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J Virol. 2012 December; 86(23): 12571–12581.
PMCID: PMC3497627

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


The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.


Coxsackieviruses are significant human pathogens that cause myocarditis, meningitis, and pancreatitis and have been implicated in the development of juvenile diabetes (58, 6064). Virulence determinants have been described throughout the genome (19, 20, 30, 51, 65), including the P1 region, which encodes the structural proteins (7, 10, 13, 25, 48, 49, 56). The capsid surface presents a topology of structural motifs that largely dictate receptor recognition and usage, directly affecting tropism and pathogenicity.

Group B coxsackieviruses (CVBs) belong to the genus Enterovirus of the family Picornaviridae. Picornaviruses are nonenveloped, positive-sense, single-stranded-RNA animal viruses with a capsid comprised of 60 protomers arranged to form an icosahedral shell ~300 Å in diameter with T=1 (pseudo-T=3) symmetry (ICTV classification) (8). In mature capsids, each protomer contains four structural proteins, VP-1, -2, -3, and -4. Structural studies have shown that capsids share common features, including a depression around the icosahedral 5-fold symmetry axes (called the “canyon”) and a hydrophobic cavity located underneath the floor of the canyon (called the “pocket”) (52). Biochemical and structural evidence indicates that the ligand within the pocket is a fatty acid (26, 55). For many picornaviruses, a receptor binds into the canyon and dislodges this “pocket factor,” initiating conformational changes that lead to the formation of “A particles” and the subsequent uncoating of the virion (1, 12, 33, 39, 66). The major CVB receptor, the coxsackievirus-adenovirus receptor (CAR), binds within the CVB3 canyon (37) and causes the formation of A particles (16, 35).

A number of CVB isolates bind a second receptor, decay-accelerating factor (DAF) (CD55), a molecule that also serves as a receptor for many other enteroviruses (2, 3, 18, 23, 24, 43, 45). DAF, which is expressed on virtually all cell surfaces, acts to protect cells from lysis by complement (34), by binding and degrading C3/C5 convertases, the central amplification enzymes of the complement cascade (5, 27, 28, 34). DAF is a 70-kDa molecule that consists of four short consensus repeats (SCRs), each about 60 amino acids long and folded into a β-structure stabilized by disulfide bridges (32, 67, 68). The 4 SCRs form a relatively rigid, bent rod with dimensions of 160 by 50 by 30 Å (32) that extends approximately 180 Å above the cell surface, supported by a serine- and threonine-rich O-glycosylated stalk and fixed to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (32). Previous structural studies of DAF complexed with echovirus 7 (E7) (22, 45) and echovirus 12 (4, 43) indicated that these viruses interact with DAF in a similar manner, with DAF oriented similarly in relation to the virus surface. In these structures, DAF does not insert into the canyon, which suggests a possible explanation for why interactions with DAF do not appear to cause the conversion of native virions to A particles (16, 35, 46). However, a variety of evidence suggests that different DAF-binding viruses interact primarily with different SCR domains (3, 11, 23, 47, 54), suggesting that several modes of virus-DAF interactions are possible.

The first DAF-binding CVB3 isolate identified was obtained by the passage of a prototype strain, CVB3 strain Nancy (CVB3-Nancy), on rhabdomyosarcoma (RD) cells (50); the RD-adapted virus, CVB3-RD, binds DAF, whereas the original CVB3-Nancy does not (3). Previously, a 14-Å-resolution cryo-electron microscopy (cryo-EM) reconstruction of the CVB3-RD isolate complexed with DAF was interpreted with the crystal structure of a non-DAF-binding CVB3 strain (CVB3 strain M [CVB3-M] [Protein Data Bank {PDB} accession number 1COV]) (18, 37). This analysis showed clashes between DAF and the virus loops at the “puff” region of VP2 (loop EF; residues 2129 to 2180) (18, 37). Here, we present the crystal structure of the CVB3-RD variant virus solved to 2.74 Å and a cryo-EM reconstruction of CVB3-RD bound to DAF refined to 9-Å resolution. The higher-resolution cryo-EM map of the virus-receptor complex made it clear that in our previous reconstruction (18), we had incorrectly assigned the handedness of the virus, resulting in an incorrect interpretation of the contact sites between CVB3 and DAF. In the new structure, each DAF molecule was found to link two adjacent protomers of the virus capsid in a mode of DAF binding different from that described previously for echoviruses 7 and 12 (4, 22, 43, 45). Here, we present evidence that the new DAF footprint on the surface of CVB3-RD includes viral residues recently reported to be critical for DAF binding (41). More than 75% of the DAF interactions involve SCR2, which is known from previous SCR deletion studies to be essential for DAF binding by CVB3-RD (3).


Virus growth and purification.

CVB3-RD (GenBank accession number JN048469) (41) was propagated in rhabdomyosarcoma (RD) cells and purified as described previously (18). Briefly, infected cells were frozen and thawed three times, and virus was concentrated by pelleting through sucrose and purified by tartrate step gradient ultracentrifugation. The virus bands were collected and exchanged into morpholineethanesulfonic acid (MES) buffer (pH 6.0), to estimate the virus concentration and quality by measuring the absorbance at 260, 280, and 310 nm.

Crystallization and data collection.

CVB3-RD crystals were obtained by using the sitting-drop vapor diffusion method at room temperature. Drops consisted of 10 μl of CVB3-RD at a concentration of 5 mg ml−1 in 50 mM MES (pH 6.0) with 0.75 M NaCl. The well contained 1 ml of 2 M (NH4)2SO4. Crystals were frozen and transported to BIOCARS beamline 14-BM-C at APS, Argonne National Laboratory, Chicago, IL. Data were collected with an oscillation of 0.2° on an ADSC Quantum 315 instrument with a detector distance of 450 cm.

Data processing and structure solution.

The data were indexed, processed, scaled, and reduced by using the HKL-2000 package (40). The crystal was determined to be in the rhombohedral crystal system and space group R32 with unit cell parameters of an a equal to 296.6385 Å and a c equal to 813.2151 Å, in the hexagonal setting. The data collection and processing statistics are given in Table 1. The orientation of the virion in the crystal unit cell was determined by using the self-rotation function in the General Lock Rotation Function (GLRF) program with kappa values equal to 72°, 120°, and 180° in the search for icosahedral 2-, 3-, and 5-fold symmetry axes, respectively, using data in the 10.0- to 5.0-Å-resolution range (59). Since the crystallographic 2- and 3-fold symmetry operators were shown to be coincident with icosahedral symmetry operators, each crystallographic asymmetric unit contained 10 protomers. To determine if these data were pseudo-R32, as seen with the CVB3 M strain (PDB accession number 1COV) (57), the GLRF program was used to examine the data in the 5.0- to 3.0-Å-resolution range. No peak splitting was observed, indicating that this crystal was in the R32 space group.

Table 1
Scaling and refinement statistics

The diffraction data were phased by using the molecular replacement method in the AMoRe package (38). The orientation of the CVB3 10-mer in the crystal unit cell was determined by a cross-rotation search, and its position was determined by a translation search, using atomic coordinates for 10 CVB3 M protomers from a previously determined crystal structure (PDB accession number 1COV) (37). This 10-mer model was oriented and positioned into the crystal unit cell on the basis of the output rotation angles and translation solutions to calculate a set of initial phases. These phases were improved by refinement using the Crystallography and NMR (nuclear magnetic resonance) System (CNS) package (6). CNS programs for simulated annealing, energy minimization, atomic position, and temperature factor refinement were used with the application of strict noncrystallographic symmetry (NCS) operators. During the first round of refinement, the residues that were different in the RD strain relative to the parental virus were mutated to their proper identity. After each refinement cycle, a single cycle of electron density Fourier map (2FoFc and FoFc, where Fo indicates the observed structure factors and Fc indicates the structures calculated from the model) averaging was carried out in CNS, with strict NCS operators, using the experimentally measured amplitudes and the improved phases. The program Coot was used for model building into averaged electron density maps between cycles of the refinement and averaging procedures (14).

Cryo-EM reconstruction.

CVB3-RD was incubated with full-length DAF molecules, as reported previously (18). Micrographs of the vitrified virus-receptor complexes used in this study were reported previously (18). Micrographs were digitized with a Zeiss Phodis microdensitometer at 7-μm intervals, averaged in boxes of 2 pixels by 2 pixels, providing a final pixel size of 3.11 Å. Using Robem, 3,011 particles were isolated from 36 of the highest-quality micrographs, with a selection area sized to 171 by 171 pixels, and preprocessed by using autopp in order to remove blemishes, linearize, normalize, and apodize (71). To correct for contrast transfer function, defocus and astigmatism values were assessed over the digitized images by using ctffind3 (36), obtaining a range of 1.04 to 5.05 μm for the defocus values and a ratio for the major/minor axis average of 1.05 ± 0.04. The reconstruction was initiated by using a random model (70). An amplitude contrast factor of 7% was applied during the reconstruction. Reconstructions initiated with several different starting models converged to the same result with auto3dem imposing icosahedral symmetry. All resulting maps had a resolution higher than 10 Å, estimated at a Fourier shell correlation (FSC) value of 0.5. Using a final delta angle of 0.25° and applying a temperature factor, B, of 400, the map improved to a 9-Å resolution and was used for subsequent analyses.

Assessment of cryo-EM map handedness.

Using the final 9-Å CVB3-DAF complex reconstruction, a map representing the stereoisomer was generated by mirroring the complex map across the x-y plane to “flip” the hand. Using Chimera, an 8-Å-resolution map calculated from the new crystal structure of CVB3-RD was fitted separately into each map, rendered at a density threshold of 1 σ (15, 42). Correct handedness was assessed by the quality of the fit, by measuring correlation coefficients (CCs) calculated about the mean data value using the Chimera protocol Fit in Map. With the correlation-about-mean option, the correlation can range from −1 to 1, as defined by Chimera. In addition, the newly solved crystal structure of the RD variant virus was fitted into the flipped and unflipped complex maps with the DAF density masked out by using the segger subroutine of Chimera to reveal the surface features of the virus (15, 42) (data not shown). This segmentation approach separated densities in the map according to local minima by using a watershed algorithm (44). In both analyses, the map in which the hand had been flipped relative to the previously reported reconstruction of the complex of CVB3-RD and DAF, EMD-1412 (18), revealed a better agreement with the virus crystal structure.

Structure fitting.

Using EMfit, the exact pixel size (2.94 Å) of the cryo-EM reconstruction was determined by scaling to a map calculated from the crystal structure (31, 53). A map calculated to 9 Å was also used to obtain a difference map by subtracting it from the virus-receptor complex map (53). The fitting of the eight different crystal forms of DAF SCR1 to SCR4 (PDB accession numbers 1OJV, 1OJW, and 1OJY) (32) into the difference map density was carried out by using Chimera. Briefly, the average map value at each fit atom position is maximized, and for each atom within the bounds of the reference map, the map value is found by trilinear interpolation from the eight corners of the enclosing data grid cell. Atoms outside the bounds of the map are not used for computing averages, and the best fit was determined by the highest relative average map value (42) (Table 2). Using the best fit of DAF, the buried surface between DAF and CVB3-RD was calculated by using the CCP4 program Surface, with the probe set to a 3.5-Å diameter (9, 29). Residues in the virus-receptor interface were identified by using the CCP4 program Contact (69).

Table 2
Statistics for fitting of the different crystal forms of DAFa

Protein structure accession numbers.

PDB accession numbers for data bank deposition are as follows: 4GB3 for the CVB3-RD crystal structure, 5475 for the cryo-EM reconstruction of CVB3-RD complexed with DAF (SCR1-SCR2-SCR3-SCR4), and 3J24 for the structure of DAF fitted into the cryo-EM reconstructions of the complex.


CVB3-RD crystal structure.

Frozen CVB3-RD crystals diffracted X rays coherently to a 2.5-Å interplanar spacing. The structure of the capsid protein was determined to 2.74 Å (Fig. 1A). As reported previously for the structure of CVB3-M (PDB accession number 1COV) (37), residues 2001 to 2007 and 4012 to 4024 were disordered and could not be built. However, four additional residues could be placed into the N terminus of VP1; whereas the CVB3-M structure is missing residues 1001 to 1012, the RD structure begins at residue 1008. The density corresponding to the pocket factor that is characteristic of picornaviruses is clearly visible. This ligand is modeled with a 16-carbon fatty acid palmitate molecule in the same orientation as that described previously for CVB3-M (37). A density corresponding to a calcium ion seen in the CVB3-M structure is present at the 3-fold icosahedral symmetry axes, although the peak is not as strong as what was reported previously for the structure of the M strain (37). CVB3-RD VP1, -2, and -3 adopt the canonical β-sandwich motif seen in many icosahedral viruses, with eight antiparallel β-strands forming two sheets, BIDG and CHEF (Fig. 1A) (52). VP3 also contains a β-cylinder structure comprised of the symmetry-related N termini forming a pore, located on the icosahedral 5-fold axis, that is highly conserved in all picornavirus structures.

Fig 1
(A) Ribbon diagram of the CVB3-RD crystal structure, with the VP1 to VP4 proteins in blue, green, red, and yellow, respectively, and the pocket factor surface rendered in orange. The RMSD of the CVB3-RD structure relative to that reported under PDB accession ...

Of the six total amino acid differences between parental strain Nancy and the variant RD strains, four map to the surface of CVB3-RD. Of the remaining two, residue 1092 (L to I) maps to the top of the pocket and is in contact with the pocket factor, and residue 2013 (A to V) maps to the protomer-protomer interface at the icosahedral 3-fold axes. In all six locations, the side chains of the substitutions could be placed without ambiguity (Fig. 1B and andC).C). There were no significant changes in local or global structure between CVB3-M and CVB3-RD. The RD crystal structure is nearly identical to the structure of CVB3-M, with an overall root mean square deviation (RMSD) of 0.4 Å or lower for each VP (Fig. 1).

Cryo-EM reconstruction of the CVB3-RD–DAF complex.

The cryo-EM reconstruction of the CVB3-RD variant complexed with all four SCRs of DAF reached a 9.0-Å resolution, calculated at an FSC value of 0.5 (Fig. 2A and andC).C). The resolution provided detailed surface features, making it possible to assign the absolute hand of the map by comparing the directionality of structural features with the fitted crystal structure of the virus (Fig. 3). In order to quantify the fit and confirm the correct hand, a calculated map made from the newly solved crystal structure of the RD variant virus was fitted into the cryo-EM complex map, generating a correlation coefficient (CC) of 0.6 (15, 42) (Fig. 3). The same fitting routine was done by using a previously reported map of the opposite hand (18), which generated a significantly worse score of 0.14 (42), thus confirming the correct handedness of the cryo-EM structure presented here (Fig. 2 and and3).The3).The magnitude of the receptor density relative to that of the capsid was nearly equal (Fig. 2B), indicating that the complex is close to full occupancy of the 60 DAF-binding sites. The radial distribution of the density differentiated into three regions, the RNA core, the virus capsid, and the bound receptor (Fig. 2D).

Fig 2
Cryo-EM structure of the complex of CVB3 strain RD with DAF SCR1 to SCR4. (A) Radially colored, surface-rendered cryo-EM reconstruction of the map density, displayed at a threshold value of 0.6. The strong DAF density lies over the virus surface as a ...
Fig 3
Determination of the handedness of the cryo-EM reconstruction. (A) An 8-Å-resolution map calculated from the crystal structure (white) was used to measure the correlation (CC) of both possible hands of the cryo-EM maps (42). The calculated map ...

CVB3-RD interactions with DAF.

To construct a pseudoatomic model of the complex, the CVB3-RD crystal structure was placed into the cryo-EM density map of the complex by superimposing the icosahedral symmetry elements. The DAF crystal structure was placed into the corresponding DAF density by using all nonhydrogen atoms (see Materials and Methods). The general shape of DAF, specifically the distinct kink between SCR1 and SCR2, readily determined the overall orientation. Eight monomers from three different DAF crystal structures (PDB accession numbers 1OJV, 1OJW, and 1OJY) (32) were fitted independently into the difference map. Molecule B from the structure reported under PDB accession number 1OJW (32) fit best into the DAF density, quantified by using relative average map value scores (see Materials and Methods) (Table 2 and Fig. 4A) (42). Atoms that potentially participate in interactions between CVB3-RD and DAF were identified by determining which atoms on the virus and receptor surfaces were within 3.6 Å of one another and had the proper geometry to form bonds (Table 3). The identified virus surface residues define a footprint of DAF on the capsid. There is 1,081 Å2 of total buried surface area in the interface of the virus-receptor complex (Table 4). Each bound DAF molecule links two adjacent viral capsid protomers by binding to the northern side of one puff (Fig. 4C, green) and the southern side of the puff in the neighboring subunit (Fig. 4C, blue). SCR1 of DAF rises above the surface of the virus, and no contacts were identified. Portions of SCR1 have a weaker density, likely due to a flexibility in the hinge that connects SCR1 to SCR2. Most virus-receptor interactions occur between SCR2 and the northern region of the viral puff of the first of the two protomers (Fig. 4A), contributing >75% of the total buried surface area (832 Å2 of the total 1,081 Å2) (Table 4). Specifically, DAF binds into a depression that exists between the northern side of the puff and the raised region of the icosahedral 5-fold vertex (Fig. 4B). Residues 2138D and 3234Q, which were shown previously to be necessary for CVB3-RD to bind DAF (41), are located on opposite sides of the depressed area where SCR2 binds (Fig. 4B). From this site of significant interactions, SCR3 crosses over to the second protomer, bridging and blocking access to the canyon. Here, DAF SCR3/4 lies across the surface of the virus in contact with the southern region of the puff located within the second protomer (Fig. 4B, blue). SCR3/4 adds 249 Å2 to the total buried surface area (Table 4). From SCR4 toward the C terminus of DAF, there is a small volume of uninterpreted density localized to the icosahedral 3-fold vertices that was reported previously (18). This density is likely filled by the C-terminal six-His tags from each of the three DAF molecules.

Fig 4
(A) Refined fit of DAF (yellow ribbon) into the difference density, displayed at 1.0 σ. (B) Capsid-DAF interaction rendered to show capsid surface topology. Five protomers of the virus capsid were colored by radius from the center of the virus, ...
Table 3
Contact residues between CVB3-RD and DAF
Table 4
Buried surface area between DAF subunits and the virus surface

DAF interactions with convertase.

Three DAF residues, R69, R96, and L171, have been shown to be critical for native DAF function. The region of the surface of DAF defined by the location of R69 and R96 is especially critical for binding to and hastening the decay of convertases in the classical and alternative complement pathways (27) (Fig. 4C). This functional face of DAF does not overlap the footprint that identifies DAF binding to CVB3-RD and is oriented away from the surface of the virus in the structure of the complex.

CVB3 DAF binding compared to the previously identified echovirus mode of binding.

Virus-DAF complex structures have been solved for echovirus 7 (E7) (22, 45) and echovirus 12 (E12) (4, 43). In the case of both echoviruses, the mode of binding is the same, with SCR2/3 nearly crossing the 2-fold axis, but the interactions are not identical (43). The new CVB3-DAF map demonstrates a second mode of DAF binding with SCR2/3 across the canyon, crossing to bind the puff near the 5-fold axis. There are fewer virus-DAF interactions than reported previously (18, 45) and less buried surface. No DAF residue makes contact with all three viruses. None of the three viruses interact with SCR1. Six SCR2 residues interact with both CVB3 and E7, making contact predominately with VP1 and VP3 residues on the viral surface. However, SCR2 interacts above the puff in CVB3 and below the puff in E7; there are no SCR2 interactions with equivalent virus residues (Fig. 5 to to7).7). E12 does not interact with SCR2. As previously noted (45), the echoviruses are similar in their SCR3 interactions but with only 2 shared DAF contacts. The new footprint on CVB3 shows that E12 shares two DAF contacts with CVB3. SCR3 interacts with VP2 residues in all three viruses (Fig. 5 and and6).6). SCR4 binds VP3 residues in the echoviruses, but no interactions were defined for CVB3.

Fig 5
(A) Two protomers of CVB3-RD, E7, and E12 with one molecule of DAF bound are surface rendered to compare the DAF contact residues of three closely related DAF-binding viruses. (B) For each virus, the DAF footprint is shown in yellow, orange, and red, ...
Fig 6Fig 6
The virus surface represented as a quilt of amino acids, shown as a projection, with the icosahedral asymmetric unit indicated by the triangular boundary. Virus residues representing two adjacent protomers are shown in light blue and green, with DAF contacts ...
Fig 7
CVB3-RD and echovirus sequence-equivalent residues from a clustaw alignment shown to compare DAF interactions (43, 45). The puff region of the virus consists of VP2 residues 129 to 180, outlined by a gray box. The four residues defined in the DAF footprint ...

An alignment of CVB3 contact residues with the sequence-equivalent residues for the two echoviruses (Fig. 7) (22, 43, 45) shows four DAF-binding virus residues in common, residues 1271, 2142, 2166, and 3063. However, the site of DAF binding for residue 1271 is located in the blue protomer (Fig. 5) of both echoviruses and binds SCR4 of DAF, whereas residue 1271, which interacts with DAF in CVB3, maps to the neighboring protomer (Fig. 5, green), where it interacts with DAF SCR2. The largest commonality in DAF interactions occurs at the lower puff region, where residues 2142 and 2166 map within all three DAF-binding footprints; this small patch on each virus interacts with SCR3 of DAF (Fig. 5 and and7).7). Finally, in both echoviruses, a common VP3 residue (residue 3063) interacts with SCR4, whereas CVB3 VP3 interacts only with DAF SCR2 in the neighboring protomer (Fig. 5).


The previous structural analysis of CVB3-RD interacting with DAF (interpreted by using the crystal structure of CVB3-M) described several clashes, suggesting that the puff region of the RD capsid might differ in structure from that of CVB3-M or that DAF binding might cause conformational changes to occur (18). In the current work, the crystal structures of CVB3-RD and CVB3-M have been shown to be virtually identical, with no differences in the conformations of the puff regions where CVB3-M and CVB3-RD differ in sequence. With the reassignment of handedness permitted by a higher-resolution cryo-EM reconstruction, there are no longer structural clashes in the fitted crystal structures of the receptor or virus. Furthermore, the new structure provides a new definition of the contact sites between DAF and CVB3.

Four of the six amino acid changes from Nancy to RD map to the surface of the virus and are located within the DAF-binding footprint and may thus contribute to DAF binding. Located at the bottom of the canyon, the substitution at residue 1092 (Nancy to RD) introduces an Ile residue into the top of the pocket. The alteration of the interaction with the pocket factor suggests that these changes may influence the stability of the virus. The final mutational difference between the structural proteins of Nancy and RD is a Val residue (residue 2013) that maps to the N terminus of VP2 at the interior surface of the capsid.

The DAF footprint on the virus is defined by two distinct areas of buried surface, one in each of two neighboring protomers. The first area involves the northern puff region, where two virus residues shown to be essential for DAF binding are located (41). Both residues interact with SCR2 of DAF (Fig. 5), and an alteration of the identity of either one of these surface residues abrogates DAF binding. At the virus-DAF interface, contacts with SCR2 account for 75% of the buried surface area, consistent with the observation that the deletion of SCR2 prevents virus attachment to DAF, whereas the deletion of other individual SCRs does not (3). The second contact area takes place in a neighboring protomer between the southern puff region and DAF SCR3. Thus, DAF crosses over the canyon to interact with two adjacent protomers. By crossing the canyon, DAF may interfere with virus attachment to CAR, which is known to bind within the canyon (21); indeed, we have found that excess soluble DAF inhibits the attachment of CVB3-RD to CHO cells expressing human CAR (J. M. Bergelson, unpublished observation). At the 3-fold vertex, the C-terminal six-His tags of three symmetry-related DAF molecules meet. This interaction effectively “staples” the DAF molecules to the virus surface. It is conceivable that interactions between the His tags might change the conformation of DAF within the complex, creating a false impression of contacts with SCR3. However, when the crystal structure of DAF was placed directly into the corresponding density in the EM model, no manipulation of hinge angles between SCRs was required, confirming that the DAF molecule is in a native state and has not undergone any conformational changes. Therefore, the His tag interactions likely did not alter the normal mode of DAF binding to the surface of the virus.

The predicted contacts of SCR3 and SCR4 had a combined buried surface area of 249 Å2 (Table 4), which represents less than 25% of the total DAF molecule binding predicted from the structure. No contacts were predicted for SCR4; thus, the buried surface of SCR4 is inaccessible but is not an area of direct interaction. Additional interactions with SCR3 defined here may not be essential to DAF binding, as these interactions potentially take place according to the background of the specific CVB3 and depend on the identity of residues in the southern puff region. This is the only site where there is some commonality among all three viruses. Perhaps, the essential CVB3-SCR2 interactions evolved after E7/12 and CVB3 diverged from each other in a split from a common ancestor (17). Regardless of when the trait was acquired, the binding of DAF is a trait that persists and likely confers an advantage to the virus.


We thank Michael G. Rossmann, Paul R. Chipman, and Valorie D. Bowman for the generous use of data originally collected at Purdue University. We also thank Mavis Agbandje-McKenna for helpful scientific discussion.

The work was supported by NIH grants K22 A179271 to S.H. and R01 AI05228 to J.M.B.

S.H. designed research; J.D.Y. processed crystal data; J.O.C. processed and analyzed the cryo-EM data, including the assignment of absolute handedness; J.P. and J.M.B. provided sequence data; and J.O.C., J.D.Y., and S.H. wrote the paper.


Published ahead of print 12 September 2012


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