Kinetic and Structural Analysis of Coxsackievirus B3 Receptor Interactions and Formation of the A-Particle

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Lindsey J. Organtini, Alexander M. Makhov, James F. Conway, Susan Hafenstein and Steven D. Carson J. Virol. 2014, 88(10):5755. DOI: 10.1128/JVI.00299-14. Published Ahead of Print 12 March 2014.

Kinetic and Structural Analysis of Coxsackievirus B3 Receptor Interactions and Formation of the A-Particle Lindsey J. Organtini,a Alexander M. Makhov,b James F. Conway,b Susan Hafenstein,a Steven D. Carsonc Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USAa; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USAb; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USAc

The coxsackievirus and adenovirus receptor (CAR) has been identified as the cellular receptor for group B coxsackieviruses, including serotype 3 (CVB3). CAR mediates infection by binding to CVB3 and catalyzing conformational changes in the virus that result in formation of the altered, noninfectious A-particle. Kinetic analyses show that the apparent first-order rate constant for the inactivation of CVB3 by soluble CAR (sCAR) at physiological temperatures varies nonlinearly with sCAR concentration. Cryo-electron microscopy (cryo-EM) reconstruction of the CVB3-CAR complex resulted in a 9.0-Å resolution map that was interpreted with the four available crystal structures of CAR, providing a consensus footprint for the receptor binding site. The analysis of the cryo-EM structure identifies important virus-receptor interactions that are conserved across picornavirus species. These conserved interactions map to variable antigenic sites or structurally conserved regions, suggesting a combination of evolutionary mechanisms for receptor site preservation. The CAR-catalyzed A-particle structure was solved to a 6.6-Å resolution and shows significant rearrangement of internal features and symmetric interactions with the RNA genome. IMPORTANCE

This report presents new information about receptor use by picornaviruses and highlights the importance of attaining at least an ⬃9-Å resolution for the interpretation of cryo-EM complex maps. The analysis of receptor binding elucidates two complementary mechanisms for preservation of the low-affinity (initial) interaction of the receptor and defines the kinetics of receptor-catalyzed conformational change to the A-particle.

C

oxsackieviruses are causative agents of myocarditis, meningitis, and pancreatitis and have also been implicated in the development of type 1 diabetes (1–4). There are six group B coxsackievirus serotypes (CVB1 to -6) within the Picornaviridae family. These viruses are icosahedral, nonenveloped, and approximately 300 Å in diameter, with a positive-sense single-stranded RNA genome (5). The crystal structures of CVB3-M and CVB3-RD strains are available in the Protein Data Bank (PDB) (PDB identifiers [ID] 1COV and 4GB3) (6, 7). The capsid has a T⫽1 (pseudo T⫽3) icosahedral architecture, is composed of four viral proteins (VP1 to -4), and has distinct topological features that are important to the function of the virus (Fig. 1A). Each 5-fold icosahedral symmetry axis is surrounded by a depression called the canyon, where a known receptor binding site is located. A hydrophobic pocket that contains a “pocket factor,” which is likely a lipid moiety, lies directly beneath the floor of the canyon. At the southern rim of the canyon, there is an elevated hypervariable region called the “puff” that is a known antigenic site (5, 8). Like other picornaviruses, CVB3 undergoes specific structural changes as it proceeds through the virus life cycle; these changes are critical for cellular entry and subsequent infection (9, 10). Initially, the virus must bind to a specific host receptor to gain entry into the cell and begin the infection. Previous studies with poliovirus (PV) and human rhinovirus (HRV) showed that there are two distinct modes for virus binding to receptor: a loweraffinity binding event that can be trapped at low temperatures and a higher-affinity binding event that is fleeting and occurs near physiological temperatures (11–13). In the picornavirus entry model, receptor binds into the virus canyon, pocket factor is lost, and the virus transitions to the altered particle, or A-particle (14,

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15). The A-particle is expanded compared to mature infectious virus, has lost VP4, has exposed the N termini of VP1, and typically has lost the ability to bind the receptor (16–18). Consequently, A-particle formed in solution has lost the ability to infect cells efficiently even though it has not yet lost the RNA genome. Previous studies have shown that mature infectious virus experiences transient structural rearrangements that are characterized by reversible partial exposure of VP4 and the N termini of VP1, a dynamic phenomenon referred to as “viral breathing” (19, 20). The purpose of viral breathing is not yet known, but it may be relevant to the high-affinity binding of the receptor (12, 21). Most picornaviruses use molecules in the immunoglobulin superfamily (IgSF) as their cell entry receptors (22–25). All CVBs use the coxsackievirus and adenovirus receptor (CAR) (26, 27), a cell adhesion molecule found in tight junctions (28). CAR is comprised of an extracellular domain folded into subdomains, D1 and D2, a single membrane-spanning sequence, and a C-terminal intracellular domain. Crystal structures for both human CAR D1 (PDB ID 1F5W and 1KAC) and murine CAR D1D2 (PDB ID 3MJ7 and 3JZ7) have been reported (29–32).

Received 30 January 2014 Accepted 5 March 2014 Published ahead of print 12 March 2014 Editor: K. Kirkegaard Address correspondence to Susan Hafenstein, [email protected], or Steven D. Carson, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00299-14

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ABSTRACT

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5-fold mesa, canyon, and puff. The compass points are used to describe the details of the canyon and the canyon rim. (B) Surface-rendered cryo-EM reconstruction of CVB3-CAR complex formed at 4°C. The map is colored radially according to the key and displayed at a sigma value of 1. One asymmetric unit is outlined in black. The cutaway view shows the interior of the reconstruction with a fully packaged genome (red). (C) Central section of CVB3-CAR complex showing strong density for CAR and labeled 2-, 3-, and 5-fold symmetry axes. (D) Fourier shell correlation versus spatial frequency of the icosahedrally averaged reconstruction indicate resolution of the map where the Fourier shell correlation curve crosses the value of 0.5 (dashed red line). (E) The D1D2 CAR structure (blue) fitted into the difference density (blue mesh) is displayed onto a surface-rendered, radially colored CVB3 map to show CAR binding to the puff region and north canyon rim.

A 22-Å resolution cryo-electron microscopy (cryo-EM) reconstruction of CVB3 complexed with CAR was previously interpreted with the CAR D1 structure (23). Here we present a 9.0-Å resolution cryo-EM map of CVB3-CAR complex interpreted with each of the four available crystal structures of CAR. This analysis provides a consensus footprint of the receptor binding site onto the virus. The receptor footprint maps to the puff region and the northern rim of the canyon, largely overlapping with known picornavirus antigenic epitopes. This mode of binding is conserved across picornavirus species. When incubated at 37°C, soluble CAR (sCAR) catalyzed virus inactivation with saturating kinetics, generating the CVB3 A-particle. The 6.6-Å cryo-EM map of the A-particle shows global expansion, loss of CAR, and extensive RNA reorganization. MATERIALS AND METHODS Virus growth and purification. CVB3 strain 28 (CVB3/28), used for the decay experiments, was passaged twice through HeLa cells after the initial transfection with the infectious cDNA plasmid (33) and isolated as described previously (34). The virus stock in 0.1 M NaCl was stored in aliquots at ⫺80°C. Infectious CVB3/28 was quantitated as 50% tissue culture infectious doses (TCID50)/ml using RDt3 cells (a rhabdomyosarcoma cell line, RD CCL-136, that expresses coxsackievirus and adenovirus receptor [CAR] with a truncated cytoplasmic domain) (35). For cryo-EM studies, CVB3/28 DNA was transfected into confluent HeLa cells using Lipofectamine 2000 (Invitrogen). The lysate was collected 36 to 48 h posttransfection and centrifuged. The supernatant was

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used as first-passage stock to expand inoculum. Virus was propagated in HeLa cells and purified as previously described (36). Briefly, infected cells were frozen and thawed three times, pelleted through a sucrose cushion, and purified by tartrate step gradient ultracentrifugation. Virus was collected and transferred to storage buffer using an Amicon centrifugal concentrator (Millipore) and four applications of 4 ml of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer with 100 mM NaCl, pH 6.0. Virus concentration and quality were assessed by measuring absorbance at 260, 280, and 310 nm. CAR production and purification. Soluble CAR (sCAR) was purified by immunoaffinity chromatography from medium conditioned by Raji cells transfected with pEF-ECAR (34). sCAR protein concentration was determined by bicinchoninic acid (BCA) assay and purity established by Coomassie-stained SDS-PAGE. These sCAR preparations have previously been shown to inhibit CVB3 infection of cell lines (34). Cryo-EM reconstruction. CVB3/28 was incubated 1 h at 4°C with excess sCAR in a 1:180 ratio of virus capsid to receptor. An aliquot was applied to a Quantifoil grid and vitrified in a 50-50 mixture of propane and ethane. The remaining virus-receptor mixture was incubated for 30 min at 37°C prior to vitrification as described for the 4°C sample. Micrographs for the two independent data sets were recorded using a FEI Tecnai TF-20 FEG electron microscope operating at 200 kV on Kodak SO-163 film. Micrographs were digitized using a Super Coolscan 9000 with a scan step size of 6.35 ␮m/pixel to generate a final pixel size of 1.25 Å/pixel. For each cryo-EM reconstruction, particles were selected using the program EMAN2 and defocus values for the micrographs were measured using the program ctffind3 (37). A random model initiated the icosahedral reconstruction. An amplitude contrast factor of 7% was applied, and phases

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FIG 1 CVB3 and virus-CAR complex maps. (A) The close-up view of the asymmetric unit of CVB3 shows the topography of the virus surface, including the

Receptor Catalyzes Formation of CVB A-Particle

TABLE 1 Cryo-EM statistics Incubation temp (°C)

No. of micrographs

Defocus range (␮m)

4 37

96 48

1.98–3.66 2.33–5.20

No. of particles selected

No. of particles used

Resolution (Å)

9,302 41,939

9,296 41,934

9.0 6.6

TABLE 2 Consensus CAR footprint onto virusa

a The consensus footprint includes all identified contacts. Virus VP1 and VP2 contact residues were predicted by fitting four crystal structures of CAR, listed with single-letter identifiers and aligned to show agreement.

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were flipped during data processing. The contrast transfer function (CTF) was fully corrected (38) during refinement. Final resolution was determined with maps with full CTF correction where the Fourier shell correlation was 0.5. Statistics for the cryo-EM reconstructions are presented in Table 1. The refined 9.0-Å CVB3-CAR complex reconstruction was mirrored across the x-y plane to “flip” the hand and generate a second map representing the stereoisomer. A calculated map was generated from the CVB3 crystal structure (PDB ID 1COV) (6) by mass weighting the atoms to simulate cryo-EM density and assigning a pixel size equal to 1 Å. Correct handedness was assessed by the quality of fit of the calculated map into the two cryo-EM density maps, by measuring correlation coefficients calculated about mean data value (cc) using the Chimera protocol Fit in Map (39). With the correlation about mean option, the correlation can range from ⫺1 to 1 as defined by Chimera. The absolute pixel size was determined by the fit that provided the highest correlation in the map of correct handedness. Difference map and structure fitting of the complex map. A difference map was made by subtracting the density of the calculated map from that of the scaled cryo-EM map of CVB3/28 complexed with sCAR (38). The CAR crystal structures (structural similarity quantified by root mean square deviation [RMSD] of 0.5 Å for the structure of D1) were fitted separately into the difference density and refined (40) (PDB ID 1F5W, 1KAC, 3MJ7, and 3JZ7) (29–32). The coordinates for each structure were superimposed to determine the similarity between structures after fitting, which raised the RMSD value between the structures to 1.2 Å (for D1). The crystal structure of CVB3 (PDB ID 1COV) was placed into density corresponding to virus, and the fit was refined (6, 40). Atoms that were ⱕ4.0 Å apart and had the correct geometry between the fitted crystal structures of CVB3 and CAR were identified as potential contacts (41). Buried surface was measured using the CCP4 program areaimol (41). Sequence alignment. All sequences were aligned using CLUSTAL W (42). Sequence sources included the following (codes in parentheses are GenBank accession numbers unless otherwise specified): poliovirus (PDB ID 2PLV), CVA21 (PDB ID 1Z7Z), HRV14 (PDB ID 1RUF), HRV16 (PDB ID 1C8M), CVB1 (AFM77200, AFM77201, and AFM77204), CVB2 (AFM77213, AFM77210, and AFM77211), CVB3 (AFD33642, AAA42931, PDB ID 1COV and 4GB3, and AFM77189), CVB4 (AAB22446, ABF19105, and AAB33885), CVB5 (AFA28144, AEX07779, and AAW71476), and CVB6 (AAF12719 and Q9QL88). For the IgSF receptors, the accession numbers are as follows: PVR, AAF69803.1; CAR, P78310.1; and ICAM, 1403404A. Virus decay analysis. Virus decay was assessed as previously reported (34). Culture medium (high-glucose Dulbecco modified Eagle medium [DMEM], 10% fetal bovine serum, 0.9 mM glutamine, 90 U/ml of penicillin, 90 ␮g/ml of streptomycin, and 67 ␮g/ml gentamicin; all GIBCO

brand, Life Technologies, Carlsbad, CA) was equilibrated overnight in a 37°C incubator with 6% CO2 and refrigerated (4°C) prior to addition of CVB3 and sCAR. The cooled medium was dispensed into sterile 1.5-ml tubes, and sCAR was added to give the final experimental concentration after addition of CVB3/28. Prior to placing the sample into the 37°C 6% CO2 incubator, an aliquot was removed and placed into a chilled 1.5-ml tube and frozen at ⫺80°C pending TCID50 assay. Subsequent samples were taken at timed intervals, placed into chilled 1.5-ml tubes, and stored at ⫺80°C until assayed for infectious virus. Total incubation time was limited to no more than 84 h. TCID50 analysis. For the TCID50 assay, samples were thawed and kept chilled. The medium for sample dilution was cold (4°C), and 96-well plates were kept on chilled aluminum blocks while the samples were serially diluted and until 20 ␮l from each dilution was placed into wells of a 96-well plate containing 100 ␮l of medium and RDt3 cells plated the previous day at 3,000 to 4,000 per well. The inoculated plates were placed into the 37°C, 6% CO2 incubator, and the microscopic cytopathic effect (CPE) was tallied every 24 h for 72 h. The final tally was used to calculate the TCID50/ml. Data points with ln(Vt/V0) (see below) values greater than ⫺10 were used in the analyses. sCAR carryover into TCID50 assays was apparent in the initial dilutions with high concentrations of sCAR but did not alter the TCID50 values obtained with sCAR up to 1,000 nM and CVB3/28 at ⱖ1.5 ⫻ 104 TCID50/ml. Each sample was evaluated twice, and the TCID50/ml results were averaged. Virus decay at each concentration of sCAR was analyzed on two separate occasions. Kinetic analysis. First-order rate constants were determined by linear regression of the equation ln(Vt/V0) ⫽ ⫺k · t ⫹ b, where V0 is the TCID50/ml at the time of first sampling, Vt is the TCID50/ml at subsequent time points, t is hours elapsed since the initial sample was taken, and k is the first-order rate constant (h⫺1). Slopes at each concentration of sCAR were averaged (weighted by the number of points in the data set); propagation of standard errors of regression was calculated per the work of P. R. Bevington (43). Nonzero values for the intercept, b, result from errors in determining V0. For graphical presentation, data sets were corrected to the equation ln(Vt/V0) ⫽ ⫺k · t. Six experiments were done with no sCAR, two of which were presented in the work of Carson et al. (34). The additional four experiments from this study were used to calculate the average and variance in the previous report. In experiments with sCAR present with the virus, starting concentrations of CVB3 ranged from 1.58 ⫻ 108 to 1.23 ⫻ 109 TCID50/ml (except for one experiment at 2.76 ⫻ 106 TCID50/ml). The lowest ratios of sCAR to number of binding sites (60 per virion) were 14.3 and 47.6 in the experiments with sCAR at 1 nM. In all other experiments, [sCAR]/[binding sites] ⬎ 200. For the analyses, [sCAR]free ⬃ [sCAR]total. The linear regression analyses of ln(Vt/V0) versus time were done with Pro-Stat v4.1 or PSI-Plot v8.1 for Windows (Poly Software International, Pearl River, NY). Nonlinear models were fit to the data using the Levenberg-Marquardt algorithm, or Powell’s method, in PSI-Plot. Structure fitting of the A-particle map. The structure of the A-particle of CVA16 (PDB ID 4JGY) (44) was fitted into the cryo-EM density map of the CVB3 A-particle using the program Situs (40), treating the protomer as a rigid body and allowing each of the 60 symmetry-related protomers freedom during the fitting process. CVB3 (PDB ID 4GB3) (7) was also fitted using the same protocol. The coordinates for the fitted

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view (A= and E=) shows that details of the capsid and receptor vary markedly with resolution of the map. With enhanced resolution, the line of demarcation between receptor and virus becomes clearer, structural details are defined, and the canyon appears unfilled.

CVA16 and CVB3 structures were superimposed and an RMSD was determined with Chimera (39). Accession numbers. Cryo-EM maps for the CVB3-sCAR complex and the A-particle are deposited in the EM data bank (www.emdatabank .org/) under accession numbers EMD-5927 and EMD-5928. Fitted structures of CAR PDB ID 1F5W, 1KAC, 3MJ7, and 3JZ7 are deposited in the PDB under ID 3J6L, 3J6M, 3J6O, and 3J6N, respectively.

RESULTS

Virus-receptor complex cryo-EM map. Cryo-electron microscopy (cryo-EM) and image analysis were used to reconstruct a 9.0-Å resolution density map of the CVB3-sCAR complex formed at 4°C (Fig. 1B). The magnitude of the CAR density was nearly equal to that of the capsid, indicating full occupancy of the receptor binding sites. The virus capsid appeared unchanged, which was verified when the crystal structure of CVB (PDB ID 1COV) was fitted into the corresponding density of the cryo-EM complex map (6). The two human CAR D1 structures (PDB ID 1F5W and 1KAC) (29, 30) and the two murine CAR D1D2 structures (PDB

ID 3MJ7 and 3JZ7) (31, 32) were fitted into the receptor density in separate experiments (40). Each fitted structure was used to interpret virus-receptor interactions, resulting in a consensus footprint of receptor on the virus (Table 2). The predicted CAR interactions mapped to the puff region of the virus (loop EF, VP2 residues 129 to 180) and to the north rim of the canyon (Fig. 1). However, there were no interactions with the canyon floor, as a separation between sCAR and the canyon could be distinguished in the 9.0-Å resolution map of the complex (Fig. 1E). The distinct separation between receptor and CVB3 was not discernible at 22 Å (Fig. 2), which was the resolution available in a previously reported map of the complex (23). Receptor footprint on the virus. The capsid protein sequence identity of group B coxsackieviruses is strongly conserved (88 to 100% sequence identity), and all CVBs use CAR as the receptor for cell entry (26, 27, 45). The sequence of the VP2 protein, which comprises the puff region, is somewhat less conserved among CVBs, ranging from 63 to 100% identity. Members of the B group of coxsackievirus were aligned to examine conservation of resi-

TABLE 3 Sequence conservation within the CVB3-CAR receptor footprinta

a The virus residues identified to be interacting with CAR were aligned with other members of the B group of coxsackieviruses and with PV, CVA21, and HRV16 (see Materials and Methods). A dash indicates each residue conserved with the CVB3 identity, whereas divergent sequences are identified with the single-letter code.

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FIG 2 Complex reconstruction rendered at various resolutions. (A to E) Maps were limited to resolutions of 22, 18, 15, 10, and 9 Å, respectively. The zoomed-in

Receptor Catalyzes Formation of CVB A-Particle

dV ⁄ dt ⫽ ⫺k1 · Vt ⫺ k2 · [VR · sCAR] · Vt

(1)

where Vt is total concentration of infectious virus at time, t, [VR · sCAR] represents the proportion of receptor binding sites occupied, k1 is the first-order rate constant without receptor, and k2 is the maximum achievable rate catalyzed by soluble mono-

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FIG 3 Comparison of virus-receptor contacts. Road maps of the virus surface are represented as a quilt of amino acids, shown as a projection with the icosahedral asymmetric unit indicated by the triangular boundary (71). For each virus—CVB3 (A), PV (B), HRV16 (C), and CVA21 (D)—the canyon is outlined in black; the receptor footprint is displayed in blue. The corresponding IgSF receptor is shown at the right, with virus contacts as red spheres. The footprints of receptor onto virus are as follows: CAR on CVB3 (A), PVR on PV (50) (B), ICAM on HVR16 (24) (C), and ICAM on CVA21 (25) (D).

meric receptor. In this equation, spontaneous decay and receptormediated decay can be concurrent (i.e., parallel reactions for which binding receptor does not prevent decay via the mechanism associated with k1). The observed rate constant is as follows: (2) kapp ⫽ k1 ⫹ k2 · [sCAR] ⁄ ([sCAR] ⫹ K) The hyperbolic relationship between kapp and [sCAR] (Fig. 5J) is described by equation 2, where k1 ⫽ 0.10 ⫾ 0.01 h⫺1 (mean ⫾ standard deviation), k2 ⫽ 1.81 ⫾ 0.07 h⫺1, and K ⫽ 50.4 ⫾ 8.9 nM (k2 and K are regression parameters ⫾ standard error). The same hyperbola is also described by a model in which the k1 and k2

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dues identified in the CAR binding site (Table 3). Ten of the 20 contact residues are identical across the CVB group. Nine of those residues are in VP1 and map to the north rim of the canyon. This result suggests that residues at the north canyon rim, many of which have been previously identified in poliovirus (PV)-receptor binding studies (46–48), make a major contribution to receptor binding (Table 3). The CAR contact residues that map to the puff are variable across the B group of coxsackieviruses and thus might be considered less likely to affect CAR binding; however, an Asp at VP2 residue 139 is conserved within the footprint across all the CVBs. VP2 residue 165, which is a known determinant for cardiovirulence and thought to be involved in receptor recognition (49), is also located within the puff portion of the CAR footprint. The CAR footprint on CVB3 was compared to the IgSF receptor footprints for PV (50), human rhinovirus (HRV) (24), and coxsackievirus A21 (CVA21) (25) (Fig. 3). Each of the four receptors interacts with its respective virus at the puff and north rim of the canyon; PV and HRV have additional receptor interactions at the south canyon rim. The common interactions with residues at the puff were unexpected because the puff is a region of high sequence variability, even among serotypes, and a known antigenic site. To investigate further, known antigenic sites for poliovirus (51, 52, 53, 54) and rhinovirus (55) were mapped on the aligned virus sequences (Fig. 4), identifying distinct clusters of receptor binding residues, three in VP1 and two in VP2 (Fig. 4A). Each of these clusters overlaps with a known antigenic epitope, except for cluster B, which consists of a group of conserved residues located at the north rim of the canyon (Fig. 4). Across the four virus species, there is conservation among the north canyon rim residues within the receptor binding footprints. HRV16 and CVA21 share more sequence-equivalent amino acids with CVB3 within the receptor footprint region than are found overall within the VP1 proteins or the VP2 puff regions (Tables 3 and 4). PV does not share this trait of increased amino acid conservation within the footprints; however, cluster C includes residues believed to be critical for PV to bind its receptor (Fig. 4) (46). The virus footprint on the receptor. A consensus footprint was also identified for the CVB3 interaction with the CAR protein. The nine CAR amino acids predicted to interact with the virus capsid map to the N-terminal region of CAR (Table 5; Fig. 3A). Receptor residues identified as critical for HRV binding map to the N-terminal region of ICAM-1 (57); however, these two canyon binding receptors appear to interact differently with their respective viruses (Fig. 3). Although the IgSF members share structural similarities (Fig. 3), they have low sequence identity (11 to 13%). Overall, there is more variation among the virus footprints on each respective receptor than is found in the receptor footprints on each virus (Fig. 3). Kinetic analysis. Picornavirus binding to IgSF receptors mediates conversion to A-particles (15–17). The conversion of CVB3 to inactive particles (A-particles), with or without receptor, proceeded at 37°C with first-order kinetics (Fig. 5A to I) as described by equation 1:

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Equivalent residues corresponding to antigenic epitopes were plotted as follows: poliovirus epitope II (yellow boxes) and NIm-II (pink line) (52); N-AgIA, N-AgIB, and N-AgII (blue boxes) (51, 53, 54); and NImIA, NImIB, and NImII (red boxes) (55). Residues interacting with the respective IgSF receptor are highlighted in yellow (24, 25, 50). Neutralization escape mutations critical to PVR binding are highlighted in cyan (46). The two overscores identify the sites of suppressing mutations that restore PVR binding to mutant PV (47). Clusters A to C map to the north canyon rim, an important PVR binding region (48). The aligned HRV14 sequence used to map the antigenic sites was removed for ease of viewing. (B to D) Road maps are shown as a surface projection with the canyon outlined (black) (71). (B) The CVB3 surface is displayed by radial height; color key indicates the lowest (red) to highest (blue) surface features. (C) Sequence-equivalent residues that comprise the antigenic epitopes are mapped onto an asymmetric unit of CVB3 with those originating from the PV and HRV antigenic sites shown in green and blue, respectively. Residues described in both N-Ag and NIm epitopes are colored cyan, with epitope II as yellow. (D) The CVB3 CAR footprint with the antigenic epitopes outlined in red defines the overlap between receptor binding and antigenicity. The CAR footprint is displayed in shades of blue representing the consensus of fitting the four different structures of CAR, from dark blue for all four in agreement to light blue for one contact (see Table 2).

TABLE 4 Percent sequence similarity between the viral capsid proteins of CVB3 and PV, HRV16, and CVA21a % Sequence similarity to region in CVB3

Virus

VP1

VP1 residues in contact with receptorb

PV CVA21 HRV16

44 39 37

36 73 64

VP2

VP2 residues in the puff regionc

VP2 residues in contact with receptor

55 56 52

14 18 8

13 25 25

a Sequence from the three different poliovirus serotypes (PDB ID 2PLV, 3EPC, and 3EPF) were also aligned. There are no differences in amino acid identity within the receptor footprint. PDB ID for the viruses listed in the table are as follows: 2PLV, 1C8M, and 1Z7Z for PV, HRV16, and CVA21, respectively. b The VP1 canyon north rim consists of VP1 residues equivalent to CVB3 VP1 residues 89 to 91, 140 to 155, and 211 to 217. c The puff consists of VP2 residues equivalent to CVB3 VP2 residues 129 to 180.

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mechanisms are mutually exclusive, with a k2 of 1.90 ⫾ 0.07 h⫺1. Use of [sCAR]a in equation 2 results in a marginally improved fit (F test, P ⬍ 0.05; using Powell’s method, k2 ⫽ 1.69 h⫺1, K ⫽ 0.06 ⫻ 10⫺9, and a ⫽ 1.39). Values of the empirical exponent, a, greater than 1 suggest positive cooperativity. Of these mathematical solutions, equation 2 is the simplest, and parallel reactions have been assumed in analyses of PV-receptor binding (12). Considering the solutions for all three mathematical models, k2 ranges from 1.69 to 1.90 h⫺1, so the maximum sCAR-catalyzed rate is 17 to 19 times higher than the spontaneous rate of virus inactivation at 37°C. The comparable k1 for poliovirus was reported as 0.5 h⫺1, and with 245 nM soluble poliovirus receptor, the kapp was 15.8 h⫺1 (58, 59). The half-maximal effect of sCAR on kapp was observed at

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FIG 4 Conservation of IgSF footprints and the overlap with known antigenic sites. (A) PV, CVA21, CVB3, HRV14, and HRV16 sequences were aligned (CLUSTAL W).

Receptor Catalyzes Formation of CVB A-Particle

TABLE 5 Consensus virus footprint onto CARa

50.4 nM, which is lower than either of the Kd (dissociation constant) values measured for poliovirus binding its receptor or the Kd reported for monomeric CAR binding to CVB3 (12, 14). The A-particle. Although heating native picornavirus has been

an acceptable surrogate for receptor binding to produce the Aparticle (9, 60, 61), for this structural study, mature CVB3 was incubated with excess sCAR at 37°C to produce the receptor-catalyzed A-particle. Global structural changes occurred during the

FIG 5 sCAR catalysis of CVB3 inactivation. Loss of infectious CVB3 with time at 37°C is first order in the absence of sCAR (slope ⫽ ⫺0.10 ⫾ 0.01 h⫺1) (A) and remains first order in the presence of sCAR from 1 nM (slope ⫽ ⫺0.12 ⫾ 0.01 h⫺1) (B) to 1,000 nM (slope ⫽ ⫺1.80 ⫾ 0.19 h⫺1) (I). Other concentrations of sCAR were 4.7 and 5 nM (slope ⫽ ⫺0.18 ⫾ 0.02 h⫺1) (C), 10 nM (slope ⫽ ⫺0.31 ⫾ 0.04 h⫺1) (D), 50 nM (slope ⫽ ⫺0.97 ⫾ 0.10 h⫺1) (E), 100 nM (slope ⫽ ⫺1.41 ⫾ 0.15 h⫺1) (F), 300 nM (slope ⫽ ⫺1.74 ⫾ 0.19 h⫺1) (G), and 500 nM (slope ⫽ ⫺1.64 ⫾ 0.15 h⫺1) (H). V0 is the concentration of infectious virus determined as TCID50/ml at the start of the 37°C incubation, and Vt is the concentration of infectious virus determined as TCID50/ml at each time sampled during the experiment. The observed first-order rate constant (kapp) varies with the concentration of sCAR (J) and can be described by the equation kapp ⫽ k1 ⫹ k2 · [sCAR]/([sCAR] ⫹ K), where k1 is the rate constant in the absence of sCAR.

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a Red font indicates residues that are disordered in the atomic structure. The structure of 1F5W was truncated (blue font) so that the N terminus was equivalent in length to that of sCAR. The yellow highlighted residues were identified from fitted CAR atoms no more than 4 Å from the fitted virus structure that had the proper geometry to be considered potential contacts. Red boxes outline the region of CAR that interacts with the virus puff and north canyon rim. The lower correlation coefficients (cc) for the D1-only structures were likely due to the lack of filled D2 density in the CAR difference density map. The four independently fitted CAR structures superimposed with an RMSD of 1.2. The X-ray structures used are from the two human D1 structures, PDB ID 1F5W (29) and 1KAC (30), and two murine D1D2 structures, PDB ID 3MJ7 (31) and 3JZ7 (32). Murine and human CARs share 90% sequence identity.

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asymmetric unit is outlined in black. (B) Cutaway view of the A-particle showing capsid separation from genomic RNA and structural changes that result from the 5% expansion of the capsid. (C) Close-up showing the columns of density connecting the RNA core to the capsid shell (white oval) and the open pores that formed at the 2-fold axes (white arrow). (D) Central section of CAR transitioned A-particle of CVB3 with symmetry axes labeled. (E) Fourier shell correlation versus spatial frequency of the icosahedrally averaged reconstruction. Resolution of the reconstruction is assessed where the Fourier shell correlation curve crosses the value of 0.5 (dotted red line).

transition to A-particle that resulted in the loss of CAR binding. The radial capsid expansion of ⬃5 Å was comparable to what has been reported for enterovirus 71 (EV71) (61), PV (62, 63), and HRV (21, 56). Notable structural changes include the formation of large open pores at each 2-fold axis of symmetry, a restructuring of the packaged RNA, and columns of density connecting the genomic RNA to the expanded capsid shell (Fig. 6). Similar density columns were reported for the EV71 A-particle structure that was formed by heat treatment (61). In the EV71 A-particle, the density columns were shown to contain the N terminus of VP1 (Fig. 6B and C). This location for VP1 was also found in a recent crystal structure of the A-particle of CVA16 (44). Thus, the A-particle formed by incubation of CVB3 with sCAR shares the important features known from studies of A-particles generated by heating other picornaviruses. The crystal structure of the CVA16 A-particle (PDB ID 4JGY) (44) (which shares 42% sequence identity with CVB3) fitted into our CVB3 A-particle cryo-EM density map with a correlation coefficient (cc) of 0.86. Since the protomer has been shown to move as a rigid body between expanded and nonexpanded forms of EV71 (64), the structure of CVB3 (PDB ID 4GB3) (7) was also fitted into the A-particle, resulting in a cc of 0.82. The experiment revealed that the 2-fold pore of the CVB3 A-particle is 20 by 31 Å in dimension, making it the largest in comparison to CVA16 and

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EV71 (44, 61). Current models propose that one of the open pores at the 2-fold axes may be selected for formation of a unique portal for release of RNA (60, 61, 64, 66). CVB3 residues in the CAR binding footprint (VP2 residues 136 to 140) align with CVA16 residues within a disordered loop (VP2 residues 137 to 141) (44). This suggests that the conformational changes that occur to form the A-particle may induce flexibility that precludes receptor binding. Furthermore, the location of the emerging VP1 N terminus of the fitted CVA16 A-particle crystal structure maps beneath the CAR binding site, suggesting that loss of CAR binding is coincident with egress of VP1. DISCUSSION

The three-dimensional structures and kinetics of the receptorcatalyzed conversion are consistent with a model that involves capsid breathing, a receptor-stabilized transition state, and conformational changes that occur on the path to genome delivery to the host. Icosahedral viruses are dynamic, with various vibrational modes; transition between antigenically distinct closed and open states of the capsid during a picornavirus breathing cycle has been documented both directly and indirectly (19, 20, 67, 68). The open capsid conformation that occurs during the virus breathing cycle is reversible and exposes segments of VP1 and VP4 that are internalized in the closed state of the capsid. Only the open-state capsid

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FIG 6 CAR-catalyzed CVB3 A-particle. (A) CVB3 A-particle is shown surface rendered and colored according to radial height (key beneath map). An

Receptor Catalyzes Formation of CVB A-Particle

ACKNOWLEDGMENTS This project is funded, in part, under a grant with the Pennsylvania Department of Health using Tobacco CURE Funds. The work was supported by NIH K22 grant A179271 (S.H.) and The Edna Ittner Foundation for Pediatric Research (S.D.C.).

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The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. L.J.O. and S.D.C. performed research, A.M.M. and J.F.C. collected cryo-EM data, L.J.O. and S.H. processed and analyzed cryo-EM data, S.D.C. processed and analyzed kinetic data, and L.J.O., S.D.C., and S.H. wrote the paper.

REFERENCES 1. Coppieters KT, Wiberg A, von Herrath MG. 2012. Viral infections and molecular mimicry in type 1 diabetes. APMIS 120:941–949. http://dx.doi .org/10.1111/apm.12011. 2. Moore M, Kaplan MH, McPhee J, Bregman DJ, Klein SW. 1984. Epidemiologic, clinical, and laboratory features of Coxsackie B1-B5 infections in the United States, 1970 –79. Public Health Rep. 99:515–522. 3. Tam PE. 2006. Coxsackievirus myocarditis: interplay between virus and host in the pathogenesis of heart disease. Viral Immunol. 19:133–146. http://dx.doi.org/10.1089/vim.2006.19.133. 4. Tracy S, Drescher KM. 2007. Coxsackievirus infections and NOD mice: relevant models of protection from, and induction of, type 1 diabetes. Ann. N. Y. Acad. Sci. 1103:143–151. http://dx.doi.org/10.1196/annals .1394.009. 5. Rossmann MG, Arnold E, Erickson JW, Frankenberger EA, Griffith JP, Hecht HJ, Johnson JE, Kamer G, Luo M, Mosser AG, Rueckert RR, Sherry B, Vriend G. 1985. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317:145–153. http://dx.doi.org/10.1038/317145a0. 6. Muckelbauer JK, Kremer M, Minor I, Diana G, Dutko FJ, Groarke J, Pevear DC, Rossmann MG. 1995. The structure of coxsackievirus B3 at 3.5 Å resolution. Structure 3:653– 667. http://dx.doi.org/10.1016/S0969 -2126(01)00201-5. 7. Yoder JD, Cifuente JO, Pan J, Bergelson JM, Hafenstein S. 2012. The crystal structure of a coxsackievirus B3-RD variant and a refined 9-angstrom cryo-electron microscopy reconstruction of the virus complexed with decay accelerating factor (DAF) provide a new footprint of DAF on the virus surface. J. Virol. 86:12571–12581. http://dx.doi.org/10.1128/JVI .01592-12. 8. Hogle JM, Chow M, Filman DJ. 1985. Three-dimensional structure of poliovirus at 2.9 Å resolution. Science 229:1358 –1365. http://dx.doi.org /10.1126/science.2994218. 9. Curry S, Chow M, Hogle JM. 1996. The poliovirus 135S particle is infectious. J. Virol. 70:7125–7131. 10. Dove AW, Racaniello VR. 1997. Cold-adapted poliovirus mutants bypass a postentry replication block. J. Virol. 71:4728 – 4735. 11. Casasnovas JM, Springer TA. 1995. Kinetics and thermodynamics of virus binding to receptor. Studies with rhinovirus, intercellular adhesion molecule-1 (ICAM-1), and surface plasmon resonance. J. Biol. Chem. 270:13216 –13224. 12. McDermott BM, Jr, Rux AH, Eisenberg RJ, Cohen GH, Racaniello VR. 2000. Two distinct binding affinities of poliovirus for its cellular receptor. J. Biol. Chem. 275:23089 –23096. http://dx.doi.org/10.1074/ jbc.M002146200. 13. Xing L, Tjarnlund K, Lindqvist B, Kaplan GG, Feigelstock D, Cheng RH, Casasnovas JM. 2000. Distinct cellular receptor interactions in poliovirus and rhinoviruses. EMBO J. 19:1207–1216. http://dx.doi.org/10 .1093/emboj/19.6.1207. 14. Goodfellow I, Evans DJ, Blow DM, Kerrigan D, Miners JS, Morgan BP, Spiller OB. 2005. Inhibition of coxsackie B virus infection by soluble forms of its receptors: binding affinities, altered particle formation, and competition with cellular receptors. J. Virol. 79:12016 –12024. http://dx .doi.org/10.1128/JVI.79.18.12016-12024.2005. 15. Milstone AM, Petrella J, Sanchez MD, Mahmud M, Whitbeck JC, Bergelson JM. 2005. Interaction with coxsackievirus and adenovirus receptor, but not with decay-accelerating factor (DAF), induces A-particle formation in a DAF-binding coxsackievirus B3 isolate. J. Virol. 79:655– 660. http://dx.doi.org/10.1128/JVI.79.1.655-660.2005. 16. Crowell RL, Philipson L. 1971. Specific alterations of coxsackievirus B3 eluted from HeLa cells. J. Virol. 8:509 –515. 17. De Sena J, Mandel B. 1977. Studies on the in vitro uncoating of poliovirus. II. Characteristics of the membrane-modified particle. Virology 78: 554 –566. 18. Fricks CE, Hogle JM. 1990. Cell-induced conformational change in

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is capable of binding the receptor with high affinity; therefore, the CVB3 open capsid is structurally distinct from the A-particle, which is incapable of high-affinity (or low-affinity) receptor binding. According to the working model, when CVB3 encounters an appropriate CAR-expressing host cell, virus in the closed conformational state can bind to CAR via the low-affinity binding site, and it is this closed-conformation capsid bound to sCAR that has been visualized (Fig. 1). The open conformation can occur in the absence of the receptor (20), and the open and closed states exist in a temperature-dependent equilibrium. It is unlikely that the lowaffinity interaction of sCAR with the virus in closed conformation induces the open conformation. However, this low-affinity interaction may allow the virus to acquire the receptor in proximity to the transient high-affinity site (presumably within the canyon) that becomes available in the open conformation. CAR occupation of the high-affinity binding site likely increases the energy required to return to the closed conformation relative to that required for conversion to the A-particle. Poliovirus receptor lowered the activation energy for virion conversion, whereas ICAM-1 was found to accelerate the decay of HRV without altering the activation energy (56, 58). For polymers with multiple ligand binding sites that have conformation-dependent affinities, the presence of ligand can shift the equilibrium toward the high-affinity conformation, often with cooperativity (65). In the current model, the CVB3 open state is an intermediate between the closed capsid conformation and the Aparticle. If this model is correct, the apparent first-order rate constant, k1⫹ k2 · [R/(R ⫹ K)] (equation 2; Fig. 5), changes with sCAR concentration because the receptor shifts the open-closed equilibrium toward the receptor-bound open intermediate. Since it is presumably the receptor-bound open intermediate that decays to A-particle, the conversion rate will be k · [open state], determined as (k1 ⫹ k2 · [R/(R ⫹ K)]) · Vt. The kapp approaches its maximum value as the equilibrium is pulled toward the maximum concentration of capsids in the open receptor-bound conformation. With this interpretation, K in equation 2 then does not represent the Kd but should correspond to the concentration of sCAR at which open and closed capsid conformations are present in equal concentrations. The receptor binding site available at low temperature is conserved within variable regions that are exposed on the virus surface, as has been described for foot-and-mouth disease virus (69). Use of the highly conserved north canyon rim residues is consistent with the canyon hypothesis that states viruses may preserve receptor binding in areas hidden from immune surveillance (70). The inaccessibility of the high-affinity binding site on the closed form of the virus indicates that this site is well hidden, perhaps deep in the canyon. The formation of the high-affinity binding site appears to be contingent on the conformational changes that take place when the capsid assumes the open conformation. The mechanisms to preserve receptor binding are conserved across species and suggest that a picornavirus takes advantage of multiple mechanisms for conserving two binding sites for the same receptor.

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19. 20.

21.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

5764

jvi.asm.org

37. 38.

39.

40. 41. 42.

43. 44.

45.

46. 47. 48.

49.

50.

51. 52. 53. 54. 55. 56.

Medof ME, Rossmann MG. 2007. Interaction of decay-accelerating factor with coxsackievirus B3. J. Virol. 81:12927–12935. http://dx.doi.org/10 .1128/JVI.00931-07. Mindell JA, Grigorieff N. 2003. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142:334 –347. http://dx.doi.org/10.1016/S1047-8477(03)00069-8. Yan X, Sinkovits RS, Baker TS. 2007. AUTO3DEM—an automated and high throughput program for image reconstruction of icosahedral particles. J. Struct. Biol. 157:73– 82. http://dx.doi.org/10.1016/j.jsb.2006.08 .007. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. 2004. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 24:1605–1612. http: //dx.doi.org/10.1002/jcc.20084. Wriggers W. 2010. Using Situs for the integration of multi-resolution structures. Biophys. Rev. 2:21–27. http://dx.doi.org/10.1007/s12551-009 -0026-3. Winn MD, Wilson KS. 2011. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67:235–242. http://dx .doi.org/10.1107/S0907444910045749. Thompson JC, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673– 4680. http://dx.doi.org/10.1093/nar /22.22.4673. Bevington PR. 1969. Data reduction and error analysis for the physical sciences. McGraw-Hill, New York, NY. Ren J, Wang X, Hu Z, Gao Q, Sun Y, Li X, Porta C, Walter TS, Gilbert RJ, Zhao Y, Axford D, Williams M, McAuley K, Rowlands DJ, Yin W, Wang J, Stuart DI, Rao Z, Fry EE. 2013. Picornavirus uncoating intermediate captured in atomic detail. Nat. Commun. 4:1929. http://dx.doi .org/10.1038/ncomms2889. Tomko RP, Xu R, Philipson L. 1997. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc. Natl. Acad. Sci. U. S. A. 94:3352–3356. http://dx.doi .org/10.1073/pnas.94.7.3352. Colston E, Racaniello VR. 1994. Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor. EMBO J. 13:5855–5862. Colston EM, Racaniello VR. 1995. Poliovirus variants selected on mutant receptor-expressing cells identify capsid residues that expand receptor recognition. J. Virol. 69:4823– 4829. Harber J, Bernhardt G, Lu HH, Sgro JY, Wimmer E. 1995. Canyon rim residues, including antigenic determinants, modulate serotype-specific binding of polioviruses to mutants of the poliovirus receptor. Virology 214:559 –570. http://dx.doi.org/10.1006/viro.1995.0067. Knowlton KU, Jeon ES, Berkley N, Wessely R, Huber S. 1996. A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3. J. Virol. 70:7811–7818. Zhang P, Mueller S, Morais MC, Bator CM, Bowman VD, Hafenstein S, Wimmer E, Rossmann MG. 2008. Crystal structure of CD155 and electron microscopic studies of its complexes with polioviruses. Proc. Natl. Acad. Sci. U. S. A. 105:18284 –18289. Epub 12008 Nov 18214. http: //dx.doi.org/10.1073/pnas.0807848105. Murdin AD, Wimmer E. 1989. Construction of a poliovirus type 1/type 2 antigenic hybrid by manipulation of neutralization antigenic site II. J. Virol. 63:5251–5257. Minor PD, Ferguson M, Evans DM, Almond JW, Icenogle JP. 1986. Antigenic structure of polioviruses of serotypes 1, 2 and 3. J. Gen. Virol. 67(Part 7):1283–1291. Fiore L, Ridolfi B, Genovese D, Buttinelli G, Lucioli S, Lahm A, Ruggeri FM. 1997. Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies. J. Virol. 71:6905– 6912. Page GS, Mosser AG, Hogle JM, Filman DJ, Rueckert RR, Chow M. 1988. Three-dimensional structure of poliovirus serotype 1 neutralizing determinants. J. Virol. 62:1781–1794. Sherry B, Mosser AG, Colonno RJ, Rueckert RR. 1986. Use of monoclonal antibodies to identify four neutralization immunogens on a common cold picornavirus, human rhinovirus 14. J. Virol. 57:246 –257. Casasnovas JM, Springer TA. 1994. Pathway of rhinovirus disruption by soluble intercellular adhesion molecule 1 (ICAM-1): an intermediate in which ICAM-1 is bound and RNA is released. J. Virol. 68:5882–5889.

Journal of Virology

Downloaded from http://jvi.asm.org/ on April 28, 2014 by ONDOKUZ MAYIS UNIVERSITESI

22.

poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 64:1934 –1945. Lewis JK, Bothner B, Smith TJ, Siuzdak G. 1998. Antiviral agent blocks breathing of the common cold virus. Proc. Natl. Acad. Sci. U. S. A. 95: 6774 – 6778. http://dx.doi.org/10.1073/pnas.95.12.6774. Li Q, Yafal AG, Lee YM, Hogle J, Chow M. 1994. Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature. J. Virol. 68:3965–3970. Xing L, Casasnovas JM, Cheng RH. 2003. Structural analysis of human rhinovirus complexed with ICAM-1 reveals the dynamics of receptormediated virus uncoating. J. Virol. 77:6101– 6107. http://dx.doi.org/10 .1128/JVI.77.11.6101-6107.2003. Belnap DM, McDermott BM, Jr, Filman DJ, Cheng N, Trus BL, Zuccola HJ, Racaniello VR, Hogle JM, Steven AC. 2000. Three-dimensional structure of poliovirus receptor bound to poliovirus. Proc. Natl. Acad. Sci. U. S. A. 97:73–78. http://dx.doi.org/10.1073/pnas.97.1.73. He Y, Chipman PR, Howitt J, Bator CM, Whitt MA, Baker TS, Kuhn RJ, Anderson CW, Freimuth P, Rossmann MG. 2001. Interaction of coxsackievirus B3 with the full-length coxsackievirus-adenovirus receptor. Nat. Struct. Biol. 8:874 – 878. http://dx.doi.org/10.1038/nsb1001-874. Kolatkar PR, Bella J, Olson NH, Bator CM, Baker TS, Rossmann MG. 1999. Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor. EMBO J. 18:6249 – 6259. http://dx.doi.org /10.1093/emboj/18.22.6249. Xiao C, Bator-Kelly CM, Rieder E, Chipman PR, Craig A, Kuhn RJ, Wimmer E, Rossmann MG. 2005. The crystal structure of coxsackievirus A21 and its interaction with ICAM-1. Structure 13:1019 –1033. http://dx .doi.org/10.1016/j.str.2005.04.011. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas AJ, Hong JS, Horwitz MS, Crowell RL, Finberg RW. 1997. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320 –1323. http://dx.doi.org/10.1126/science.275.5304.1320. Martino TA, Petric M, Weingartl H, Bergelson JM, Opavsky MA, Richardson CD, Modlin JF, Finberg RW, Kain KC, Willis N, Gauntt CJ, Liu PP. 2000. The coxsackie-adenovirus receptor (CAR) is used by reference strains and clinical isolates representing all six serotypes of coxsackievirus group B and by swine vesicular disease virus. Virology 271:99 –108. http://dx.doi.org/10.1006/viro.2000.0324. Cohen CJ, Shieh JTC, Pickles RJ, Okegawa T, Hsieh J-T, Bergelson JM. 2001. The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction. Proc. Natl. Acad. Sci. U. S. A. 98:15191– 15196. van Raaij MJ, Chouin E, van der Zandt H, Bergelson JM, Cusack S. 2000. Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution. Structure 8:1147–1155. http://dx.doi.org/10 .1016/S0969-2126(00)00528-1. Bewley MC, Springer K, Zhang YB, Freimuth P, Flanagan JM. 1999. Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR. Science 286:1579 –1583. http://dx.doi.org/10.1126 /science.286.5444.1579. Verdino P, Witherden DA, Havran WL, Wilson IA. 2010. The molecular interaction of CAR and JAML recruits the central cell signal transducer PI3K. Science 329:1210 –1214. http://dx.doi.org/10.1126 /science.1187996. Patzke C, Max KE, Behlke J, Schreiber J, Schmidt H, Dorner AA, Kroger S, Henning M, Otto A, Heinemann U, Rathjen FG. 2010. The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic interactions on neural cells. J. Neurosci. 30:2897–2910. http://dx .doi.org/10.1523/JNEUROSCI.5725-09.2010. Tu Z, Chapman NM, Hufnagel G, Tracy S, Romero JR, Barry WH, Zhao L, Currey K, Shapiro B. 1995. The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5= nontranslated region. J. Virol. 69:4607– 4618. Carson SD, Chapman NM, Hafenstein S, Tracy S. 2011. Variations of coxsackievirus B3 capsid primary structure, ligands, and stability are selected for in a coxsackievirus and adenovirus receptor-limited environment. J. Virol. 85:3306 –3314. http://dx.doi.org/10.1128/JVI.01827-10. Cunningham KA, Chapman NM, Carson SD. 2003. Caspase-3 activation and ERK phosphorylation during CVB3 infection of cells: influence of the coxsackievirus and adenovirus receptor and engineered variants. Virus Res. 92:179 –186. http://dx.doi.org/10.1016/S0168-1702(03)00044-3. Hafenstein S, Bowman VD, Chipman PR, Bator Kelly CM, Lin F,

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64. Wang X, Peng W, Ren J, Hu Z, Xu J, Lou Z, Li X, Yin W, Shen X, Porta C, Walter TS, Evans G, Axford D, Owen R, Rowlands DJ, Wang J, Stuart DI, Fry EE, Rao Z. 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 19:424 – 429. http://dx.doi.org/10.1038/nsmb.2255. 65. Hermans J, Lentz B. 2013. Equilibria and kinetics of biological macromolecules. John Wiley and Sons, Hoboken, NJ. 66. Bostina M, Levy H, Filman DJ, Hogle JM. 2011. Poliovirus RNA is released from the capsid near a twofold symmetry axis. J. Virol. 85:776 – 783. http://dx.doi.org/10.1128/JVI.00531-10. 67. Dykeman EC, Sankey OF. 2010. Normal mode analysis and applications in biological physics. J. Phys Condens. Matter 22:423202. http://dx.doi .org/10.1088/0953-8984/22/42/423202. 68. Lin J, Cheng N, Chow M, Filman DJ, Steven AC, Hogle JM, Belnap DM. 2011. An externalized polypeptide partitions between two distinct sites on genome-released poliovirus particles. J. Virol. 85:9974 –9983. http://dx .doi.org/10.1128/JVI.05013-11. 69. Jackson T, Sharma A, Ghazaleh RA, Blakemore WE, Ellard FM, Simmons DL, Newman JW, Stuart DI, King AM. 1997. Arginine-glycineaspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin alpha(v)beta3 in vitro. J. Virol. 71:8357– 8361. 70. Rossmann MG. 1989. The canyon hypothesis. Hiding the host cell receptor attachment site on a viral surface from immune surveillance. J. Biol. Chem. 264:14587–14590. 71. Xiao C, Rossmann MG. 2007. Interpretation of electron density with stereographic roadmap projections. J. Struct. Biol. 158:182–187. http://dx .doi.org/10.1016/j.jsb.2006.10.013.

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57. Register RB, Uncapher CR, Naylor AM, Lineberger DW, Colonno RJ. 1991. Human-murine chimeras of ICAM-1 identify amino acid residues critical for rhinovirus and antibody binding. J. Virol. 65:6589 – 6596. 58. Tsang SK, McDermott BM, Racaniello VR, Hogle JM. 2001. Kinetic analysis of the effect of poliovirus receptor on viral uncoating: the receptor as a catalyst. J. Virol. 75:4984 – 4989. http://dx.doi.org/10.1128/JVI.75.11 .4984-4989.2001. 59. Tsang SK, Danthi P, Chow M, Hogle JM. 2000. Stabilization of poliovirus by capsid-binding antiviral drugs is due to entropic effects. J. Mol. Biol. 296:335–340. http://dx.doi.org/10.1006/jmbi.1999.3483. 60. Levy HC, Bostina M, Filman DJ, Hogle JM. 2010. Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy. J. Virol. 84:4426 – 4441. http://dx.doi .org/10.1128/JVI.02393-09. 61. Shingler KL, Yoder JL, Carnegie MS, Ashley RE, Makhov AM, Conway JF, Hafenstein S. 2013. The enterovirus 71 A-particle forms a gateway to allow genome release: a cryoEM study of picornavirus uncoating. PLoS Pathog. 9:e1003240. http://dx.doi.org/10.1371/journal.ppat.1003240. 62. Belnap DM, Filman DJ, Trus BL, Cheng N, Booy FP, Conway JF, Curry S, Hiremath CN, Tsang SK, Steven AC, Hogle JM. 2000. Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus. J. Virol. 74:1342–1354. http://dx.doi.org/10.1128/JVI.74.3 .1342-1354.2000. 63. Bubeck D, Filman DJ, Cheng N, Steven AC, Hogle JM, Belnap DM. 2005. The structure of the poliovirus 135S cell entry intermediate at 10angstrom resolution reveals the location of an externalized polypeptide that binds to membranes. J. Virol. 79:7745–7755. http://dx.doi.org/10 .1128/JVI.79.12.7745-7755.2005.