Human Enterovirus 71 Uncoating Captured at Atomic Resolution Ke Lyu, Jie Ding, Jian-Feng Han, Yu Zhang, Xiao-Yan Wu, Ya-Ling He, Cheng-Feng Qin and Rong Chen J. Virol. 2014, 88(6):3114. DOI: 10.1128/JVI.03029-13. Published Ahead of Print 18 December 2013.

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Human Enterovirus 71 Uncoating Captured at Atomic Resolution Ke Lyu,a Jie Ding,a Jian-Feng Han,b Yu Zhang,b Xiao-Yan Wu,b Ya-Ling He,a Cheng-Feng Qin,b,c Rong Chena Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, Chinaa; Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, Chinab; Graduate School, Anhui Medical University, Hefei, Chinac

ABSTRACT

IMPORTANCE

Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children. EV71 contains an RNA genome protected by an icosahedral capsid shell. Uncoating is essential in EV71 life cycle, which is characterized by conformational changes in the capsid to facilitate RNA release into host cell. Here we present the atomic structures of the full virion and an uncoating intermediate of a clinical C4 strain of EV71. Structural analysis revealed drastic conformational changes associated with uncoating in all the capsid proteins near the junction at the quasi-3-fold axis and protein-RNA interactions at the bottom of the junction in the uncoating intermediate. Significant capsid rearrangements also occur at the icosahedral 2- and 5-fold axes but not at the 3-fold axis. Taking the results together, we hypothesize that the junction and nearby areas are hot spots for capsid breaches for the exit of polypeptides and viral RNA during uncoating.

H

uman enterovirus 71 (EV71) is the leading causative agent for severe hand-foot-and-mouth diseases (HFMD) in infants and young children (1), and EV71 infection has caused significant morbidity and mortality in the Asia-Pacific regions. During 2008 to 2012, numerous outbreaks of HFMD occurred in mainland China, with over 2,000 fatal cases reported (www.chinacdc.cn). However, vaccines and effective drugs remain unavailable. EV71 belongs to Enterovirus species A within the Enterovirus genus of the Picornaviridae (2). The EV71 virion contains a singlestranded positive-sense RNA genome of 7.5 kb. The viral capsid, which has a diameter of approximately 300 Å, is composed of 60 copies each of VP1 to VP4 (3, 4) that are organized onto a quasiT⫽3 icosahedral lattice. The capsid proteins VP1, VP2, and VP3 each possess a ␤-sandwich jelly roll fold and form the outer surface of the capsid shell, whereas VP4 is situated inside the shell. The capsid surface is characterized by a depression encircling each 5-fold symmetry axis, which is referred to as the “canyon” and often contains the receptor-binding site in picornaviruses (5, 6). The hydrophobic pocket of VP1, which is located at the bottom of the canyon, contains a lipid moiety termed the “pocket factor,” which likely stabilizes the mature virion. As observed in poliovirus and certain picornaviruses, receptor binding at the junction site triggers the uncoating process, which is characterized by the delivery of the viral genome into the host cell compartment for replication and transcription (7). The uncoating is considered to be a

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multistep process, including the conversion of the mature virion (160S) into an expanded intermediate or A-particle (135S), characterized by the release of the pocket factor and the opening of the 2-fold symmetry axis channels, and the generation of the empty capsid (80S) from the A-particle, characterized by the release of the RNA genome (8–11). The structural basis of picornavirus uncoating has been extensively studied using both cryo-electron microscopy (cryo-EM) and X-ray crystallography. The uncoating products can be obtained in vitro from virions by receptor binding and/or exposure to low pH (5.5 to 6.0) or elevated temperature (3, 12–16). Upon receptor binding, the poliovirus converts to the A-particle, which is associated with irreversible conformational changes, including shifts of the capsid protein ␤-barrels and externalization of VP4 and the N terminus of VP1 (8, 17, 18). During coxsackievirus A7 (CVA7) uncoating triggered by heat, both VP4 and RNA are re-

Received 15 October 2013 Accepted 9 December 2013 Published ahead of print 18 December 2013 Editor: L. Hutt-Fletcher Address correspondence to Rong Chen, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03029-13

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Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterization of EV71 during its life cycle can aid in the development of therapeutics against HFMD. Here, we present the atomic structures of the full virion and an uncoating intermediate of a clinical EV71 C4 strain to illustrate the structural changes in the full virion that lead to the formation of the uncoating intermediate prepared for RNA release. Although the VP1 N-terminal regions observed to penetrate through the junction channel at the quasi-3-fold axis in the uncoating intermediate of coxsackievirus A16 were not observed in the EV71 uncoating intermediate, drastic conformational changes occur in this region, as has been observed in all capsid proteins. Additionally, the RNA genome interacts with the N-terminal extensions of VP1 and residues 32 to 36 of VP3, both of which are situated at the bottom of the junction. These observations highlight the importance of the junction for genome release. Furthermore, EV71 uncoating is associated with apparent rearrangements and expansion around the 2- and 5-fold axes without obvious changes around the 3-fold axes. Therefore, these structures enabled the identification of hot spots for capsid rearrangements, which led to the hypothesis that the protomer interface near the junction and the 2-fold axis permits the opening of large channels for the exit of polypeptides and viral RNA, which is an uncoating mechanism that is likely conserved in enteroviruses.

EV71 Uncoating Revealed by X-Ray Structures

MATERIALS AND METHODS Virion purification. EV71 (strain AH08/06 [GenBank accession no. HQ611148], with one amino acid change at residue 227 of VP3 [K to Q]) was isolated from an HFMD patient in Anhui, China, in 2008. The virus was grown in RD cells in Dulbecco modified Eagle medium (DMEM) containing 10% fetal (FBS) until 90% of cells exhibited a cytopathic effect (CPE). About 0.5 to 1 liter of cell lysate was frozen and thawed three times and then subjected to low-speed centrifugation at 8,228 ⫻ g for 30 min to remove cell debris. The virus supernatant was mixed with 50% polyethylene glycol 8000 (PEG 8000) and 2 M NaCl-phosphate-buffered saline (PBS) (pH 7.2) to final concentrations of 5% and 200 mM, respectively, and stirred overnight at 4°C. Ultracentrifugation at 27,000 rpm in a SW28 rotor was carried out to spin down virus particles into a 40% sucrose cushion, followed by no-brake ultracentrifugation at 26,000 rpm in an SW28 rotor for 4 h onto a noncontinuous 10 to 65% sucrose gradient to separate virions from naturally occurring empty particles. Fractions obtained after ultracentrifugation were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis. Those fractions

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containing virions were collected and concentrated by one more round of ultracentrifugation at 28,000 rpm in an SW28 rotor for 4 h. The pelleted virions were resuspended in PBS buffer to a final concentration of about 3 mg/ml. The quality of the virions was examined by negative-staining electron microscopy, which showed a high homogeneity. Crystallization and diffraction data collection. The purified EV71 virions were subjected to screening for crystallization conditions. Crystals were obtained by the vapor diffusion method in hanging drops at 16°C by mixing 2 ␮l of virus (⬃3 mg/ml) and 2 ␮l of the reservoir solution. The crystallization condition for crystals of full virions was 0.1 M sodium acetate (NaAc) (pH 4.5) containing 3.5 M sodium formate. Another specific crystallization condition triggered the conversion of the full virion to an uncoating intermediate (for unknown reasons) and formation of crystals. The crystallization condition for crystals of this uncoating intermediate was 0.1 M sodium cacodylate (pH 7.0) containing 1.6 M sodium acetate. Prior to data collection, cryoprotection of the crystals was achieved by resuspending the crystals in the mother liquor with increasing concentrations of glycerol through four steps, i.e., 5, 10, 15, and 20% (vol/vol). The equilibration time at each concentration was at least 30 s. These crystals were flash-frozen in liquid nitrogen and used for diffraction data collection on an ADSC Quantum-315 charge-coupled device (CCD) detector at the beamline BL17U1 at the Shanghai Synchrotron Radiation Facility. For the full virion, a data set at 3.3-Å resolution was collected with monochromatic X-rays (␭ ⫽ 0.97930 Å) and a detector-to-crystal distance of 350 mm using an oscillation angle of 0.3° and an exposure time of 2 s. For the uncoating intermediate, a data set at 3.8-Å resolution was collected with monochromatic X-rays (␭ ⫽ 0.97914 Å) and a detector-to-crystal distance of 400 mm using an oscillation angle of 0.3° and an exposure time of 1 s. Indexing, integration, scaling, postrefinement, and reduction of the data were carried out using the HKL2000 software package (25). Totals of 50 and 30 diffraction images were used for data processing for the full virion and the uncoating intermediate, respectively. Structure determination and refinement. Crystals of the uncoating intermediate belong to space group P4232 with 5 copies of the protomer as the asymmetric unit. Thus, 5-fold noncrystallographic symmetry was employed during structure determination and refinement. The program GLRF (26) was used to calculate the self-rotation function and combined with crystal packing analysis to determine the position and orientation of the particle. The structure of poliovirus type 1 empty capsid (PDB accession no. 1POV) was positioned at the origin with the determined orientation in the unit cell and was used to calculate a 10-Å density map as an initial phase model. The phase refinement and extension were carried out using real-space averaging, taking advantage of the 5-fold noncrystallographic symmetry. The phase extension from 10 Å was carried out gradually in steps of one reciprocal lattice point by iterative cycles of molecular averaging with noncrystallographic 5-fold symmetry, solvent flattening, back transformation using RAVE (27), and CCP4 (28). Crystals of EV71 virions belong to space group I23 with 20 copies of the protomer as the asymmetric unit. Thus, 20-fold noncrystallographic symmetry was employed during structure determination and refinement. Self-rotation function calculation and crystal packing analysis were preformed, which showed that the particle center is not at the origin. The virion structure was then determined by the molecular replacement method using the program package PHENIX (29). To avoid model bias, the crystal structure of a more distantly related picornavirus, coxsackievirus A9 (PDB accession no. 1D4M), was taken as the search model, and a single solution was obtained with a log-likelihood gain (LLG) of 11,234 and a translation function Z score (TFZ) of 53.2. A single solution with an LLG of 9,098 and a TFZ of 74.2 was obtained if the structure of poliovirus type 1 empty capsid (PDB accession no. 1POV) was taken as the search model. After obtaining the initial density map, model building and refinement were carried out iteratively using the COOT (30) and PHENIX (29) programs, respectively. The final refinement statistics are summarized in

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leased, and VP1 is rotated, which causes major conformational changes at the interfaces of the capsid proteins VP1, VP2, and VP3 (19). The crystal structure of the empty capsid of human rhinovirus type 2 (HRV2), obtained after release of the viral genome, exhibited a key movement around the hydrophobic pocket of VP1 that allowed a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the interprotomer interfaces, resulting in particle expansion and the opening of 2-fold channels in the capsid, which facilitates RNA egress (11). The structures of different strains of EV71 have been determined (20–22). By comparing the structures of the mature virion and a naturally occurring empty particle (sedimentation coefficient of 80S), Wang et al. proposed a sensor-adaptor mechanism for EV71 uncoating (21) and found that the largest movement of the polypeptide occurs in residues 230 to 233 of VP1 at the end of the GH loop and at the beginning of strand H. They referred to this polypeptide region as the adaptor-sensor, which is directly downstream of a region of the GH loop that is external and structurally variable in poliovirus (21). However, the naturally occurring empty particle is composed of VP0 (the precursor of VP2 and VP4), VP1, and VP3, whereas the uncoating empty capsid is composed of VP1, VP2, and VP3. Whether the structure is identical for the two is unknown. Based on cryo-EM studies, Shingler et al. proposed that the EV71 A-particle forms a gateway to allow genome release (23) and found that the diameter of the 2-fold channel in the A-particle is approximately 10 Å, whereas that in the empty capsid is only 6.4 Å. They thus proposed that the 2-fold channel shrinks after RNA release. Recently, a coxsackievirus A16 (CVA16) A-like particle was captured in atomic detail (24), which revealed that a portion of the N-terminal extensions of VP1 is extruded through the capsid, thereby providing novel insights into picornavirus uncoating. In this study, we determined the crystal structures of the full virion and an uncoating intermediate of a clinical C4 strain of EV71 at high resolution. Comparison of these two structures enabled us to map the conformational changes associated with uncoating and provide a more detailed picture to understand the early steps of EV71 uncoating. Furthermore, fitting of the crystal structure of the uncoating intermediate into the cryo-EM reconstruction obtained by Shingler et al. (23) revealed the location of the VP2 N terminus and the interaction between capsid proteins and the RNA genome.

Lyu et al.

TABLE 1 Data collection and refinement statistics Valueb for: Parametera

Refinement Resolution (Å) No. of reflections Rwork/Rfree (%) No. of atoms Avg B-factors RMSD Bond lengths (Å) Bond angles (°)

Uncoating intermediate

I23 591.375

P4232 352.416

49.84–3.30 (3.38–3.30) 314,603/15,490 21.6 (70.1) 3.7 (0.93) 62.2 (46.2) 1.5 (1.2)

49.84–3.80 (3.87–3.80) 50,101/2,652 38.4 (83.9) 3.3 (1.3) 68.0 (73.4) 5.0 (4.6)

49.84–3.30 314,499 22.80/26.09 130,720 50.47

49.84–3.80 50,069 24.54/27.41 26,295 65.65

0.003 0.728

0.002 0.641

n

a

Rmerge ⫽

兺 兺 |Ii共hkl兲⫺I៮共hkl兲| hkl i⫽1 n

兺 兺 I 共hkl兲 hkl i⫽1 兺 |F 共hkl兲⫺F 共hkl兲| R⫽ i

hkl

obs

calc

Fobs共hkl兲 兺 hkl

The R for the larger “working” set of reflections is referred to as Rwork. |Fobs共h,k,l兲|⫺k|Fcalc共h,k,l兲| Rfree T ⫽

兺 |

共h,k,l兲僆T

|

兺 |F

共h,k,l兲僆T

obs共h,k,l兲

|

T, set of reflections. b Values in parentheses refer to the highest-resolution shell.

Table 1. Figures were drawn and rendered with PyMol (31) and Chimera (32). Capsid expansion calculation. For the uncoating intermediate, the distances from all the C␣ atoms to the particle center were averaged to obtain the capsid radius. For the full virion, these same C␣ atoms were used to calculate the capsid radius. To calculate the capsid expansion at the 5-fold axes, residues 142 to 149 in the DE loop and 183 to 187 in the FG loop of VP1 were taken for calculation. For the 2-fold axes, residues 90 to 98 and 249 to 254 of VP2 and 137 to 152 of VP3 were considered. For the 3-fold axes, residues 224 to 232 in the HI loop of VP2 and 203 to 209 in the HI loop of VP3 were considered. For the junction, residues 193 to 231 in the GH loop of VP1, residues 130 to 180 in the EF loop of VP2, and residues 173 to 192 in the GH loop of VP3 were considered. All these residues were chosen based on visual inspection of the capsid structures. Cryo-EM. Aliquots of sample from the hanging drop containing crystals of the uncoating intermediate were blotted onto glow-discharged holey carbon grids and plunged into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark III freezing robot (FEI). Data were collected in an FEI electron microscope operating at 300 kV with a magnification of ⫻89,000. Electron micrographs were recorded at a dosage of about 10 electrons/Å2, with focal settings ranging from 0.9 to 3 ␮m underfocus. Samples of purified naturally occurring empty particles were also used for cryo-electron microscopy (cryo-EM) experiments to show different internal structure features.

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RESULTS

Crystallization of two forms of EV71. A clinical EV71 C4 strain isolated from Anhui, China, was used in this study. The purified virions contained all four structural proteins, VP1 to VP4 (Fig. 1A), and retained infectivity, as demonstrated by a cell infection study (data not shown). Crystallization experiments were performed, and crystals were obtained under two different conditions. Unexpectedly, the two determined structures exhibited significant differences, as described in detail below. Structure of the full virion. The crystal structure of the first crystal form, the full virion, was determined at a resolution of 3.3 Å (Fig. 2). The capsid is composed of 60 copies of the protomer. Each protomer is composed of VP1, VP2, VP3, and VP4 (Fig. 3A to E). The quality of the resulting electron density maps enabled the modeling of the polypeptides of VP1 (residues 1 to 297), VP2 (residues 9 to 254), VP3 (residues 1 to 242), VP4 (residues 12 to 69), and the pocket factor (Fig. 3B to F). The structure of the full virion was compared with that reported for the C4 strain (PDB accession no. 3VBS) (21). Superposition of equivalent C␣ atoms in the VP1, VP2, VP3, and VP4 proteins resulted in root mean square deviation (RMSD) values of 0.94 Å, 0.48 Å, 0.68 Å, and 1.28 Å, respectively, indicating similar capsid protein structures. Amino acid sequence alignment indicated differences in five residues within the capsid protein regions (Fig. 4). Residue 225 of VP1 in the GH loop near the pocket factor region is Cys (Fig. 3F), rather than Met as in 3VBS. All of the clinical EV71 C4 strains contain Cys at residue 225 of VP1. The virus strain used to obtain the crystal for structure determination of 3VBS was a vaccine strain that had been adapted into Vero cells. During this adaptation process, mutation occurred in the viral genome, leading to a mutation at residue 225 of VP1. Conversely, the virus strain used here was propagated in RD cells, which more closely resemble the natural host of EV71. Additionally, residue 98 of VP1 in the BC loop is Glu rather than Lys in 3VBS, and residue 144 of VP2 in the EF loop is Ser rather than Thr in 3VBS. Both loops are involved in the formation of neutralization epitopes (33–35). VP3 also contains two different residues: residue 93 is Ser rather than Asn in 3VBS, and residue 227 is Gln rather than Lys in 3VBS. Residue 93 is located on the surface loop (Fig. 5A). None of these mutations cause significant local structural variations.

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Data collection Space group Cell dimensions, a ⫽ b ⫽ c (Å) Resolution (Å) No. of unique reflections Rmerge (%) I/␴I Completeness (%) Redundancy

Full virion

Characterization of the uncoating intermediate. The proteolytic sensitivities of the full virion and the uncoating intermediate were assessed by adding 0.5 ␮g of trypsin to a 5-␮l solution containing 0.5 ␮g of the corresponding particles. Samples with or without trypsin were incubated at 37°C. The digestion was stopped by dilution of the samples with 1/5 volume of 5⫻ SDS loading buffer followed by boiling of the mixture for 10 min. The samples were subjected to 12% SDS-PAGE, and VP1 was visualized by Western blotting using a VP1 antibody (Abnova, catalog no. PAB7631-D01P). To detect the presence of VP4 in the uncoating intermediate, 0.025U of ␣-chymotrypsin (Sigma, catalog no. C4129) was added to a 10-␮l solution containing 1.2 ␮g of the corresponding particles. Samples with or without ␣-chymotrypsin were incubated at 25°C. The digestion was stopped by dilution of the samples with 1/5 volume of the 5⫻ SDS loading buffer followed by boiling of the mixture for 10 min. The samples were subjected to 15% SDS-PAGE, and VP4 was visualized by Western blotting using a VP4 antibody (Biorbyt, catalog no. orb10624) with VP2 as a loading control. Protein structure accession numbers. The coordinates and structure factors of the uncoating intermediate and the full virion have been deposited in the Protein Data Bank (PDB accession no. 4N43 and 4N53).

EV71 Uncoating Revealed by X-Ray Structures

which was purified as described in Materials and Methods, was determined using 15% SDS-PAGE analysis. Lane 1, molecular mass marker; lane 2, purified full virions. The calculated molecular masses of VP1, VP2, VP3, and VP4 are 32.6 kDa, 27.7 kDa, 26.4 kDa, and 7.5 kDa, respectively. The purified full virions contain all four structural proteins, i.e., VP1, VP2, VP3, and VP4. (B) Cryo-EM images of samples from the crystallization drops containing crystals of the uncoating intermediate (left) and purified naturally occurring empty particles (right). The internal density corresponded to the RNA genome in the uncoating intermediate, whereas the empty particle was completely devoid of internal density. The presence of the RNA genome inside the particle suggests that these are uncoating intermediates. (C) The uncoating intermediate is more protease sensitive than the full virion. The proteolytic sensitivities of the full virion and the uncoating intermediate were assessed by trypsin digestion as described in Materials and Methods. The digested samples were analyzed by Western blot using a VP1 antibody. Lane 1, hanging drops containing crystals of the uncoating intermediate. Lane 2, hanging drops containing crystals of the uncoating intermediate digested with trypsin for 1 h at 37°C. The truncated VP1 is indicated with an arrow. Lane 3, hanging drops containing crystals of the full virion. Lane 4, hanging drops containing crystals of the full virion digested with trypsin for 1 h at 37°C. VP1 in the full virion is resistant to trypsin digestion. (D) The VP4 in the uncoating intermediate is protected from ␣-chymotrypsin digestion. The uncoating intermediate was treated by ␣-chymotrypsin digestion as described in Materials and Methods. The digested samples were analyzed by Western blotting using a VP4 antibody, with VP2 as a loading control. Lane 1, hanging drops containing crystals of the uncoating intermediate were incubated at 25°C. Lane 2, hanging drops containing crystals of the uncoating intermediate were digested with ␣-chymotrypsin for 1 h at 25°C. The VP4 in the uncoating intermediate was resistant to protease digestion.

Structure of an expanded particle. The crystal structure of the second crystal form was determined at a resolution of 3.8 Å (Fig. 2). The average radius in the capsid was found to increase from 130.9Å (the full virion) to 138.5 Å (the expanded particle), with an enlargement of approximately 5.8%. The refined model includes residues 72 to 296 of VP1, 16 to 47 and 54 to 250 of VP2, and 1 to 175 and 189 to 236 of VP3 (Fig. 3B to D). Residues 48 to 53 in VP2 and 176 to 188 in VP3 are disordered. The N-terminal region (residues 1 to 71) of VP1 becomes disordered in the expanded particle, and the pocket region is empty (Fig. 3F). The disposition of the VP1 to VP3 subunits is largely maintained in the expanded particle in comparison to those in the full virion. The largest structural changes were mapped to the terminal regions and some loops connecting the strands of the ␤-barrel. Superpositions of the individual VP1, VP2, and VP3 proteins in these two particles resulted in RMSD values of 2.47 Å, 1.69 Å, and 2.07 Å for equivalent C␣ atoms, respectively (Fig. 3B to D), whereas superpositions of the individual ␤-barrels of VP1, VP2, and VP3 resulted in RMSD values of 2.06 Å, 1.67 Å, and 1.98 Å, respectively. Thus, VP1 and VP3 undergo more substantial conformational changes than VP2.

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In VP1, in addition to the disordered N-terminal extensions, drastic conformational changes also occur in the C-terminal region (residues 266 to 297) and the GH loop (residues 191 to 230), with RMSD values of 2.20 Å and 2.66 Å, respectively. The C-terminal regions wrap around the VP3 of the same protomer, whereas the GH loop lies near the junction at the quasi-3-fold axis (Fig. 5C). In VP2, the overall structure is rather conserved, except that both of the N-terminal extensions and residues 48 to 53 that precede the ␤-barrel core become disordered. In VP3, a 310-helix in the GH loop (residues 173 to 192) of VP3 in the full virion located near the junction becomes disordered (Fig. 3C and D). Moreover, significant changes occur in the FG loop (residues 159 to 164), with an RMSD value of 2.74 Å. Identification of the expanded particle as an uncoating intermediate. The mature virions of some enteroviruses, including poliovirus and human rhinovirus 14, exist in a metastable state, transiently exposing VP4 and the N terminus of VP1 through hot spots in the capsid near the junction or 2-fold axis in a process called “breathing” (36, 37). Typically, the exposed polypeptides rapidly retract into the particle, and the breathing is a reversible process

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FIG 1 Purification of EV71 virions and characterization of the uncoating intermediate. (A) Purification of EV71 virions. The protein composition of the virion,

Lyu et al.

under physiological conditions. However, under certain conditions, such as upon receptor binding and/or with acidic pH or elevated temperature, the conformational changes associated with breathing become irreversible (3). The structural features that we identified suggest that the first crystal form corresponded to the full virion, whereas the second crystal form corresponded to a capsid-rearranged product. Because the crystals were grown at 16°C, breathing, which occurs only at 37°C (36, 37), should not have occurred. Therefore, we speculate that the second crystallization condition triggered the uncoating process of EV71. Generally, enterovirus produces two forms of particles during uncoating: an altered “A-particle” (containing RNA) and an empty capsid (without RNA) (3). To determine whether the second crystal form contained RNA in the interior of the particle, samples from the crystallization drops that contained crystals of the uncoating intermediate were analyzed using cryo-EM. In the cryo-EM images, the internal density corresponded to the RNA genome, whereas the purified naturally occurring empty particle was completely devoid of internal density (Fig. 1B). The presence of the RNA genome in the interior of the particle suggests that this is an uncoating intermediate. The specific crystallization condition triggered the uncoating process and converted the full virion into the uncoating intermediate. In poliovirus, the A-particle is more susceptible to proteolysis than the intact virion and is characterized by the irreversible externalization of the N terminus of VP1 (38–40). To assess and compare the proteolytic sensitivities of the full virion and the uncoating intermediate, we performed trypsin digestion experiments. The crystallization hanging drops containing crystals of the uncoating intermediate and the full virion were digested with trypsin (Fig. 1C). In hanging drops containing crystals of the full virion, VP1 was resistant to trypsin digestion, whereas in hanging drops containing crystals of the uncoating intermediate, a truncated VP1 product was observed after trypsin digestion. This suggests that the uncoating intermediate obtained in this study has increased protease sensitivity. To determine whether VP4 is still present, hanging drops containing crystals of the uncoating intermediate were digested by ␣-chymotrypsin and VP4 was visualized by Western blotting (Fig. 1D). Unexpectedly, most of the VP4 was resistant to ␣-chymotrypsin digestion.

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Similar results were obtained when trypsin was used for digestion. These studies suggest that most of the VP4 is still associated with the uncoating intermediate and protected from proteolysis. Channel expansion at the 2-fold, but not 5-fold, axes in the uncoating intermediate. In the virion structure, at the top of each 5-fold axis, a channel with a diameter of approximately 13 Å runs from the particle surface toward the particle center. At the top opening of the channel, the BC, EF, and HI loops of VP1 form a large protrusion (Fig. 5A). An elaborate network of hydrogen bonds established between Lys182 and Asp185 from a neighboring VP1 maintains the channel in the open conformation and stabilizes this conformation at the 5-fold axes (Fig. 5B). Both of these residues (Lys182 and Asp185) are conserved among enteroviruses in species A and D, whereas they are divergent in species B and C (data not shown). In the structure of the uncoating intermediate, despite significant enlargement of the capsid along the 5-fold axes, no apparent expansion of the 5-fold channel was observed, and the hydrogen bonding network between the 5-foldrelated Lys182 and Asp185 is maintained. Conversely, a clear expansion of the 2-fold channels was observed in the uncoating intermediate (Fig. 6). This channel, which is located at the interface of two neighboring pentamers and is surrounded by segments of VP2 and VP3, expands from dimensions of 7.0 Å by 27.6 Å in the full virion to 11.7 Å by 27.7 Å in the uncoating intermediate, with the channel dimension calculated between the 2-fold-related C␣ atoms (Fig. 6). The top of this pore is formed by the C-terminal segment and one ␣-helix from the CD loop of VP2, an additional ␣-helix from the EF loop of VP3, and their 2-fold symmetry mates in the particle. The bottom of the pore is formed by two segments from VP2, including a sharp turn at residues 17 and 18 and a loop region at residues 54 to 58, both of which precede the ␤-barrel core structures. This pore opening at the 2-fold axes was also observed in the uncoating intermediate of CVA16 (24) and the empty capsid of HRV2 that resulted from the release of the genomic RNA (11). Structural rearrangements during uncoating. Capsid expansion implies rearrangements of the interactions at the interfaces between and within protomers. The junction, which is surrounded by VP1 and VP2 from one protomer and VP3 from a neighboring protomer that is related by the 5-fold symmetry, undergoes drastic

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FIG 2 Overall structures of EV71 full virions and the uncoating intermediate. (Left) Radius-colored surface representation of the EV71 full virion viewed along the 2-fold axis. The surface is colored from blue to red according to the distance from the particle center (blue represents the closest). (Middle) Ribbon representations of the full virion (colored red) and the uncoating intermediate (colored blue). Only half of each capsid shell is represented, as an ⬃80-Å slab, to illustrate the expansion of the uncoating intermediate with respect to the full virion. The position of the 2-fold axis of the particle is indicated. (Right) Radius-colored surface representation of the uncoating intermediate viewed along the 2-fold axis. The surface is colored as in the left panel.

EV71 Uncoating Revealed by X-Ray Structures

Downloaded from http://jvi.asm.org/ on March 16, 2014 by UNIV OF NEWCASTLE FIG 3 Structural changes in the protomer and individual capsid proteins during uncoating. (A) Structural comparison of the protomer in the full virion and the uncoating intermediate. A surface representation of the EV71 full virion viewed along the 2-fold axis is shown, with VP1, VP2, and VP3 colored magenta, yellow, and cyan, respectively. Cartoon representations of the protomer with VP1, VP2, and VP3 in the uncoating intermediate are colored magenta, yellow, and cyan, respectively, whereas their counterparts in the full virion are colored gray. The positions of the icosahedral symmetry elements are indicated. (B) Superposition of VP1. Residues 1 to 297 are modeled in the full virion and colored red, whereas residues 72 to 296 are modeled in the uncoating intermediate and colored blue. (C) Superposition of VP2. The proteins are colored as in panel B. Residues 9 to 254 and residues 16 to 47 and 54 to 250 are modeled in the full virion and the uncoating intermediate, respectively. (D) Superposition of VP3. The proteins are colored as in panel B. Residues 1 to 242 and residues 1 to 175 and 189 to 236 are modeled in the full virion and the uncoating intermediate, respectively. (E) Structure of VP4, colored as in panel B. Residues 12 to 69 of VP4 were modeled from well-defined electron density in the full virion. (F) Comparison of the VP1 pockets in the full virion (red, with the pocket factor shown in green) and the uncoating intermediate (blue). Cys225 near the pocket region is shown in yellow.

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conformational changes, leading to rearrangements in the interactions between protomers around the 5-fold axes and across the 2-fold axes (Fig. 5C). The junction also undergoes expansion during uncoating, as the distance to the particle center increases from 137.95 Å to 143.06 Å, with a change of approximately 3.7%. Additionally, significant structural changes occur at the junction, as observed in the GH loops of VP1 and VP3 (Fig. 5C). The overall organization of the uncoating intermediate is similar to that of the full virion. The distances from the particle center to the surface along the 5-, 3-, and 2-fold axes are 149.49 Å, 146.44

FIG 6 Structural changes at the 2-fold axis channel. VP1, VP2, and VP3 are colored magenta, yellow, and cyan, respectively. The 2-fold channel in the full virion is shown on the left, whereas the 2-fold channel in the uncoating intermediate is shown on the right. The 2-fold axis channel expands during uncoating.

Å, and 137.45 Å, respectively, in the uncoating intermediate, in comparison to 143.11 Å, 146.14 Å, and 135.12 Å, respectively, in the full virion. These distances imply an expansion of approximately 4.5%, 0.2%, and 1.7% in the capsid along the 5-, 3-, and 2-fold axes, respectively, indicating a substantially smaller enlargement along the 3-fold axes. The absolute distance shift for every C␣ atom between the full virion and the uncoating intermediate was also calculated and mapped onto the full virion structure (Fig. 7A). The most dramatic movement lies in VP4 and the terminal regions of VP1, VP2, and VP3 (colored red) (Fig. 7B), with modest changes in VP1 around the 5-fold axes (colored green). The protomer interfaces along the 2-fold axes (predominantly VP2/VP2) and the junction between neighboring 2- and 5-fold axes, in addition to the intraprotomer interfaces (VP1/VP2 and VP1/VP3) (colored cyan), exhibit few changes, and the VP2/VP3 portions around the 3-fold axes (colored blue) exhibit the fewest changes (Fig. 7B). Thus, capsid regions around the 2- and 5-fold axes and the junction undergo significant structural changes, in comparison to relatively minor variations around the 3-fold axes. The uncoating process appears to be a coordinated process, with major rearrangements around the 2- and 5-fold axes resulting in local capsid expansion without obvious changes around the 3-fold

FIG 4 Structure-based sequence alignments of the capsid proteins VP1, VP2, and VP3 from different EV71 strains. Capsid protein sequences used for the alignment include those of the clinical EV71 C4 strain used in this study (4N53) and of two other EV71 strains (denoted 3VBS and 4GMP) whose capsid proteins have been structurally determined. The secondary structure elements for the EV71 full virion and the uncoating intermediate are shown at the top and bottom of the sequence alignment, respectively. The residue numbers are those in the EV71 full virion. Conserved residues are shown in white with a red background. Helices and strands are labeled according to standard picornavirus nomenclature and are represented by coils and arrows, respectively. The blue triangles indicate the residues that are variable between 4N53 and 3VBS. The black triangles indicate the residues that are variable between 4N53 and 4GMP. The VP1 GH loop, VP3 GH loop, and residues 48 to 53 of VP2 are boxed with blue rectangles and correspond to the disordered regions in the structure of the uncoating intermediate. This figure was produced using ESPript (50).

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FIG 5 Structural changes at the protomer interface. (A) Top view of the 5-fold axis channel in the virion. The surfaces are colored from blue to red according to their distance from the particle center (blue represents the closest). Four mutations between the clinical C4 strain and 3VBS in the capsid proteins are exposed on the viral surface as indicated. The triangle is drawn around the quasi-3-fold axis (surrounded by VP1 and VP2 from one protomer and VP3 from a neighboring protomer). The variable residues between the clinical C4 strain and 3VBS that are exposed on the capsid surface are colored blue (Glu98 and Cys225 of VP1), cyan (Ser144 of VP2), and black (Ser93 of VP3). (B) Hydrogen bonding network around the 5-fold axis. The amino group in the side chain of Lys182 (colored black) interacts with Asp185 (colored red) of a neighboring VP1 through hydrogen bonds. This interaction network around the 5-fold axis channel is likely conserved among human enterovirus species A and D. (C) Structural changes at the junction during uncoating. The structure of the virion is shown on the left, whereas that of the uncoating intermediate is shown on the right. VP1, VP2, and VP3 are colored magenta, yellow, and cyan, respectively. The GH loop of VP1 is colored green, residues 48 to 53 of VP2 are colored blue, and the GH loop of VP3 is colored orange. During uncoating, conformational changes occur in the GH loop of VP1, whereas residues 48 to 53 in VP2 and the GH loop in VP3 become disordered.

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axes. A similar analysis was conducted using structures of the full virion and the empty capsid after HRV2 uncoating, which exhibited similar structural changes (Fig. 7C). Fitting of the crystal structure into the cryo-EM reconstruction of the uncoating intermediate. Previously, the cryo-EM structure of the EV71 A-particle of the 1095/Shiga strain was determined at a resolution of 6.3 Å (EMDB accession no. 5465) (23). Five residues that were mapped to capsid proteins were divergent between the 1095 strain (PDB accession no. 4GMP) (22) and our clinical C4 strain (Fig. 4), three of which are linked to EV71 antigenicity (34). Residue 22 of VP1 is His rather than Arg in 4GMP, and residue 145 of VP1 in the surface loop is Glu rather than Gly in 4GMP. Nishimura et al. recently found that the PSGL-1 binding phenotype of EV71 strains is regulated by a single residue (residue 145 of VP1) that maps to the center of the 5-fold mesa (41). Additionally, this residue has been shown to be a determinant for the strain-specific antigenicity of EV71 (42). Residue 126 of VP2 in the ␤-strand E is Val rather than Ile in 4GMP. VP3 also contains two divergent residues: residue 29 is His rather than Tyr in 4GMP, and residue 227 is Gln rather than Lys in 4GMP. Residue 29 is located on the capsid interior surface. None of these residue variations cause significant local structural variations. The crystal structure of our uncoating intermediate was fitted into the A-particle cryo-EM density map (EMDB accession no.

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5465) (␴ ⫽ 1) (23). The crystal structure agreed well with the cryo-EM structural features except for a few protruding loops, the most distinct of which is a region in the EF loop of VP2 (residues 136 to 145) that lies near the junction. Additionally, residues 54 to 59 of VP2 in the inner side of the 2-fold channel lie outside the electron density. Interestingly, residues 48 to 54 of VP2 are disordered in the crystal structure of the uncoating intermediate. We observed a relatively large 2-fold channel, and the capsid interior surface exhibits large patches of negative charge at the 2-fold axis with minimal interspersed positive charge in both the uncoating intermediate and the EV71 A-particle cryo-EM structures. One novel observation is that the visible N terminus of VP1 (residue 72) and residues 32 to 36 of VP3 in the interior of the capsid, both of which are adjacent to the bottom of junction, interact with the inner RNA density (Fig. 8A). In the full virion, VP4 and the N-terminal extensions of VP1, VP2, and VP3 are packed in layers in the interior of the capsid and interact with the RNA genome. In the uncoating intermediate, it appears that only the N-terminal extensions of VP1 and residues 32 to 36 of VP3 interact with the RNA instead. A structural comparison indicated a lack of obvious shift of this VP3 region during uncoating. Moreover, we observed electron density extending from the visible N terminus of VP2 (residue 16) into the interior of the capsid, but interaction of VP2 with the RNA genome was not observed (Fig. 8B).

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FIG 7 Capsid rearrangements during uncoating. (A) The absolute distance shift for every C␣ atom (⫻10) during EV71 uncoating was mapped onto the full virion structure. The surface is colored from red to blue according to the relative distance shift (blue represents the lowest shift). (B) The inner surface of the full virion is colored as in panel A, showing the internal rearrangements during uncoating. (C) The absolute distance shift for every C␣ atom (⫻10) during HRV2 uncoating was mapped onto the full virion structure. The surface is colored from red to blue according to the relative distance shift (blue represents the lowest shift).

EV71 Uncoating Revealed by X-Ray Structures

EV71 A-particle, showing density extending from the capsid shell that interacts with the viral RNA genome. The fitted uncoating intermediate crystal structure is depicted in a ribbon representation with VP1, VP2, and VP3 colored magenta, yellow, and cyan, respectively. The A-particle cryo-EM density is depicted as a gray mesh. Residue 72 of VP1 is colored in green, and residues 32 to 36 of VP3 are colored in orange, both of which interact with the inner RNA density. (B) The fitted uncoating intermediate crystal structure is depicted in a ribbon representation with VP1, VP2, and VP3 colored magenta, yellow, and cyan, respectively. The A-particle cryo-EM density is depicted as a gray mesh. Residue 16 of VP2 is colored red and does not interact with the inner RNA density.

The N terminus of VP1 is externalized at the base of the canyon, as observed from cryo-EM reconstruction analysis of the Aparticle. The ordered region of the EV71 uncoating intermediate begins at Ser72 and is located in the center of the quasi-3-fold axis in the top view. Based on the correlation between the visible VP1 N termini in the uncoating intermediate and the A-particle electron density map, we speculate that the VP1 N-terminal extensions (residues 1 to 71) may externalize from the capsid surface in the junction channel. Uncoating in EV71 compared with CVA16. Recently, the structure of the CVA16 A-like particle was determined at a resolution of 3.0 Å (PDB accession no. 4JGY) (24). Superposition of equivalent C␣ atoms in the VP1, VP2, and VP3 proteins in our uncoating intermediate and 4JGY resulted in RMSD values of 1.27 Å, 0.89 Å, and 1.54 Å, respectively, suggesting similar structures for the EV71 uncoating intermediate and the CVA16 A-like particle (Fig. 9A to D). In both expanded particles, the GH loop of VP3, which is facing the junction channel, becomes disordered (Fig. 9D). Structural differences between the two particles are all mapped to regions near the junction or the 2-fold axis channel. One major

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FIG 9 Structural comparison between the EV71 uncoating intermediate and the CVA16 A-like particle. (A) Structural comparison of the protomer in the EV71 uncoating intermediate with that in the CVA16 135S-like particle. VP1, VP2, and VP3 in EV71 are colored magenta, yellow, and cyan, respectively, and those in CVA16 are colored gray. Thr175 and Tyr189 in VP3 mark the beginning and end of the disordered regions in the determined uncoating intermediate structure. Ala47 and Thr54 in VP2 mark the beginning and end of the disordered region in the determined uncoating intermediate structure. (B) Superposition of VP1. Residues 72 to 296 of VP1 in EV71 (colored blue) and residues 62 to 210 and 219 to 297 in CVA16 (colored orange) are modeled. The ordered region in VP1 begins at Ser72 (colored blue) of the determined uncoating intermediate structure. The ordered region in VP1 of the CVA16 135Slike particle begins at Asn62 (colored orange). (C) Superposition of VP2. The proteins are colored as in panel B. Residues 16 to 47 and 54 to 250 in EV71 and residues 6 to 136 and 142 to 249 in CVA16 are modeled. Ala47 and Thr54 indicate the beginning and end of the disordered regions in EV71. Ala136 and Glu142 indicate the beginning and end of the disordered regions in CVA16. (D) Superposition of VP3. The proteins are colored as in panel B. Residues 1 to 175 and 189 to 236 in EV71 and residues 1 to 179 and 185 to 236 in CVA16 are modeled. Tyr185 and Ala179 (orange) indicate the beginning and end of the disordered regions in CVA16. (E) Side views showing different positions of the VP1 N terminus in the EV71 uncoating intermediate (left) and the CVA16 135S-like particle (right). EV71 is colored blue, whereas CVA16 is colored red. The gray surface representation shows the surface of the capsid pentamer from the side view. A stretch of polypeptide was observed to traverse the capsid in CVA16, whereas this region is disordered in EV71. (F) Structures at the junction in CVA16. VP1, VP2 and, VP3 are colored magenta, yellow, and cyan, respectively. The GH loop in VP1 is colored green, and residues 142 to 146 in VP2 are colored blue. Portions of the GH loop of VP1 and the EF loop of VP2 (residues 137 to 141), both facing the junction channel, are disordered.

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FIG 8 Capsid-RNA interactions in the EV71 uncoating intermediate. (A)

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variation occurs in the N-terminal extensions of VP1 (residues 62 to 71) (Fig. 9B and E). This region of the polypeptide was found to traverse the capsid in the CVA16 A-like particle, whereas it is disordered in the EV71 uncoating intermediate (Fig. 9B and E). Additionally, in the EV71 uncoating intermediate, a loop region (residues 48 to 53) of VP2 that precedes the ␤-barrel core structure and runs between the 2-fold channel and the junction is disordered, whereas in the CVA16 A-like particle, the GH loops of both VP1 and a portion of the EF loop of VP2 (residues 137 to 141), which both face the junction channel, are disordered (Fig. 9F).

Previous studies demonstrated that the A-particles and the empty capsids of some enteroviruses can be produced through incubation with the corresponding receptors or after treatment under specific physical conditions. Human scavenger receptor class B member 2 (SCARB2), which is an identified uncoating receptor for EV71, has been shown to convert the EV71 virion into either the 135S particle (43, 44) or an empty capsid that lacks both genomic RNA and VP4 (45) after incubation under acidic conditions. Shingler et al. (23) demonstrated that a mixture of the Aparticle and empty capsid can be produced from purified EV71 virions by heating. In this study, we obtained crystals of an uncoating intermediate of EV71 from the purified infectious virions of a clinical EV71 C4 strain under a specific crystallization condition. This intermediate shares some structural features in the capsid with the naturally occurring empty particle composed of VP1, VP3, and VP0, which is the precursor for VP2 and VP4, such as the opening of pores at the 2-fold axes, loss of ordered structures in the VP1 N-terminal extensions, and expansion of the capsid. The junction at the quasi-3-fold axis (Fig. 6) in the intermediate structure exhibited drastic conformational changes from that of the full virion. Furthermore, fitting of the crystal structure into the EM density map revealed that the electron density observed between the RNA genome and the capsid proteins (particularly the N-terminal extensions of VP1) is attached to the bottom of the junction. A previous electron tomography analysis of poliovirus during RNA release indicated that the footprint of RNA on the capsid outer surface was located approximately 20 Å away from a 2-fold axis (18). Collectively, these observations suggest that large pores that are temporarily open at the junction and the 2-fold axes are hot spots for polypeptide and RNA release. In the poliovirus virion, the N terminus of VP1 is located near the 5-fold axis (46). EM analysis using a Fab directed against the N terminus of VP1 indicated that the full virion “breathes” with the N terminus of VP1 (residues 1 to 53) transiently exposed (47). In the poliovirus A-particle, antibody labeling studies indicated that the location of the N terminus of VP1 shifts toward the tips of the 3-fold propeller, which lies near the junction, and becomes externalized (48). In the EV71 virion, however, the N termini of VP1 are located near the 2-fold axis (20, 21). In the EV71 A-particle (EMDB accession no. 5465), cryo-EM analysis suggested that the N terminus of VP1 is externalized at the base of the canyon (Fig. 3A) (23). The crystal structure of a CVA16 135S-like particle (24) revealed that residues 62 to 71 of VP1 penetrate through the junction. The present crystal structure of the EV71 uncoating intermediate revealed that the visible N termini of VP1 lie at the bottom of the junction, implying that the N-terminal extensions of VP1 may be externalized through the junction. Collectively, these observa-

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DISCUSSION

tions suggest that the externalization mechanism of the N termini of VP1 from different enteroviruses may undergo function-driven convergence. A novel observation from this study is that the capsid expands significantly around the 2- and 5-fold axes and the junction between the quasi-3-fold axes, in contrast to the much lower expansion observed around the 3-fold axes. Additionally, capsid regions around the 3-fold axes undergo fewer structural perturbations, in contrast to regions around the 2- and 5-fold axes and the junction. Such differences during capsid rearrangement may arise from the significantly fewer interactions present around the 2-fold axes and the quasi-3-fold axes, which makes these regions hot spots for capsid breaches. Because the junction is located between neighboring 2- and 5-fold axis pairs, structural changes may easily propagate into regions around the 5-fold axes. It is possible that the appropriate protomer interface near the 2-fold axis channel and the junction may open further to permit RNA release. Another novel observation is that in the EV71 uncoating intermediate, only the visible N terminus (residue 72) of VP1 and residues 32 to 36 of VP3 in the interior capsid near the junction interact with the inner RNA density. In the full virion, VP4 and the N-terminal regions of VP1, VP2, and VP3 are packed inside the capsid and directly interact with the RNA genome. The disposition of residues 32 to 36 in VP3 is not greatly altered during uncoating. Such protein-RNA interactions in the uncoating intermediate may facilitate communication from outside the capsid to the RNA to trigger RNA release or provide an anchored site for RNA release. Enterovirus uncoating occurs in multiple steps. One of the early steps involves the formation of an expanded, altered “Aparticle” that is primed for genome release. One of the late steps involves an unknown trigger that results in RNA expulsion, generating an empty capsid. The expanded particle analyzed in this study likely represented an intermediate at the initial stage of uncoating, likely prior to the formation of the A-particle, as shown by the increased protease sensitivity of VP1 and the retaining of VP4. We addressed the question of how the EV71 capsid conformation changes in the initial stage by comparing the crystal structures of the full virion and the uncoating intermediate and observed specific protein-RNA interactions in the uncoating intermediate, whereas Shingler et al. (23) found that the 2-fold channel regulates genome release in the late stage of uncoating. Collectively, these findings provide a more complete understanding of EV71 uncoating. In summary, together with previous studies, the results presented here provide a more complete model for enterovirus uncoating. Under certain conditions, conformational changes that are associated with breathing become irreversible to initiate the uncoating process. Conformational changes in the junction region (such as the GH loops of VP1 and VP3), particularly the expulsion of the pocket factor that is buried inside the hydrophobic pocket of VP1, propagate into other regions of the capsid proteins, and the C-terminal region of VP1 shifts away from the VP3 surface to allow more dramatic conformational changes in VP3 to occur. These structural changes permit the opening of the 2-fold channel, leading to rearrangements and expansion at the junction and the 5-fold axes. The extreme N termini of VP1 and later VP4 are externalized through the capsid breaches at the protomer interface near the 2-fold axis and the junction, anchoring the amphipathic helices into the membrane and resulting in the forma-

EV71 Uncoating Revealed by X-Ray Structures

ACKNOWLEDGMENTS Research in R. Chen’s group was supported by the 100 Talents Program of the Chinese Academy of Sciences, a Shanghai Pu-Jiang Career Development Award (grant no. 09PJ1411400), the 973 Project (grant no. 2010CB912403), and a Frontier Research Award from the Shanghai Institutes for Biological Sciences-Chinese Academy of Sciences (grant no. 2008KIP105). Research in C.-F. Qin’s lab was supported by the Beijing Natural Science Foundation (grants no. 7122129 and no. 7112108), the National Science Foundation of China (grant no. 31270195), and the Beijing Nova Program of Science and Technology (grant no. 2010B041). We thank the staff at the Shanghai Synchrotron Radiation Facility (beamline BL17U1) for on-site assistance and the staff at Institute of Biophysics, Chinese Academy of Sciences, for cryo-EM data collection. We also thank Felix A. Rey (Institut Pasteur) for helpful suggestions and discussions.

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tion of the A-particle. The RNA genome, through its interaction with the N-terminal extensions of VP1, is thus poised at the bottom of the junction, awaiting the trigger for its release to complete the uncoating process. One of our future studies will be to further characterize the conformational changes at high resolution during the late stages of EV71 uncoating. During revision of our manuscript, Butan et al. presented a high-resolution cryo-EM structure of the 135S particle of type 1 poliovirus (49), which revealed externalization of the N-terminal regions of VP1 near the quasi-3-fold axis. Their study also provided evidence that uncoating is a succession of stepwise change. Thus, different stages of uncoating may have been sampled in their study and in our study, which explains different structural features. Nonetheless, their structural analysis on the 135S particle also supported our observation that the junction at the quasi-3fold axis and the GH loops in VP1 and VP3 play important roles during uncoating.

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Human enterovirus 71 uncoating captured at atomic resolution.

Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterizat...
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