The structure of echovirus type 12 bound to a two-domain fragment of. its cellular attachment protein decay-accelerating factor (CD 55)

Transcription

1 JBC Papers in Press. Published on November 1, 00 as Manuscript M10 1 The structure of echovirus type 1 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD ) David Bhella * MRC Virology Unit, Church Street, Glasgow, G JR, UK Ian G. Goodfellow 1 Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow, G JR, UK Pietro Roversi Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 QU, UK David Pettigrew Yasmin Chaudhry 1 David J. Evans Susan M. Lea Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 QU, UK Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow, G JR, UK Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow, G JR, UK Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 QU, UK 1 * Corresponding author: d.bhella@vir.gla.ac.uk Current address: School of Animal and Microbial Sciences, University of Reading, P.O. box, Reading, Berkshire, RG AJ, United Kingdom. 1 Running Title: Structure of the echovirus 1 receptor complex 1 Copyright 00 by The American Society for Biochemistry and Molecular Biology, Inc.

2 Summary Echovirus type 1 (EV1), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV1 bound to a fragment of DAF, consisting of short consensus repeat domains and, from cryo-negative stain electron microscopy data (EMD #). This shows that, as for an earlier reconstruction of the related echovirus type bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV. Fitting of the crystallographic co-ordinates for DAF and EV into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV1 is distinct from that seen for EV raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses. Keywords Echovirus/Electron microscopy / Decay-accelerating factor/picornavirus /Receptor 0

3 Introduction Echoviruses along with the coxsackieviruses and polioviruses comprise the Enterovirus genus within the Picornaviridae family. Echovirus infection is usually mild although these viruses are sometimes associated with severe disease such as aseptic meningitis, encephalitis, haemorrhagic conjunctivitis and myocarditis. The Picornaviridae also include a number of other important human and animal pathogens including rhinoviruses and foot and mouth disease virus (FMDV). Picornaviruses are small (approximately 00 Å in diameter), non-enveloped icosahedral viruses that have a single-stranded positive-sense RNA genome of between.0 and. kb. The genome encodes a single polyprotein, which is co- and post-translationally processed by viral proteases to yield the capsid and non-structural proteins required for virus replication. The capsid assembles as a pseudo T= icosahedral shell from four protein species, VP1-VP. VP1- occupy the three quasi-equivalent positions in the icosahedral lattice, while VP is located at the inner surface of the capsid (1). The rhinoviruses and enteroviruses share a common and distinctive morphology, consisting of a raised, star-shaped, platform at the pentameric apices of the capsid. Surrounding this is a narrow cleft, termed the canyon (). For many picornaviruses (the polioviruses, the major receptor group rhinoviruses, coxsackie A virus type 1 and coxsackie B virus type ), it has been shown that the interaction with their cellular receptors (all of which are members of the Ig-superfamily of proteins) occurs in the canyon (-). Binding of these viruses to soluble monomeric receptor protein in vitro triggers an irreversible conformational change in the virus particle that manifests as a change in the sedimentation characteristics of the virus (from

4 the S mature virion to the 1S A-particle) (). Similar changes in sedimentation coefficient are seen when the virus interacts with the receptor at the cell surface and such transformations are thought to be essential for successful entry and infection (,). The structural rearrangements that lead to this change in sedimentation include loss of VP and externalisation of the N-terminus of VP1, which is normally located at the inner surface of the capsid surrounding the pentameric apices in the mature virion. The extruded terminus of VP1, which is hydrophobic, becomes membrane associated and is thought to form a pore through which the genome enters the cell cytoplasm (). Within the Rhinovirus and Enterovirus genera, other types of receptor interactions have also been identified. The minor receptor group rhinoviruses, for example, bind the very low-density lipoprotein receptor (VLDL-R) (1). This interaction does not lead to the irreversible conformational changes outlined above, and recent studies have demonstrated that this receptor binds to the virion around the five-fold symmetry axes, not in the canyon (1). The lack of virus uncoating upon receptor binding suggests the existence of another, as yet unknown, receptor or co-factor that induces uncoating. Many enteroviruses have been shown to bind decay-accelerating factor (DAF; CD), a member of the regulator of complement activity (RCA) protein family (1-1). DAF is a 0 kda glycosylphosphatidylinositol (GPI) anchored protein, present on the surface of the majority of serum-exposed cells, which functions to protect them from complementmediated lysis by accelerating the decay of both the classical and alternative pathway C and C convertases. The RCA protein family are defined by the presence of one or more, short consensus repeat (SCR) domains, each of about 0 residues. DAF has four such domains and these are linked to the C-terminal GPI anchor by a heavily O-glycosylated

5 serine/threonine/proline rich region (1). Complement control, enterovirus binding and other known interactions, including binding of the cellular ligand CD and bacterial adhesins, only involve the SCR domains (1,0-). Within the DAF-binding enteroviruses there is a range of different interactions. Coxsackie A virus type 1 (CV- A1) and enterovirus type 0 (ENV0) bind the membrane-distal domain SCR 1 (1,), whereas the DAF-binding echoviruses all interact with SCR, with additional binding to SCR and/or SCR (1). Enterovirus binding to soluble monomeric DAF does not, however, lead to the conformational changes normally associated with uncoating, that these viruses are observed to undergo at the cell surface (). The status of DAF as a sole cellular receptor has therefore been subject to question. Although other molecules have been implicated in cell binding and entry by DAF-binding enteroviruses (-), candidates that induce irreversible conformational changes in the virion have yet to be identified. Structural analysis of variants of echovirus type that exhibit different cell tropism has, however, provided evidence for an as yet undiscovered canyon binding receptor, while implicating a number of residues in the binding interaction with DAF, both around the five-fold symmetry axis and in the EF loop of VP (). The recent analysis of DAF binding in echovirus type, by cryomicroscopy and image reconstruction, has shown that DAF does not bind in the canyon; rather it binds to the hyper-variable region of VP just outside of the south rim of the canyon and also to another hyper-variable region of VP (0). In related viruses these regions have been shown to be antigenic and capable of eliciting a neutralising antibody response, suggesting that the paradigm of segregation of receptor-binding regions from those parts of the capsid surface subject to immune surveillance is not strictly adhered to in the

6 enteroviruses. Similarly, receptor binding to hyper-variable epitopes has been demonstrated for the minor group rhinoviruses and FMDV. In these viruses a strategy is employed whereby conserved receptor-binding domains are embedded in hyper-variable regions capable of mutation, to escape the host immune response (1,1). In the EV- receptor complex, bound DAF is located close to, and lies across the two-fold symmetry axes, such that symmetry related molecules are in steric collision (0). This complicates the interpretation of this reconstruction in terms of the known atomic structures for DAF (-). There are, however substantial data regarding specific residues both on the virus capsid, and in the SCR and domains of DAF that are involved in receptor binding in the echoviruses (,). Echovirus type 1 (EV1) exhibits a distinct interaction with DAF, which predominantly involves SCR domains and, a feature shared only with echovirus type (1). A recent mutagenic analysis of DAF structure and function demonstrated that EV1 binding involves a different face of the receptor than that bound by other echoviruses (). We have therefore conducted a structural investigation of the virus-receptor complex, and present here a reconstruction of echovirus type 1 bound to SCR domains and of DAF, determined to 1 Å resolution by cryo-negative stain transmission electron microscopy and image reconstruction. Similarly to echovirus type, DAF binds to this virus outside the south rim of the canyon close to (but not over) the two-fold symmetry axes. The distribution of density in our reconstruction is quite different however, supporting the previous findings that there are at least three distinct virus-daf interactions within the enteroviruses. By labelling the virus with only two domains of DAF, we have been able to determine an unambiguous density for the receptor that is

7 consistent with the crystallographic co-ordinates for DAF. Docking of these data into the reconstructed protein envelope reveals that DAF binds to EV1 mainly via interactions between SCR and VP. The orientation determined for the DAF fragment allows us to superimpose the crystallographic data from a DAF fragment comprising all four SCR domains (DAF 1 ) onto our model. We find that 0 copies of this protein may be modelled onto the surface of the EV1 capsid without any significant molecular clashes between symmetry related molecules or the virion itself. This further highlights the differences between the complex seen here and that seen in the earlier investigation of the EV-DAF interaction.

8 Experimental procedures Virus and DAF preparation Echovirus type 1 was routinely passaged in rhabdomyosarcoma (RD) cells. Ten cm roller bottles (Corning) of confluent RD cells were infected with EV1 at high multiplicity ( or higher) and the infection allowed to proceed for hours at o C. Cell associated virus was released by cycles of freeze thawing and the resultant supernatant was clarified by low speed centrifugation (000 g for minutes) and filtered (0. µm) to remove particulates. Sodium chloride was added to a final concentration of.% (w/v), followed by % polyethylene glycol 000 and the preparation was stirred at o C overnight. The precipitated virus was collected by centrifugation (000 g for 0 minutes) and resuspended in mls of DMEM using an 1-gauge needle and syringe. Large particulate material was removed by low speed centrifugation and the remaining virus was pelleted through a 0% sucrose cushion by centrifugation at 0,000 rpm for six hours (TH1, Sorval) and resuspended in 1 ml of DMEM using an 1-gauge needle and syringe. The partially purified virus was subsequently layered onto a -% sucrose gradient and centrifuged at 0,000 rpm for one hour (TH1, Sorval); S virus was extracted from the gradient and pelleted by centrifugation for six hours at 0,000 rpm (TLS ). Purified virus was resuspended in 00 µl of PBS and stored at 0 o C. Purity of virus preparations was assessed by SDS PAGE. The two SCR domain fragment of decay accelerating factor (DAF ), was expressed in Pichia pastoris and purified as previously described (,)

9 Electron microscopy EV1 preparations were labelled with DAF by incubation overnight at o C. Unlabelled and labelled virus was then prepared for electron microscopy, using the cryo-negative stain technique (). In brief, a µl droplet of virus or labelled virus was loaded onto a freshly glow-discharged Quantifoil holey carbon support film (Quantifoil Micro Tools GmbH, Germany) for 0 seconds. Grids were then transferred to a droplet of ~0% (w/v) ammonium molybdate solution (ph.) for ten seconds. Finally grids were blotted and plunged into liquid nitrogen-cooled ethane slush. Vitrified virus preparations were then imaged in a JEOL 0 EX II transmission electron microscope, equipped with an Oxford instruments cryo-transfer stage. Focalpair images were recorded at a nominal magnification of 0,000 and defocus of between 00 and,000 nm, on Kodak SO-1 film, under low-electron dose conditions. Image reconstruction In total 1 focal-pairs of unlabelled EV1 and focal-pairs of DAF labelled EV1 were selected for processing. Micrographs were digitised on a Dunvegan Hi-Scan drum scanner (Dunvegan SA, Lausanne, Switzerland) at a raster step-size corresponding to. Å/pixel in the specimen. Particles were selected from each pair of micrographs using the program Xd, and deconvolution of the CTF was accomplished using the CTFMIX program, at which point the focal pairs were merged (). Orientations and origins were determined for the unlabelled EV1 dataset using the polar-fourier transform method (), using a starting model that was generated from the crystal structure of echovirus type 1 (), by the EMAN PDBMRC program (0). Subsequent iterations of three-

10 dimensional reconstruction (1) and PFT refinement led to the calculation of a 0Å resolution reconstruction that was used to determine initial origins and orientations for the DAF labelled dataset. In both cases, origins and orientations were further refined until they ceased changing significantly. Final reconstructions were calculated to 1 Å and in both cases all inverse eigenvalues were less than 0.01, indicating that the data adequately filled reciprocal space to this resolution. Final resolutions for each reconstruction were estimated by randomly dividing the data sets into two sub-sets and calculating independent reconstructions, for which a number of measures of agreement were calculated, including the Fourier-shell correlation, and the spectral signal to noise ratio. Reconstructions were deposited in the EM databank (accession numbers EMD # - DAF labelled virus and EMD # - unlabelled virus). Isosurfaces calculated from three-dimensional reconstructions were visualised in Iris Explorer (Numerical Algorithms Group, Oxford UK), using a radial depth cueing module (). To draw a direct comparison between our reconstructions and the previously determined model for EV bound to DAF 1, a density map was calculated at 1 Ångstroms resolution using the EMAN PDBMRC program from the deposited coordinates of He et al. (0) PDB #1M. Fitting of the EV and DAF crystal structures to the cryo-negative stain reconstruction. Visual inspection of an EV virus model (PDB #1HT) overlapped with the EV1- DAF density map confirmed the correctness of the original hand assignment for the reconstruction. The reconstructed volume was then placed at the origin of a cubic cell with a cell edge a=. Å, oriented so that its threefold and twofold axes coincided

11 with the symmetry axes through the origin of space group P, and was rendered periodic with P symmetry. The X-ray model for a pentameric asymmetric unit of EV was oriented and placed around the origin in the same cell. The overall correlation coefficients in real space between the experimental and model density was 0.. The X-ray model for DAF domains and (PDB #1H0) with all B factors set to was manually placed in the EM density in an initial orientation with domain closer to the virus (placement A). A second placement of the model was also generated, by rotating the first so as to swap the positions of domains and (placement B). Alternative orientations of both placements A and B were sampled by rotation of each model around its major axis (defined by the midpoints of the disulphide bonds) in steps of 0 degrees. Figure shows the real space correlation coefficients for the main chain atoms in each of the rotated models. Placement A in its original orientation gave the best correlation coefficients suggesting that both the domain assignment and the DAF face assignments are correct. Maximum-likelihood rigid body refinement of the domain model (residues 1-1) targeting the phases computed from the reconstruction (program CNS ()) further improved the main chain atoms correlation coefficients (Figure ) and yielded the final model (Figure ). The Cα-Cα distance for the domain - junction is only 0. Å away from the distance given by ideal geometry a reasonable level of accuracy for a fit at 1 Å resolution. DAF 1 was overlaid on the membrane-proximal fragment using the CCP program LSQkab (,). Molecular models were visualised using PyMol (DeLano Scientific,

12 San Carlos, CA, USA), Molscript () and RasterD (). Co-ordinates for virus receptor complexes were deposited in the protein data bank (PDB #1UPN). 1

13 Results and discussion Cryo-negative stain imaging and three-dimensional reconstruction of EV1 labelled with DAF The cryo-negative stain approach yielded high contrast images of virus preparations in a frozen hydrated state (Figure 1). Analysis of the incoherently averaged power-spectra, calculated from particle images cut from focal pair micrographs, showed that Thon rings were clearly visible out to Å in these data. In total, 0 focal pair images of unlabelled EV1 virions and paired images of DAF labelled EV1 were corrected for the effects of the microscope s contrast transfer function and processed to calculate three-dimensional image reconstructions. Final reconstructions were calculated from 1 unlabelled particle images and 0 DAF labelled images (Figure ). The resolutions of these reconstructions were determined to be 1 Å and 1 Å respectively. The reconstruction of the DAF bound virion has clear regions of contiguous density decorating the exterior of the capsid that can be attributed to the DAF fragment. This density is linear in appearance and consists of two readily defined domains. The density attributed to the DAF molecule is much weaker than that of the capsid itself, suggesting a low level of receptor occupancy. A low density-threshold setting, approximately equal to the average density of the reconstructed volume, was therefore necessary to calculate an isosurface that encompassed the correct molecular volume for the DAF component of this reconstruction. Fortunately the high contrast in the reconstruction, brought about by the use of the cryo-negative stain technique, prevented the expansion of the virion density or incorporation of noise in the final representation of the volume. The apparent low 1

14 occupancy in the virus-receptor complex is consistent with biochemical data and previously published reconstructions (1,0,) EV1 decorated with DAF shows a different receptor orientation for DAF compared to EV The reconstruction of labelled EV1 shows a distinct distribution of density compared to the previously published structures for EV bound to the three or four SCR domain fragments of DAF; DAF and DAF 1 (0). In our reconstruction, two symmetryrelated DAF molecules are oriented such that they are in a position corresponding to the ten past eight setting of hands on a clock, when viewed along the two-fold symmetry axis (Figure B). In the published reconstruction of the EV-DAF complex, the linear density most likely to correspond to the DAF component is in a five to five position, when viewed along the same axes (Figure D). The principle point of contact, however, is similar, located at a puff of density just outside the southern lip of the canyon, approximately equidistant between the five-fold and three-fold symmetry axes. Our data reveals only one point of contact between virus and receptor fragment, whereas for EV there appears to be three. In our analysis, the use of a two-domain fragment and the different mode of receptor binding in EV1 have overcome the problems of interpretation associated with the orientation of the DAF molecule relative to the icosahedral symmetry of the virion experienced by He et al. (0). Inspection of the DAF labelled EV1 reconstruction shows a clear region of density that comprises two domains. Two symmetry-related DAF fragments approach each other across a two-fold symmetry axis without coming into steric collision with each other. We are, therefore, able to interpret 1

15 the reconstructed density unambiguously in the context of the published crystallographic co-ordinates for SCR domains and of DAF () Determination of a high-resolution model of receptor binding for DAF-binding Echoviruses by docking crystallographic co-ordinates into the low-resolution reconstruction. As the structure of EV1 has not been determined at atomic resolution, we have fitted the known crystallographic co-ordinates for EV (PDB #1HT, ()) into our reconstruction. EV should provide a good model for EV1, as these two viruses are % sequence identical in the capsid proteins. In fact we see that positioning of the EV co-ordinates in the EV1 density gives a close fit to the surface structure of the reconstruction, reflected in the fact that a map calculated at 1 Å from the X-ray structure contains density that correlates well with the experimental density observed in the reconstruction (real space correlation of 0. in the correct hand). Initial manual positioning of the DAF fragment (PDB #1H0, ()) was found to give an optimal fit, as verified by calculation of the real-space correlation coefficients between the density for the receptor and differently oriented copies of the atomic model (Figure ). Further rigid-body refinement of the SCR component led to the final calculation of the fully fitted virus-receptor complex. A robust fit of crystallographic data has been calculated, allowing us to unambiguously determine that the SCR domain lies closest to the icosahedral two-fold symmetry axes and contains the largest proportion of the area of contact with the virion surface. We were also able to determine which face of the receptor provides the primary site of interaction (Figure ). The highest correlation for 1

16 the optimally positioned receptor fragment was 0. with the occupancy of the receptor set to 0.1, confirming that only a small percentage of the potential receptor binding sites are occupied under these conditions. This is perhaps not surprising given that the affinity of DAF for EV (the only virus-daf interaction which has been measured quantitatively) is known to be in the µm range (). This is a relatively weak interaction by comparison to those virus-receptor interactions previously investigated by cryomicroscopy and image reconstruction, such as those of the polioviruses and rhinoviruses, which have receptor affinities in the nm range (,0). The high quality of fit for the DAF X-ray model in our reconstruction provides evidence of the relative rigidity of these two domains of the DAF molecule and contrasts strikingly with the flexibility observed in studies of a DAF domain pair by NMR (). Analysis of the atomic-resolution virus-receptor complex, produced by fitting the X-ray models to the reconstruction, shows that residues in the capsid protein VP are mainly involved in DAF interactions (Figure ). In fact, despite the different receptor orientation, just under half of the capsid residues buried in the EV1-DAF complex were found to also contribute to binding of DAF in the EV-DAF complex - although the part of the receptor contacted by any of these residues varies due to the different mode of DAF association (1,, and figure C). A single virus residue, VP, contributes just over % of the total surface area buried in the interaction and is clearly critical to this binding 0 1 event. In terms of the key DAF residues buried in the complex it is noteworthy that E 1 in SCR is amongst the three residues found to contribute more than % to the buried surface area, since our earlier mutational studies implied that this residue was critical for EV1 binding but not involved in binding EV (). The other two residues 1

17 contributing more than % of the buried surface area are Q 1 (also in SCR ) and R 1 (in SCR ). All three of these residues lie in close proximity on the surface of DAF close to the SCR / interface and their participation in the binding site suggests that recognition of DAF by EV1 is likely to be highly dependent on the relative orientation of these two SCR modules. Further evidence supporting our model of this virus-receptor interaction includes the observation that EV1 binds a range of primate DAF proteins; of the 1 DAF residues buried in this EV1-DAF complex, 1 are conserved across all primate and human DAFs that are known to bind EV1. In fact the two variable residues within the virus footprint on the receptor contribute less than % to the total binding area. Other DAF binding echoviruses interact with less conserved domains of the receptor, and generally do not bind a wide range of primate DAF proteins (1). By superimposing SCRs and from the recently determined structure of all four DAF SCR domains (DAF 1 ) () onto our docked DAF model, we are able to produce a model for EV1 in complex with DAF 1. In contrast to the EV-DAF reconstruction this procedure produces a model in which the only atomic clashes between symmetry related DAF molecules occur between the flexible loop that links strands b and a of SCR (as defined in Williams et al. Ref. ) and the bulge that interrupts strand in SCR (Figure ). Since the SCR loop is the only portion of the DAF structure seen to vary between different crystal structures, and since a minor rearrangement of this loop would prevent any steric clashes between the two copies of DAF, we believe this superposition is likely to represent a reliable model for the interaction between DAF 1 and EV1. The interaction seen between the two-fold symmetry related copies of DAF is 1

18 intimate, and is reminiscent of the head to tail interactions seen in crystals of DAF 1 () the linkers between SCRs and appear to form a rough sheet across the two-fold axis. In agreement with earlier studies (1,,1) this model predicts that SCR will be minimally, and SCR 1 not at all, involved in virus binding (Figure b) Implications for the evolution of a DAF-binding phenotype in the Enteroviruses. We have presented the first data that unambiguously delineates the nature of the interaction between an echovirus (type 1) and its receptor DAF. Our finding that this interaction is substantially different from that suggested by previous studies (both structural and mutational) has implications for the significance of the DAF-binding phenotype. It has been shown that both EV and EV bind to DAF in a substantially different manner to EV1 and possibly each other, despite their conserved characteristic of interacting with the membrane-proximal SCR domains (1,0). This is surprising given the high level of sequence identity between these viruses, particularly EV and 1. It is striking, however, that of the 1 residues that contribute significantly to the binding site in our atomic model of the EV1-DAF interaction, only are conserved between EV and EV1. This is consistent with the suggestion that these viruses observe a strategy of maintaining their receptor binding sites in a hyper-variable region, possibly as a means of evading antibody neutralisation. These findings, along with the results of previous investigations showing that the more distantly related DAF-binding enteroviruses CV-A1 and ENV0 bind to SCR domain 1, suggest a considerable selective pressure for these viruses to utilize DAF as their cellular receptor. 1

19 Several possible explanations might be postulated to explain the diversity of interactions that exist between the enteroviruses and DAF. It has been suggested that DAF binding could have evolved independently in these viruses rather than being present as an ancestral trait inherited through many generations from a distant, common ancestor (1). Convergent evolution of DAF binding in these viruses from non-daf binding picornavirus progenitors is an attractive means of explaining the radically different modes of binding found in these viruses, particularly between the SCR 1 binding viruses and those that bind SCR. Alternatively the wide range of virus-daf interactions found in the enteroviruses may reflect the ancient divergence of this trait, and co-evolution of these viruses with their receptor, such that the progenitor virus bound a single domain complement control protein, and as this domain evolved into a number of shortconsensus repeats, capable of serving different functions, the viruses evolved to bind these newly adapted domains differently. While these explanations may account for the more distantly related enteroviruses interactions with DAF SCR domain 1, they seem less likely in the case of EV and 1, given the close phylogenetic relatedness of these viruses. Therefore, we are forced to consider the possibility that the different modes of binding in these viruses may represent a comparatively recent adaptation of a pre-existing trait on the part of one or other virus. Rapid evolution of novel receptor-binding mechanisms is found throughout the picornaviruses, such as culture adaptation to bind heparan sulphate in FMDV, the in-vitro evolution of rhinoviruses capable of infecting cells in an ICAM-1 independent manner and the reversion of non-daf binding EV mutants to a DAF binding phenotype (,,). Although the evolution of a novel receptor-binding site might seem intuitively improbable for a virus that has an already 1

20 functional interaction with this receptor, consideration of the high mutation and recombination rates in these viruses, as well as the numbers of virions produced in the course of an infection, makes such events plausible. It therefore seems conceivable that selective pressure to retain the DAF utilizing phenotype, but alter the receptor-binding site to evade immune surveillance, might give rise to such alterations in this interaction. Clearly, regardless of the evolutionary route these viruses took to arrive at the situation we find today, where several viruses bind the receptor by very different mechanisms, the fact that this situation exists indicates that this phenotype is of biological significance and must confer considerable evolutionary advantages. 0

21 Acknowledgements The authors wish to thank Duncan McGeoch for critical reading of the manuscript, Marc Adrian for advice and instruction on the application of the cryo-negative stain technique and James Conway for provision of the CTFMIX software and advice on its application. DB is supported by the United Kingdom Medical Research Council. IGG and YC were funded by an MRC programme grant (G0) and Wellcome trust project grant (00) to DJE. DP is funded by an MRC studentship award to SML and PR by BBSRC grant # /B1. 1

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27 Figure Legends Figure 1. Focal pair, cryo-negative stain images of unlabelled (A and B) and DAF labelled (C and D) EV1 virions. The defocus values for these images were determined to be (A) 1. µm, (B) µm, (C) 1. µm and (D). µm. Figure. Stereo pairs of surface rendered three-dimensional reconstructions of (A) unlabelled EV1 virions, and (B) DAF labelled virions. Isosurfaces of these reconstructions are merged and rendered in their respective colour schemes to highlight the differences in density attributed to the two SCR domain fragment of DAF (C). A low-resolution representation of EV bound to DAF 1, derived from PDB #1M (0), highlights the differently oriented densities in these two complexes (D). In this model the densities of two copies of DAF 1 are superimposed, laying across the virions two-fold symmetry axes, giving rise to a hybrid density representing the two possible positions for the molecule. A radial depth-cue colour scheme is used to indicate distance from the centre of the virion (see key). Figure. Plot of the real-space correlation coefficients for the main chain atoms of DAF rotated about the molecules long axis, in both placement A (SCR forming the major contacts shown as circles) and placement B (SCR forming the major contacts shown as triangles). The square indicates the CC after rigid-body refinement of SCR to yield the final model shown in all other figures. Correlation values were calculated from the

28 receptor densities only. The optimal placement of the receptor in the modelled virusreceptor complex gave a correlation of 0. with the reconstructed density at an occupancy of 0.1. Figure. Ribbon diagrams showing the final fit of crystallographic co-ordinates for EV (PDB #1HT) and DAF (PDB #1H0) into the surface-contoured envelope of the EV1- DAF reconstruction. DAF is coloured yellow, VP1 is blue, VP is green and VP is red. A discontinuity between SCR domains and is present at residues 1 and 1. This is due to the displacement of SCR, induced by rigid body refinement, which results in the Cα-Cα distance being 0.Å greater than the ideal geometry. Figure. A comparison of the low-resolution three-dimensional reconstruction of EV1-DAF (A) and a space-filling representation of the EV1-DAF complex (B), generated using the crystallographic co-ordinates for EV and DAF. Radial depth-cueing emphasises the distance between atoms or regions of density and the centre of the virion, such that dark colours are close to the centre and light colours are further away. EV1 (and EV) is coloured in shades of blue, while DAF is coloured in green. A space-filling representation of the EV-DAF 1 complex (0) (C) highlights the different orientation of DAF bound to these two viruses. The model deposited under PDB #1M contains alpha-carbon atoms only this view is therefore rendered with the atomic radii for each atom set to.å. EV is coloured in shades of purple and the receptor in red. A closeup view of DAF shown as in panel (B), but rotated o about a vertical axis, exposes the residues buried in the virus-receptor complex (D). Residues are coloured according

29 1 1 1 to their contribution to the total contact area (~0Å ); yellow (1<%), orange (<%) and red (%+). A close-up view of EV without the receptor in place exposes buried residues on the surface of the capsid that are coloured according to the same scheme (E), the biological protomer is indicated. Figure Close-up views of the model for the complex formed by superposition of DAF 1 onto the EV1-DAF complex. The virus surface is shown in grey with the receptor molecules shown as cartoon representations above it. Each copy of DAF 1 is independently coloured and the views are centred on a pair of DAF molecules (cyan and green) interacting across the viral two-fold symmetry axis. (A) View perpendicular to the surface of the capsid. Portions of four copies of DAF are shown and the SCR domains are labelled. The clash between the flexible loop and bulge is denoted by *. (B) Side view of the virus-receptor complex. Two copies of the DAF 1 molecule are shown oriented across a two-fold symmetry axis.

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36 The structure of echovirus type 1 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD ) David Bhella, Ian G. Goodfellow, Pietro Roversi, David Pettigrew, Yasmin Chaudhry, David J. Evans and Susan M. Lea J. Biol. Chem. published online November 1, 00 Access the most updated version of this article at doi:./jbc.m10 Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts