTRACT 25 Enterovirus 71 (EV71) is an important emerging human pathogen with a global distribution and 26 presents a disease pattern resembling poliomyelitis with seasonal epidemics that include cases of severe 27 neurological complications such as acute flaccid paralysis. EV71 is a member of the Picornaviridae 28 family, which consists of icosahedral, non-enveloped, single-stranded RNA viruses. Here we report 29 structures derived from X-ray crystallography and cryo-electron microscopy (cryo-EM) for the 1095 30 strain of EV71, including a putative precursor in virus assembly, the procapsid, and the mature virus 31 capsid. The cryo-EM map of the procapsid provides new structural information on portions of capsid 32 proteins VP0 and VP1 that are disordered in the higher resolution crystal structures. Our structures 33 solved from virus particles in solution are largely in agreement with those from prior X-ray 34 crystallographic studies, however, we observe small but significant structural differences for the 1095 35 procapsid compared to a structure solved by Wang et al, 2012, for a different strain of EV71. For both 36 EV71 strains, the procapsid is significantly larger in diameter than mature capsid, unlike any other 37 picornavirus. Nonetheless, our results demonstrate that picornavirus capsid expansion is possible 38 without RNA encapsidation and that picornavirus assembly may involve an inward radial collapse of 39 the procapsid to yield the native virion. 40

41 KEY WORDS: picornavirus, enterovirus 71, hand foot and mouth disease, cryo-EM, single particle 42 reconstruction, assembly, conformational change 43 44 Running Head: The structures of EV71-1095 procapsid and mature virion 45 46 INTRODUCTION 47

Enteroviruses are the causative agents of disease in both developed and in developing countries 48 (32). Enterovirus 71 (EV71) was first isolated in California, USA, from patients with central nervous 49 system diseases (43) and is responsible for hand-foot-and-mouth disease (HFMD), which is a common 50 viral illness of infants and children that causes fever, sore throat, blisters, and skin rash. There is no 51 specific therapy for HFMD and most patients recover without special treatment. However, EV71 52 infection may lead to severe neurologic diseases including meningitis, poliomyelitis-like disease, and 53 fatal cases of encephalitis (45). Since first reported, EV71 has been implicated in an increasing number 54 of outbreaks throughout the world and is now classified as an emerging infectious disease with 55 pandemic potential (21, 51). A thorough structural and mechanistic understanding of the virus and its 56 interactions with host cells will support efforts at developing anti-viral therapies and we focus here on 57 establishing a structural basis for this knowledge. 58

EV71 is classified as a Human enterovirus A (HEV-A) (39), one of four species, A-D, within 59 the genus Enterovirus of the Picornaviridae family. EV71 has been divided into three genogroups, 60 designated A, B and C, according to nucleotide sequence analysis between different isolates (22). The 61 mature virus has one copy of a single stranded positive sense RNA genome packaged inside a 62

non-enveloped icosahedral capsid with T=1 (quasi T=3) symmetry (41, 53). The mature capsid is built 63 of 60 structural subunits or protomers, which are comprised of four structural proteins, VP1-4. Several 64 key residues that map to the five-fold symmetry vertex, VP1 98 and 145, have been linked to positive 65 selection, virulence, and receptor binding (8, 21, 27, 49). 66

Five host receptors have been reported for EV71: P-selectin glycoprotein ligand-1 67 (PSGL-1)(36), the Scavenger Receptor B2 (SCARB2)(58), sialylated glycans (46, 61), annexin II (62), 68 and heparin sulfate (47). Currently, no receptor footprint on the virus capsid is known, but there is 69 evidence that SCARB2 binds into the depression surrounding each five-fold axis, known as the 70 “canyon” (9). The model for infection of many picornaviruses begins with a specific capsid-receptor 71 interaction that triggers a conformational change in the capsid to form the 135S, or “A” particle. These 72 changes are necessary for the release of the viral genome into the host cell cytoplasm. The viral RNA is 73 translated into a single polyprotein that is subsequently cleaved to generate the initial structural proteins 74 (VP0, VP1, VP3). These viral proteins associate into pentamers that self-assemble into empty protein 75 shells, sometimes referred to as procapsids due to the lack of RNA and the presence of uncleaved VP0 76 (26). Two pathways to the provirion are proposed: 1) packaging of a progeny genome into the 77 preformed procapsid (4, 13, 64) or 2) incorporation of the RNA genome during the self-assembly of 12 78 pentamers. Either way, the association of RNA with capsid leads to the cleavage of VP0 into VP2 and 79 VP4 (2, 23) by an autocatalytic mechanism (5, 10) yielding the mature virus. 80 Recently, crystal structures have been reported of the EV71 Fuyang (53) and MY104 (41) 81 strains representing two different genogroups, C4 and B3, respectively. Here we present structures of 82 EV71 from the 1095 strain that belongs to genogroup C2 (33), including cryo-EM reconstructions of the 83 procapsid and mature virion at resolutions of 8.8Å and 9.5Å, respectively, and a 3.9Å crystal structure of 84 the procapsid. The cryo-EM structures solved from particles in solution agree with the crystal structures. 85 In addition our cryo-EM reconstruction of the EV71 procapsid shows additional density corresponding to 86

the N-terminus of VP1 and the VP4 region of VP0, both of which are disordered in the crystal structure. 87 A detailed comparison between the Fuyang and 1095 crystal structures show differences at the five-fold 88 vertex, specifically in residues that determine virulence, antigenicity and receptor use (8, 27, 47)(Lee et 89 al, manuscript in preparation). Finally, we discuss our new structures in the context of uncoating and 90 genome release (20, 53) and their impact on the current models for capsid assembly and the final stages 91 of EV71 morphogenesis. 92 93 MATERIALS AND METHODS 94 Virus propagation and purification. The EV71 strain 1095 (inoculum kindly provided by Yorihiro 95 Nishimura) that binds receptors PSGL-1 and SCARB2 (36, 58) was propagated by infecting HeLa cells 96 at a MOI of 0.1 in culture with DMEM supplemented with 2.5% fetal bovine serum. After 24 hours, 97 cells and media were collected and processed by three cycles of freezing and thawing. The lysate was 98 centrifuged at 13K rpm in a Sorvall centrifuge using a SLA1500 rotor at 4°C for 15 min to remove 99 cellular debris. Supernatant was collected and precipitated by incubation overnight with PEG 8K and 100 NaCl to final concentrations of 8% and 0.5M, respectively. The precipitate was collected by Sorvall 101 centrifugation in a SLA1500 rotor at 4°C at 13K rpm for 45 minutes. The pellets were resuspended in 102 10mM Tris - HCl, 200mM NaCl, 50mM MgCl buffer and incubated with DNase (0.05 mg/ml) while 103 gently rocking for 30 min at room temperature. Initially trypsin was added (0.5 mg/ml) and incubated 104 for 10 min at 37°C; however, this step was omitted in later purifications, resulting in more stable 105 particles. EDTA 0.1M, pH 8.0 and n-lauroyl sarcosine were added, each to 10% of total volume. If 106 necessary, pH was adjusted with ammonium hydroxide. A slow speed centrifugation was used to clear 107 supernatant and before pelleting by ultracentrifugation through a 30% sucrose-buffer cushion using a 108 50.2ti rotor at 48K rpm at 4°C for 90 minutes. Pellets were collected and resuspended in the 109 purification buffer, 10mM Tris - HCl, 200mM NaCl, 50mM MgCl, and loaded onto 10-35% tartrate 110

step gradients for ultracentrifugation at 36K rpm for 2 hours at 4°C in a SW41 rotor. Two distinct 111 bands formed in the gradient and were collected independently by side puncture and washed three 112 times with purification buffer in a spin filter (Milipore) to change the buffer. The samples were 113 characterized by SDS-PAGE and electron microscopy. The upper band corresponded to procapsids 114 devoid of genomic material and containing uncleaved VP0 proteins, whereas the lower band contained 115 particles consisting of mature viral capsid proteins with a packaged genome. 116 117 Crystal structure determination of the procapsid. EV71 procapsid crystals were obtained using the 118 hanging drop vapor diffusion method at room temperature. Drops consisted of 5 µl of purified EV71 119 procapsid at a concentration of 10 mg/ml in 10mM Tris pH 7.5, 200mM NaCl, and 50mM MgCl2 120 mixed with 5µl of well solution. Crystals were obtained from 100mM Tris pH 7.5, 0.6M NaCl, 0.2M 121 NaCl, 0.4M PEG 8000, 0.4% glycerol. Crystals were frozen using 12% PEG400 and 19% glycerol as 122 cryoprotectants and transported to the F1 beamline at CHESS (Cornell, Ithaca NY). Data were 123 collected with an oscillation of 0.2˚ on an ADSC Quantum 315 detector at a distance of 450 cm. 124 Data were indexed, processed, scaled, and reduced using the HKL-2000 package (37). The 125 crystal was determined to be in the cubic crystal system and space group P4[2]32 with unit-cell 126 parameters of a=b=c= 350.248Å. The data collection and processing statistics are provided in Table 1. 127 Each crystallographic asymmetric unit contained 5 protomers. The diffraction data were phased using 128 the molecular replacement method in the AMoRe program (35) implemented in the Phenix software 129 suite (1). 130

For phasing purposes, a three-dimensional structure of the EV71 protomer was calculated 131 through the UCSF Chimera interface (63) using MODELLER (42). All four structural proteins VP1-4 132 were modeled independently as follows: the protein sequence was acquired from the UniProt database 133

(accession number E5RPG1) for the EV71 strain 1095-LPS1. Using the function Match-Align from 134 Chimera (38) the known crystal structures of nine other picornaviruses from the PDB database were 135 superimposed using atomic coordinates as a reference, and aligned using the global alignment 136 algorithm (Needleman-Wunsch) with the BLOSUM 62 matrix. To prevent bias, structures representing 137 as many different enterovirus groups as possible were used:: poliovirus 1 (1ASJ) (54), bovine 138 enterovirus 1 (1BEV) (44), coxsackievirus B3 (1COV) (34), coxsackievirus A9 (1D4M) (19), 139 echovirus 1 (1EV1) (14), swine vesicular disease virus (1MQT) (52), coxsackievirus A21 (1Z7S) (56), 140 echovirus 7 (2X5I) (40), and human rhinovirus 14 (4RHV) (3). In addition, individual EV71-1095 141 structural proteins were aligned to the corresponding picornavirus proteins using the same algorithm. 142 Crystal structures for the individual proteins were used as a template for modeling the aligned sequence 143 of EV71-1095 with MODELLER. Once the independent proteins models were calculated, VP1-4 144 models were combined in a single PDB file using MOLEMAN (25). 145

This homology model of the protomer was fit into the cryo-EM procapsid reconstruction to 146 generate a pentamer model used as a search model in molecular replacement. The orientation of the 147 pentamer in the crystal unit cell was determined by a cross-rotation search and its position was 148 determined by a translation search. The pentamer model was positioned accordingly into the crystal 149 unit cell to calculate a set of initial phases. These phases were improved by refinement using the 150 Crystallography and NMR System (CNS) package (6). CNS programs for simulated annealing, energy 151 minimization, atomic position, and temperature factor refinement were used with the application of 152 strict non-crystallographic symmetry (NCS) operators. After each refinement cycle, a single cycle of 153 electron-density Fourier map (2Fo-Fc and Fo-Fc, where Fo are the observed structure factors and Fc 154 are those calculated from the model) averaging was carried out in CNS, with strict NCS operators, 155 using the experimentally measured amplitudes and the improved phases. The program Coot (12) was 156 used for model building into averaged electron-density maps between cycles of the refinement and 157

averaging procedures. During the process of refinement, an X-ray crystal structure was published for an 158 EV71 empty particle of the Fuyang strain, which differed in the identity of seven amino acid residues 159 (PDB accession 3VBU) (38). Our working model (Rfree of 32.6) superimposed onto 3VBU with an 160 rmsd of 2.4Å, with differences mapping almost exclusively to loop tips and termini. Using the newly 161 available 3VBU structure, altered to the EV71-1095 identity at the sites of the seven amino acid 162 differences, two more rounds of refinement were completed, interspersed with manual building, 163 resulting in the final model with an Rfree of 28.4% and an overall rmsd of 1.0Å compared to 3VBU. 164 165 Cryo-EM reconstruction. 3µl of purified virus were blotted onto Quantifoil grids (Quantifoil Micro 166 Tools GmbH, Jena, Germany) and plunge-frozen into a liquid ethane/propane mixture (50) by a 167 Vitrobot (FEI, Hillsboro, Oregon). Data were collected under low dose conditions on Kodak SO-163 168 film (Kodak, Rochester, New York) in an FEI TF-20 electron microscope operating at 200KV and a 169 nominal magnification of 50,000x, and equipped with a Gatan 626 cryoholder (Gatan, Pleasonton, CA). 170 Films were scanned using a Nikon Super Coolscan 9000 (Nikon, Melville, NY) giving a final pixel size 171 of 1.27 Å/pixel for both the EV71-1095 procapsid and the mature virus particle. Programs suites 172 AUTO3DEM (60) and EMAN2 (48) were used for reconstruction and image processing. 173 Semi-automatic particle selection was performed using e2boxer.py to obtain the particle coordinates, 174 followed by particle boxing, linearization, normalization, and apodization of the images using Robem 175 (60). Defocus and astigmatism values to perform contrast transfer function (CTF) correction were 176 assessed using Robem for the extracted particles (Table 2). The icosahedrally averaged reconstructions 177 were initiated using a random model generated with setup_rmc (59) and reached better than 10Å 178 resolution estimated at a Fourier Shell Correlation, FSC=0.5. For the last step of refinement, the final 179 maps were CTF corrected and sharpened with an inverse temperature factor of (1/300 Å2). 180

181 Assessment of cryo-EM map handedness and pixel size. Using the final 9.5Å resolution EV71 mature 182 particle reconstruction, a density map representing the stereo-isomer was generated by mirroring the 183 complex map across the x-y plane to “flip” the hand. A map calculated from the crystal structure of 184 EV71, 3VBS (53) was fitted as a rigid body separately into each stereo-isomer map rendered at a density 185 threshold of 1σ (17, 38). Correct handedness was assessed by the quality of fit, by measuring correlation 186 coefficients (cc) using the UCSF Chimera protocol “Fit in Map” (38); for procapsid cc=0.959 (as 187 opposed to 0.935 for the opposite hand) and native virus the cc=0.973 (versus 0.956) In addition, the 188 highest cc fit also allowed calibration of the spatial scale of the cryo-EM reconstruction at 1.25Å/pixel. A 189 similar procedure was followed for the structure of the final 8.8Å resolution EV71 procapsid using the 190 crystal structure of EV71-1095 strain reported here. 191 192 Difference map and volume assessment. A simulated cryo-EM map was also made from our 193 EV71-1095 procapsid crystal structure using pdb2vol and limiting the resolution to 9.0Å (55). A 194 difference map was made by subtracting the simulated map of procapsid from the cryo-EM map using 195 Situs which applies zero-padding to scale the lower density map (7) . The crystal structure of the mature 196 virus (3VBS) (53) was superimposed on the fitted procapsid structure to provide the approximate 197 position for the disordered sections of the procapsid, which are ordered in the mature capsid. The 198 N-terminal 72 residues of VP1 and all of VP4 were used to make a density map that was subsequently 199 surface rendered and measured to compare the volume to that of the difference density map, using 200 Chimera to report the total surface area and enclosed volume (38). 201 202

Accession numbers: EMD5557 for the cryo-EM reconstruction of EV71-1095 procapsid; EMD5558 203 for the reconstruction of the EV71-1095 native virus. The crystal structure of the EV71-1095 procapsid 204 is 4GMP 205 206 RESULTS 207 Purification of two particle types. Two distinct bands formed in the final gradient purification of 208 EV71-1095 and were characterized by SDS PAGE (data not shown) and electron microscopy (Figure 209 1A). As seen previously (29, 57) , the upper band contained empty, ~30nm diameter spherical particles 210 comprised of intact VP0, VP1 and VP3. These empty particles are characteristic of procapsid (4, 11), a 211 naturally occurring type of empty particle that forms during virus assembly and which contains 212 uncleaved VP0, the precursor of VP2 and VP4. The lower band was comprised of particles with 213 homogeneously dark centers due to the presence of the genome (Figure 1A), and were consistent with 214 mature viral capsids since they contained all four structural proteins, VP1-4. 215 216 Cryo-EM reconstruction. The 3D reconstructions achieved resolutions of 8.8Å for the procapsid and 217 9.5Å for the mature virus, estimated at FSC=0.5 (Table 2, Figure 1). The electron density for the maps 218 clearly indicates that the procapsid consists of an empty protein shell, whereas the mature virus 219 contains density corresponding to a filled genomic core (Figure 1). Compared to other enteroviruses, 220 the EV71 mature virus has a smoother, nearly featureless exterior, due to shorter loops that decorate the 221 external surface. The overall shape and diameter of the EV71 procapsid is different compared to the 222 virus, as the procapsid is larger and more angular with protruding five-fold plateaus. In contrast, the 223 virus appears as a smaller, rounder, and smoother particle, which likely explains the lower resolution 224 achieved for it in our reconstruction. 225

226

A superimposition of the central sections of the procapsid and capsid maps shows the radial 227 difference at external and internal protein shell surfaces (Figure 2). The procapsid is larger than the 228 capsid with the greatest external radial difference of 11Å observed at the five-fold axis and smaller 229 differences seen at the two-fold and three-fold axes. Conversely, the internal radial surface differences 230 are most significant near the two-fold axis of symmetry. Differences in the distribution of the structural 231 proteins are also reflected in the capsid shells, with the procapsid walls being thinner at the two-fold 232 axis and thicker at the five-fold, whereas the mature capsid has an evenly packed protein distribution 233 and thickness throughout. The icosahedral radial projections illustrate the specific differences in protein 234 packing in the procapsid and capsid (Figure 2B). At a spherical shell radius value of 135Å the density 235 is packed tighter and more evenly distributed in the mature capsid. In addition there is a clear rotation 236 of the density around the three-fold axis, corresponding to the external changes in the three-fold plateau 237 (Figure 2B). 238 239 Crystal structure of the procapsid. The crystal structure of the EV71-1095 procapsid (Figure 3A) was 240 solved to 3.9Å and shows the canonical jelly-roll β-barrel fold for each protein, VP0, VP1, and VP3, that 241 is typical for picornaviruses. As seen in other procapsid structures (4, 11) some peptides on the internal 242 surface of the particle are disordered, including the N-termini of VP0 (first 81 residues) and VP1 (first 72 243 residues). The 81 disordered residues of VP0 correspond to all 69 residues of VP4 and the first 12 244 residues of VP2, the proteolytic products of VP0 found in mature capsids. The GH loop of VP1 (residues 245 211-217) is also disordered. 246 247

The sequence and structural differences between EV71-1095 and the Fuyang strain at the five-fold 248 vertex. The previously solved structure from Wang et al (PDB: 3VBU) was of EV71 genotype C4, 249 isolated from Fuyang, Anhui Province, China. Seven-amino acids mapping to structural proteins were 250 different between the Fuyang and 1095 strains (Figure 3C)(Table 4), four of which have been linked to 251 antigenicity of EV71 (15, 28). The crystal structure of the Fuyang strain procapsid superimposed onto 252 our procapsid structure with an overall rmsd value of 0.5Å (Figure 3B). The most significant differences 253 in the C-α chains (larger than 2Å apart) were found in the GH loop of VP1, AB and EF loop of VP0, and 254 the GH loop of VP3 (Figure 3B). There were no discernible differences between the two structures at the 255 site of VP1-172, a determinant of SCARB2 receptor binding (9, 57). 256

We also found differences in the conformations of the five-fold HI-loops and the side-chain 257 positions of two lysine residues (Figure 3 C). Specifically these differences affect the positively-charged 258 patches around the five-fold axes, which have been implicated in the receptor binding and antigenicity of 259 the virus (47, 53). Among the residues contributing to the positively-charged patches.(41), Lys 98 in 260 Fuyang strain has been replaced by Glu in EV71-1095 strain. Thus, in 1095 strain Lys 242 and Lys 244 261 of the VP1 HI-loop and Arg 166 of the VP1 DE-loop contribute most to form the positively-charged 262 patches around the five-fold axes. The side-chains of VP1 Lys 242 and Lys 244 are exposed to the 263 surface whereas the side-chain of Arg 166 is buried in both procapsid strain structures. Additionally, 264 EV71-1095 has a Gly at residue VP1 145 whereas the Fuyang-strain has Glu at this position. (Figure 3D). 265 This region at the five-fold vertex represents a functional “hot-spot” since these residues have been 266 implicated in receptor binding and have been shown to determine virus virulence and affect 267 positive-selection (8, 21, 27, 47, 49). 268 269

Evaluation of the cryo-EM density maps. The cryo-EM 3D structures were evaluated by fitting the 270 X-ray structures from our procapsid and the previously reported mature virus (PDB: 3VBS)(53) inside 271 the cryo-EM density maps. The crystal structures agreed well with the cryo-EM structural features and 272 the fitting generated correlation coefficients of 0.96 and 0.98 for procapsid and capsid, respectively 273 (38) (Figure 4). However, due to disordered density in our procapsid crystal structure, a significant 274 amount of cryo-EM density remained unfilled inside the procapsid. To investigate these densities, a 275 simulated cryo-EM map was calculated from the crystal structure and subtracted from the cryo-EM 276 map. To determine how well the difference density corresponded to the disordered regions of the 277 crystal structure, the VP4 and VP1 structures that are ordered in the mature capsid (PDB: 3VBS) (53) 278 were fitted into the difference map together by superimposing 3VBS onto the fitted crystal structure of 279 the procapsid (53). This approximate positioning of VP4 and the N-terminus of VP1is reasonable since 280 the protomer moves as a rigid body between expanded capsid and collapsed virion (53). The combined 281 structures of VP4 and VP1 satisfactorily filled the difference density (Figure 5). 282 To quantify this placement for the missing portions of the VP0 and VP1 proteins, we tested how 283 well the volume of the difference map compares to a density volume that would encompass the structures 284 of VP4 and VP1. The N-terminal 72 residues of VP1 and all of VP4 were used to make a model map. 285 This map was surface rendered and the total surface area and enclosed volume were measured (38). The 286 volume of the VP1-VP4 model density is about 7% smaller than the volume represented by the difference 287 map (Table 3). 288 289 DISCUSSION 290

Strain specific structures. Several structures of EV71 capsids have now been solved, 291 providing opportunities for understanding similarities in assembly and function as well as differences 292

that affect virulence, antigenicity, and receptor use. Our crystal structure of the EV71 genogroup C2 293 strain 1095 procapsid and cryo-EM map of the mature particle joins crystal structures of a genogroup 294 B3 capsid (41) and the procapsid and mature capsid of the C4 Fuyang strain (53). We observe that 295 fitting the corresponding Fuyang strain crystal structures into our cryo-EM maps confirms the 296 correspondence between particles in solution with those from the crystallized form (Figure 5), at least 297 to the lower resolution of the EM data. Further, this successful fitting of the Fuyang empty expanded 298 particle into our procapsid map, together with our identification of the uncleaved VP0 to confirm the 299 procapsid state, strongly indicates that the structures represent the same particle, i.e., the Fuyang empty 300 expanded particle is the procapsid form. 301

The differences between the structures of EV71-1095 and the Fuyang strain are very minor, but 302 likely have consequences in virulence, antigenicity, and receptor use. Recent studies revealed that the 303 basic residues at the five-fold axes are responsible for binding heparin sulfate (31, 47) and we have 304 found a strain-specific neutralizing epitope that maps to this patch of basic residues (Lee et al, 305 manuscript in preparation). Changes in this patch such as the charge difference observed between the 306 two strains for residues occupying the VP1 98 position, may be expected to significantly alter the 307 functional properties of the capsid surface. Also, the effect of the slight conformational differences of 308 the side-chains of two basic amino acids and the VP1 HI-loop could be enhanced in the native virus 309 since those two residues have significantly different conformations in mature virus compared to 310 procapsid (53). Consequently, it is reasonable to suspect that differences in PSGL-1 receptor 311 interaction and antigenicity are related to the structure and character of the five-fold loop region. 312 313

Peptide locations assigned by cryo-EM complete the transition of procapsid to capsid. The 314 incomplete visualization of capsid subunits in the procapsid crystal structures was addressed by 315

examining cryo-EM density that was unoccupied by the subunit atomic models. In particular, the 316 N-terminus of VP0, which after proteolysis forms VP4 and the N-terminus of VP1 could largely be 317 accounted for in the unoccupied cryo-EM density. The VP4 residues 14-31, observed to form a loose 318 spiral in the X-ray model of the mature capsid (53), fit well into the cryo-EM density on the inside of 319 the procapsid surrounding the five-fold vertex, whereas the N-terminus of VP1 fills unoccupied density 320 around the icosahedral three-fold axis. Some density directly on the five-fold axis remains unfilled, 321 although its high level of noise suggests less certainty in its occupancy and reliability. Nonetheless, the 322 positions of these peptides supports rigid body movement of an entire protomer as the conformational 323 switch between expanded (procapsid) and non-expanded (mature capsid) forms of EV71. 324

The EV71 VP4 subunit in the mature capsid occupies a different position relative to the rest of 325 the protomer than is found in other picornaviruses, lying under the adjacent protomer instead of being 326 directly located under its own biological protomer (53). Our cryo-EM procapsid structure is consistent 327 with this observation, with the VP4 precursor - the N-terminal portion of VP0 - occupying the same 328 position within the procapsid as does VP4 in the mature capsid. This unique positioning of VP4 relative 329 to the protomer has unknown consequences, but possibly this could be related to the expanded nature 330 of the procapsid. The typical location of VP4 under the protomer is known for both poliovirus and 331 FMDV, the only other picornaviruses for which a procapsid structure exists, and for both of which the 332 procapsid is the same dimension as the virus. 333

Assembly model. An expanded procapsid, i.e., an empty, VP0-containing capsid, has not been 334 observed in any other picornaviruses, although few structures have yet been obtained (4, 11). Two 335 alternative pathways have been proposed for genome packaging (4, 13, 64) (Figure 6) during virus 336 morphogenesis. Twelve pentamers may assemble into a ‘true’ procapsid into which the RNA is 337 packaged by an unknown mechanism (23, 24), or alternatively the genome may recruit pentamers, 338 which subsequently condense into a capsid around the genome (16, 30). In either case, since cleavage 339

of VP0 into VP4 and VP2 occurs after RNA-packaging, an expanded particle containing RNA may 340 exist as a provirion that would mature by proteolysis, collapsing upon the packaged genome to form 341 virus. We note that such a maturation step has been established for another small icosahedral virus (18). 342 Such a model also implies that the pentamer assembly intermediate exists in a conformational state 343 similar to that of the procapsid, rather than the conformation of the pentamer in the mature virion. 344 345 Acknowledgements: The work was supported by NIH K22 A179271 and 2011 C. Max Lang Junior 346 Faculty Research Scholar Award to SH. We gratefully acknowledge the use of the Core Facility of the 347 Penn State College of Medicine. X-ray crystallographic datasets were collected at the Cornell High 348 Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and the 349 National Institutes of Health/National Institute of General Medical Sciences under NSF award 350 DMR-0936384, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is 351 supported by award GM-103485 from the National Institute of General Medical Sciences, National 352 Institutes of Health. 353 354

Table 1. X-ray crystallography statistics for scaling and refinement 355 Parameter Value or description

Space group P4(2)32

Unit cell dimensions (Å)

A=B=C 350.25

α=β=γ 90°

Resolution limits (high-resolution bin) (Å) 50.0-3.90 (3.93-3.90)

No. of unique reflections 66880

Completeness (%) 99.9 (100)

Rmerge 18.9 (73.8)

Avg redundancy 7.1 (6.2)

<I>/<sI> 10.3 (2.4)

Rcryst 26.8 (35.5)

Rfree 28.4 (38.0)

Avg B factor 127

% Ramachandran plot outliers 0.5

% Most-favored regions in Ramachandran plot 70.5

RMSD for bonds 0.01

RMSD for angles 1.46

356 357 358 Table 2. Cryo-EM data 359

Particle type Films Defocus Range (µ) Particles total Particles used Resolution (Å) 360 Procapsid 20 0.79-3.91 12579 8805 8.8 361 Virion 24 2.36-3.94 8812 6168 9.5 362 363 364 Table 3. Volume and area of procapsid-procapsid difference map compared to VP1-VP4 model 365 map 366

Difference map * Model Map** 367 Volume (Å)3 629 x 103 580 x 103 368 Area (Å)2 281 x 103 310 x 103 369 *Difference map = (Cryo-EM) – (Simulated cryo-EM Map calculated from crystal structure) 370 **Model Map = N-terminal 72 residues of VP1 + all of VP4 (3VBS). 371 372 Table 4. Differences between EV71 Fuyang and 1095 strains that map to the structural proteins 373 VP1 VP0 VP3 374 K98E V126I(a) H29Y(a) 375 E145G T144S(b) N93N 376 M225C(b) 377 The single letter code for Fuyang is listed before the amino acid number followed by the 1095 residue 378 identifier 379

(a) and (b) residues map to a synthetic peptide that elicited an immune response, Gao et al and Liu et al, 380 respectively (15, 28). 381 382 References 383 1. Adams, P. D., P. V. Afonine, G. Bunkoczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L. W. 384

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Figure Legends 559 Figure 1. Cryo-EM reconstructions of EV71-9105 procapsid and capsid, A) Representative areas of 560 cryo-micrographs used for the reconstructions with procapsid at the top and mature virion below. Bar = 561 300Å. B) Fourier shell correlation vs spatial frequency. Resolution of the reconstructions is assessed 562 where the FSC curve crosses below a correlation value of 0.5. C) Radial density profiles revealing that 563 the radius of the procapsid is greater than that of the virion capsid. D&E) Surface renditions colored by 564 radius of the procapsid and mature capsid density maps, respectively, illustrate the differences in size and 565 angularity between the procapsid and capsid forms. The regions at the five-fold vertices are distinctly 566 raised in the procapsid by ~11Å compared to the mature capsid. F&G) Corresponding central sections 567 from the cryo-EM maps of the procapsid and the virion, respectively, show the arrangement of the capsid 568

density without and with a packaged RNA genome, and before and after cleavage of VP0. Locations of 569 the two-, three-, and five-fold axes of symmetry are indicated. 570 571 Figure 2. Central surface projections and radial density projections. A) A radial plot of a central section 572 in the standard orientation for the capsid surfaces, displayed centered to a circle with r=135Å and 573 labeled positions for two-, three, - and five-fold axes of symmetry that cross the section. Red surfaces 574 for the procapsid are rendered at 1sigma. For the virion (blue) the external surface is rendered at 1 575 sigma and the inner surface is depicted at 2.35 sigma, which is the level at which the RNA core can be 576 distinguished from the protein capsid shell. The figure shows the rearrangement of both capsid surfaces 577 between procapsid and virion with notable difference in the width of the capsid wall, which is more 578 regular and has more densely packed protein in the virion. B) The radial density projection is a 2D 579 representation of the distribution of density of a spherical shell of the map at a given radius , r=135Å. 580 Density is white and black represents the absence of density. The asymmetric unit is marked by a 581 yellow line, with the icosahedral axes indicated. Comparison of the density at the same radii, shows 582 intricate rearrangement of the capsid structural proteins in virion relative to the procapsid. Specifically 583 at the three-fold, rearrangement introduces directionality and fills the virion capsid density; at the 584 two-fold of the procapsid there is notably less density in the procapsid; differences around the five-fold 585 are also noted due to the rearrangement of the density from movement of the protomers in the virion 586 compared to the procapsid. 587 588 Figure 3. A) EV71 1095 procapsid crystal structure. Peptide chains are colored using the canonical 589 picornavirus coloring scheme with VP1 in blue, VP0 in green, and VP3 in red. The first 81 N-terminal 590 residues of VP0 are disordered, hence displayed here is the structure of VP0, residues 82-318 that 591 correspond to VP2 residues 13-249, post cleavage. B) The sites where the EV71-1095 superimposed 592

over the crystal structure of 3VBU (shown in gold) with a rmsd greater than 2Å: residues VP1 218-219 593 (GH-loop, including disordered residues 211-217), residues VP0 43-51 (η1-η2-loop or AB-loop) 594 and138-139 (βE-α2-loop or EF-loop), and VP3 residues 180-183 (βG-η3-loop or GH-loop) (specific 595 nomenclature of loops from Wang et al 2012)(53). C) The zoomed view shows the EV71-1095 structure 596 (blue) superimposed with the Fuyang structure, 3VBU (gold) to show the VP1 BC-, DE-, and HI-loops of 597 the five-fold vertex. Side-chains of the specific residues were depicted by sticks and labeled to show the 598 slight but significant differences in residues known to affect virulence and PSGL-1 receptor binding. 599 600 Figure 4. The cryo-EM reconstructions of the procapsid (A) (red) and the virion (B) (blue), displayed at 1 601 sigma, showing the fitting of crystal structures depicted as ribbons and colored according the canonical 602 picornavirus coloring: VP1 in blue, VP0 in green, and VP3 in red for the procapsid and VP1 in blue, VP2 603 in green, VP3 in red, and VP4 in yellow for the virion. Full map and cutaway views are shown for each 604 with close-up views of the docked crystal structures adjacent. In panel A, the procapsid reconstruction 605 fitted with the crystal structure of the empty particle (procapsid) 4GMP and VP4 from 3VBS(53).The 606 front hemisphere of the reconstruction is displayed in transparent red, and the back hemisphere of the 607 reconstruction, shows the inner and outer surfaces. Panel B, the same views of the virion reconstruction 608 (less RNA) fitted with the crystal structure 3VBS (53), with the reconstruction density displayed in 609 transparent blue. The outer surface was rendered at 1 sigma and the inner surface at 2.35 sigma, as 610 described in Figure 2. 611 612 Figure 5.Detail of the difference map in which the density corresponding to an 8Å map simulated from 613 the EV71-1095 procapsid X-ray crystal structure was subtracted from the cryo-EM map of procapsid, 614 resulting in an internal five-fold plateau of density. The EV71-1095 procapsid structure was fitted with 615

additional VP1 and VP4 portions of mature virus (3VBS) spliced in to provide the structure of those parts 616 that are disordered in the procapsid crystal structure. The first 81 N-terminal residues of VP0 disordered 617 in the procapsid correspond to all of VP4 (yellow ribbon) and five residues of VP2 (not shown). The 618 N-terminal 72 residues of VP1, which are also disordered in the crystal structure, are shown in blue 619 ribbon. The view is centered on a five-fold axis of symmetry and shows the inner portion of the internal 620 difference density map surface in translucent grey (radii 0-140 Å). Non-colored portions of the 3VBS 621 structure appear in white ribbon. Scale bar = 30Å. 622 623 Figure 6. Picornavirus assembly flowchart incorporating the new EV71 structural information and 624 displaying the structural proteins, intermediates of assembly, and protein stoichiometry. The single 625 RNA transcript is translated into a polypeptide that is proteolysed to produce the structural proteins 626 VP0, VP1, and VP3. These proteins assemble into protomers, five of which self-assemble to form a 627 pentamer. Pentamers (14S) assemble into the provirion either by procapsid self-assembly followed by 628 progeny genome packaging (A) or by capsid condensation around a progeny genome (B). The latter 629 model suggests that the empty procapsids are an off pathway structure. The final step of maturation 630 involves the cleavage of VP0 to generate VP2 and VP4, which is induced by the packaged RNA. In the 631 flowchart the observed and hypothetical paths are depicted by solid and dashed arrows, respectively. 632

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