Structure of a Neutralizing Antibody Bound Monovalently to Human Rhinovirus 2

The structure of a complex between human rhinovirus 2 (HRV2) and the Fab fragment of neutralizing monoclonal antibody (MAb) 3B10 has been determined to 25-Å resolution by cryoelectron microscopy and three-dimensional reconstruction techniques. The footprint of 3B10 on HRV2 is very similar to that of neutralizing MAb 8F5, which binds bivalently across the icosahedral twofold axis. However, the 3B10 Fab fragment (Fab-3B10) is bound in an orientation, inclined at approximately 45° to the surface of the virus capsid, which is compatible only with monovalent binding of the antibody. The canyon around the fivefold axis is not directly obstructed by the bound Fab. The X-ray structures of a closely related HRV (HRV1A) and a Fab fragment were fitted to the density maps of the HRV2–Fab-3B10 complex obtained by cryoelectron microscope techniques. The footprint of 3B10 on the viral surface is largely on VP2 but also covers the VP3 loop centered on residue 3064 and the VP1 loop centered on residue 1267. MAb 3B10 can interact directly with VP2 residue 2164, the site of an escape mutation on VP2, and with VP1 residues 1264 to 1267, the site of a deletion escape mutation. Deletion of these residues shortens the VP1 loop, moving it away from the MAb binding site. All structural and biochemical evidence indicates that MAb 3B10 binds to a conformation epitope on HRV2.     

Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.Picornaviruses are among the largest of animal virus families and include the well-known poliovirus, rhinovirus, foot-and-mouth disease virus (FMDV), coxsackievirus, and hepatitis A virus. The rhinoviruses, of which there are more than 100 serotypes subdivided into two groups, are major causative agents of the common cold in humans (42). The viruses are nonenveloped and have an ∼300-Å-diameter protein shell that encapsidates a single-stranded, plus-sense RNA genome of about 7,200 bases. The human rhinovirus 14 (HRV14) capsid exhibits a pseudo-T=3 (P=3) icosahedral symmetry and consists of 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, with VP4 at the RNA-capsid interface (40). An ∼20-Å deep canyon lies roughly at the junction of VP1 (forming the north rim) with VP2 and VP3 (forming the south rim) and surrounds each of the 12 icosahedral fivefold vertices. The canyon regions of HRV14 and HRV16, both major receptor group rhinoviruses, were shown to contain the binding site of the cellular receptor, intercellular adhesion molecule 1 (ICAM-1) (8, 24a, 37). Four major neutralizing immunogenic (NIm) sites, NIm-IA, NIm-IB, NIm-II, and NIm-III, were identified by studies of neutralization escape mutants with monoclonal antibodies (MAbs) (46, 47) and then mapped to four protruding regions on the viral surface (40).Several mechanisms of antibody-mediated neutralization have been proposed. Perhaps the simplest is based on aggregation of virions (5, 53, 54), which generally occurs over a narrow range of antibody/virus ratios. This limited range has raised questions about the role of aggregation in vivo. Alternative suggestions are that antibodies may neutralize virions by inducing extensive conformational changes in the capsid (15, 29), abrogate virus attachment to the host cell (8, 14), or prevent uncoating (57). There is no universal acceptance of a single neutralization mechanism, and the various MAbs may neutralize with different combinations of these mechanisms.Neutralizing MAbs against HRV14 have been divided into three groups: strong, intermediate, and weak neutralizers (26, 34). All strongly neutralizing antibodies bind to the NIm-IA site, which was defined by natural escape mutations at residues D1091 and E1095 of VP1 on the loop between the β-B and β-C strands of the VP1 β-barrel (the letter designates the amino acid, the first digit identifies the viral protein, and the remaining three digits specify the sequence number). Because strongly neutralizing antibodies form stable, monomeric virus-antibody complexes with a maximum stoichiometry of 30 antibodies per virion, it was concluded that they bind bivalently to the virions (26, 34). Weakly neutralizing antibodies form unstable, monomeric complexes with HRV14 and bind with a stoichiometry of ∼60 antibodies per virion (26, 52). The remaining antibodies, all of which precipitate the virions, are classified as intermediate neutralizers (26, 34).The structures of two complexes, the strongly neutralizing antibody MAb17-IA and its Fab fragment, Fab17, bound to HRV14, were determined by means of cryo-transmission electron microscopy (cryo-TEM) and three-dimensional image reconstruction (51, 52) and interpreted on the basis of model-building studies that used the atomic structures of HRV14 (40) and Fab17 (28). These studies showed that no observable conformational changes were induced in the viral capsid upon Fab or MAb binding. Modeling and site-directed mutagenesis studies demonstrated that electrostatic interactions play a key role in the binding of Fab17 to HRV14 (52). In the complex, the loop of the NIm-IA site on HRV14 sits clamped in the cleft between the heavy- and light-chain hypervariable regions and forms complementary electrostatic interactions with Lys58^H (on the heavy chain) and Arg91^L (on the light chain) of Fab17. In addition, a cluster of lysines on HRV14 (K1236, K1097, and K1085) interact with two acidic residues, Asp45^H and Asp54^H, in the CDR2 (CDR stands for complementarity-determining region) of the Fab heavy chain (49). Earlier modeling studies also suggested that bidentate binding of MAb17-IA to HRV14 is facilitated by rotation of the Fab constant domains about the elbow axes towards the viral twofold axes (51). This suggested that the flexibility of the elbow region (the junction between the variable and constant domains) plays a role in the bivalent binding process, which in turn increases antibody avidity. Finally, the 4-Å-resolution crystal structure of the Fab17-HRV14 complex clearly showed that the virion does not undergo conformational changes upon Fab binding (49). This crystal structure determination also revealed that the earlier docking of the HRV14 and Fab17 atomic structures into the 22-Å cryo-TEM density map (50) yielded a pseudo-atomic model that was very close to the real structure of the complex.We have expanded our complementary X-ray crystallography and cryo-TEM microscopy studies to examine the structures of two more Fab-virus complexes, using Fab fragments from two other NIm-IA antibodies, MAb1-IA (MAb1) and MAb12-IA (MAb12), bound to HRV14. MAb1 and MAb12 are weak and strong neutralizing antibodies, respectively. Image reconstructions of these two complexes are interpreted on the basis of pseudo-atomic models, which substantiate the previous hypothesis that neutralizing efficacy and binding valency are interrelated (34). Electrostatic interactions at the epitope-paratope interface are highly conserved and apparently important for the antibody binding to the virion surface. Like Fab17, Fab1 and Fab12 penetrate the canyon. There are, however, differences between the orientations of the strongly and weakly neutralizing antibodies and in the contacts made with the receptor binding region of the canyon. Finally, data suggesting that antibody binding to HRV14 is alone sufficient for neutralization and that other possible mechanisms are not required are presented.     

The concerted conformational changes during human rhinovirus 2 uncoating

Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The structure of the empty capsid has been determined to a resolution of better than 15 A by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 A diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.     

Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism.     

Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.     

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.     

Antibodies to the Buried N Terminus of Rhinovirus VP4 Exhibit Cross-Serotypic Neutralization

Development of a vaccine for the common cold has been thwarted by the fact that there are more than 100 serotypes of human rhinovirus (HRV). We previously demonstrated that the HRV14 capsid is dynamic and transiently displays the buried N termini of viral protein 1 (VP1) and VP4. Here, further evidence for this “breathing” phenomenon is presented, using antibodies to several peptides representing the N terminus of VP4. The antibodies form stable complexes with intact HRV14 virions and neutralize infectivity. Since this region of VP4 is highly conserved among all of the rhinoviruses, antiviral activity by these anti-VP4 antibodies is cross-serotypic. The antibodies inhibit HRV16 infectivity in a temperature- and time-dependent manner consistent with the breathing behavior. Monoclonal and polyclonal antibodies raised against the 30-residue peptide do not react with peptides shorter than 24 residues, suggesting that these peptides are adopting three-dimensional conformations that are highly dependent upon the length of the peptide. Furthermore, there is evidence that the N termini of VP4 are interacting with each other upon extrusion from the capsid. A Ser5Cys mutation in VP4 yields an infectious virus that forms cysteine cross-links in VP4 when the virus is incubated at room temperature but not at 4°C. The fact that all of the VP4s are involved in this cross-linking process strongly suggests that VP4 forms specific oligomers upon extrusion. Together these results suggest that it may be possible to develop a pan-serotypic peptide vaccine to HRV, but its design will likely require details about the oligomeric structure of the exposed termini.Rhinoviruses are the major causative agents of the common cold and cost the United States economy approximately $40 billion per year (6). Therefore, it is of great interest to prevent or ameliorate the symptoms of the common cold. The rhinovirus genus is a member of the picornavirus family and is characterized by nonenveloped capsid with a diameter of ∼300 Å containing a single-stranded, plus-sense RNA genome (19). Other members of the picornavirus family include foot-and-mouth disease virus, poliovirus, encephalomyocarditis virus, and hepatitis A virus. The capsids exhibit pseudo T = 3 icosahedral symmetry and are composed of 60 copies of the four capsid proteins VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 have an eight-stranded antiparallel beta-barrel motif structure and form the outer surface of the capsid, while VP4 lies at the interface between the capsid and the interior genomic RNA (22). VP4 is approximately 70 amino acids in length and is myristoylated at the N terminus (3, 14).Antibodies are the major line of defense against picornavirus infections. In the case of human rhinovirus 14 (HRV14), a number of studies have been performed to detail the antibody recognition and neutralization processes (25). While it had been long suggested that antibodies neutralize viral infectivity by inducing large conformational changes in the capsid, both cryo-transmission electron microscopy (cryo-TEM) (2, 28) and crystallographic analysis (27) clearly demonstrated that this was not the case. Further, it was shown that antibody recognition is more plastic than previously thought in that it is able to bind into the relatively narrow receptor-binding region of the canyon (27). These results suggested that the major in vivo role of antibodies is to bind to virion and work synergistically with other immune system components (26). This hypothesis has gained further support from studies of other pathogens (1) and implies that vaccines need only to elicit antibodies that bind to the authentic pathogen with high affinity.While these results simplified the goal of creating a synthetic vaccine by focusing on capsid recognition rather than possible antibody-induced conformational changes, developing synthetic vaccines against all 100 serotypes of HRV remains a daunting task. As shown in the structures of HRV14/antibody complexes, the antibodies make extensive contacts with the surface of the capsid that is not limited to a single antigenic loop (2, 27). Further evidence for this extensive contact is that antibodies to peptides corresponding to antigenic NIm loops fail to neutralize the virions (17, 29), and antibodies raised against intact capsids do not bind effectively to peptides corresponding to NIm-IA loop (T. J. Smith, unpublished results). One notable exception is the case of HRV2, where there is cross-reactivity between the NIm-II site of the virion and a synthetic peptide (30). Nevertheless, developing a repertoire of peptides representing the entire antigenic ensemble of HRVs is not only impractical but also unlikely to elicit neutralizing antibodies.All of the studies described above were performed with the antibodies that were raised against intact particles or to peptides representing epitopes that reside on the outer surface of the capsid. In the case of poliovirus, however, antibodies were raised against VP4 and the N termini of VP1 of poliovirus serotype I (15, 21). It was shown that these antibodies are capable of neutralizing the virion despite the fact that those portions of the capsid protein are buried in the interior of the capsid at the capsid-RNA interface (8). These results suggested that the poliovirus capsid was more dynamic than indicated by the crystal structure and that these termini are presented to the exterior of the virion in a temperature-dependent and reversible manner. While the role of capsid dynamics in the viral life cycle was not clear, it was suggested that the N termini of VP1 and VP4 might facilitate cell membrane attachment and subsequent entry of the virus into the host cell (3, 4).More recently, evidence for capsid dynamics has been found in other viruses as well. In the cases of swine vesicular disease virus (10) and coxsackievirus A9 (18), antibodies were raised against the whole virus in pigs and rabbits, respectively. These polyclonal antibodies demonstrated a strong reaction to the peptides corresponding to the N termini of VP1 and VP3 of swine vesicular disease virus and coxsackievirus A9, respectively. In a similar study, antibodies from the plasma of patients suffering from type I diabetes were found to target VP4 protein of coxsackievirus B3, again suggesting the exposure of VP4 peptide during coxsackievirus infection (23). These results imply that capsid “breathing” may be a phenomenon common to many proteinaceous capsids.Using a very different approach, the dynamic nature of HRV14 was analyzed using limited proteolysis and mass spectrometry (matrix-assisted laser desorption ionization [MALDI]) analyses (14). In these experiments, the virus was treated with both matrix-bound and soluble forms of trypsin for various periods of time, and the resulting proteolytic fragments were identified by MALDI. Surprisingly, the N termini of VP4 and VP1 were found to be the most proteolytically sensitive portions of the capsid in spite of being buried inside the viral capsid. As an additional control, the antiviral “WIN” compounds, which had been previously shown to stabilize the virions against thermal and acid denaturation, were added during digestion. While these WIN compounds did not affect the intrinsic proteolytic activity of trypsin, they nearly completely protected the VP1 and VP4 termini from proteolysis for an extended period. Together, these results suggested that HRV14 is transiently exposing these termini in a “breathing” process and that the empty hydrophobic drug-binding region apparently plays an important role in facilitating these dynamics.In this study we further examined HRV14 capsid dynamics by raising polyclonal antibodies against several peptides representing the N termini of VP1 and VP4. In these experiments, only the antibodies against the VP4 N terminus were found to successfully neutralize viral infectivity in vitro. Further, we demonstrate that the HRV14 VP4 antiserum cross-reacts with other serotypes of rhinovirus (HRV16, and HRV29), which is likely due to the high degree of conservation of VP4. Antibody neutralization closely parallels the MALDI analysis in that antibody neutralization and proteolysis are enhanced at 37°C in the case of HRV16 whereas the elevated temperatures are not required for either phenomenon in the cases of HRV14 and HRV29. Epitope mapping of the N-terminal 30 residues of VP4 suggests that it adopts a nonlinear conformation, and this is further substantiated by results showing that all of the copies of VP4 in the Ser5Cys HRV14 mutant at room temperature form cysteine cross-linked dimers. This cysteine cross-link does not form at 4°C, suggesting that capsid breathing is essential for VP4 exposure and interactions. Since VP4 dimerization does not affect viral infectivity, it seems likely that VP4 extrusion is a normal part of the cell attachment and entry process of rhinovirus. Together, these results suggest that VP4 might be useful as a pan-serotypic rhinovirus vaccine, but it seems likely that better understanding of the VP4 oligomeric structure will be necessary for further optimization.     

The recognition of parvovirus capsids by antibodies

The parvoviruses are small, non-enveloped icosahedral viruses which infect many animals, including vertebrates and arthropods. Vertebrate parvoviruses can be classified into the autonomous and the adeno-associated viruses — the autonomous parvoviruses have been examined in detail for antigenic structure. The protective immunity against parvoviruses in animals appears to be primarily antibody-mediated. The capsid of the autonomous parvoviruses is assembled from two proteins, VP1 and VP2, which overlap in sequence, with VP1 having additional N-terminal residues. Empty capsids can be assembled from VP2 alone.The structures of canine parvovirus (CPV) and feline panleukopenia virus (FPV) have been solved to better than 3·5 Å resolution, and the structure of human parvovirus, B19, has been solved to 8 Å resolution. In each case the T = 1 icosahedron is made up to 60 copies of a structural motif common to VP1 and VP2, consisting of an eight-stranded anti-parallel β-barrel. The surface of the capsid is made up primarily of large elaborate loops which connect the β-strands that make up the barrel. Antigenic epitopes have been mapped utilizing escape mutants, natural variants, peptide analysis and by expression of viral proteins. In CPV two major antigenic determinants were defined by escape mutant analysis, while peptide analysis revealed antigenic determinants in many different regions of the capsid protein, including the amino terminus of VP2. Neutralizing epitopes of B19 were found by peptide analysis in the VP1-unique region and in sequences common to VP1 and VP2. Other antigenic, but non-neutralizing, epitopes were found in the VP1–VP2 junction, as well as various other parts of the VP2 protein.The binding of a Fab derived from one neutralizing anti-CPV Mab has been examined by cryo-electron microscopy image reconstruction, which showed that 60 copies of the Fab were bound per virion. The Fab footprint covered approximately 796 Ųof the capsid surface, in a region where escape mutations to that Mab had been previously shown to cluster. The mechanism of neutralization was not clear, but could involve interference with cell attachment, cell entry or uncoating during the process of cell infection.     

Solution conformation of an immunogenic peptide from HRV2: comparison with the conformation found in a complex with a Fab fragment of an anti-HRV2 neutralizing antibody

The conformation of a [15]-peptide (H-VKAETRLNPDLQPTE-NH₂) from VP2 of rhinovirus HRV2 complexed with a Fab fragment was previously shown by X-ray crystallographic studies to be similar to the one found in the corresponding region of HRV1A. Antibodies raised against this peptide bind to and neutralize HRV2. In order to identify structural features preserved in solution that may explain the ability of this short peptide to mimic the structure of the protein surface, the peptide has been studied by NMR in aqueous solution as well as under denaturing conditions. The peptide is shown to be a random coil in solution. However, the sequence forming a 3₁₀ helix in the complex is biased into a helical conformation according to NOE intensity data as well as from urea and pH titrations. This sequence adopts the same conformation in an unrelated protein. NOE data suggest that a β-turn found in the complex may be sampled in solution. Also, Glu4, interacting with Arg6 in the crystal, has a reduced pK_a value in solution. It is concluded that the local structure present in the random coil state of VP2(156–170) contains enough information to direct the production of antibodies that bind to and neutralize HRV2. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2.

The crystal structure of the antigen-binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X-ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross-reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R-factor of 18.9% at 2.8 A resolution. The elbow angle, relating the variable and constant modules of the molecule is 127 degrees, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen-binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus.     

Structure of a neutralizing antibody bound bivalently to human rhinovirus 2.

The structure of a complex between human rhinovirus serotype 2 (HRV2) and the weakly neutralizing monoclonal antibody 8F5 has been determined to 25 A resolution by cryo-electron microscopy and 3-D reconstruction techniques. THe antibody is seen to be bound bivalently across the icosahedral 2-fold axis, despite the very short distance of 60 A between the symmetry-related epitopes. The canyon around the 5-fold axis is not obstructed. Due to extreme flexibility of the hinge region the Fc domains occupy random orientations and are not visible in the reconstruction. The atomic coordinates of Fab-8F5 complexes with a synthetic peptide derived from the viral protein 2 (VP2) epitope were fitted to the structure obtained by cryo-electron microscope techniques. The X-ray structure of HRV2 is not unknown, so that of the closely related HRV1A was placed in the electron microscopic density map. The footprint of 8F5 on the viral surface is largely on VP2, but also covers the VP3 loop centred on residue 3060. C alpha atoms of VP1 and 8F5 come no closer than 10 A. Based on the fit of the X-ray coordinates to the electron microscope data, the synthetic 15mer peptide starts and ends in close proximity to the corresponding amino acids of VP2 on HRV1A. However, the respective loops diverge considerably in their overall spatial disposition. It appears from this study that bivalent binding of an antibody directed against a picornavirus exists for a smaller spanning distance than was previously thought possible. Also bivalent binding does not ensure strong neutralization.     

Structural studies on the mechanisms of antibody-mediated neutralization of human rhinovirus

Antibodies represent a major component of the mammalian immunological defense against picornavirus infection. The work reviewed here examines structural details of antibody-mediated neutralization of human rhinovirus 14 (HRV14) using a combination of crystallography, molecular biology and electron microscopy. The atomic structures of the Fab fragment from a neutralizing monoclonal antibody (Fab17-IA) and HRV14 were used to interpret the ∼25Å resolution cryo-electron microscopy structure of the Fab17-IA/HRV14 complex. While there were not any observable antibody-induced conformational changes in the HRV14 upon antibody binding, there was evidence that charge interactions dominate the paratope-epitope interface and that the intact antibody might bind bivalently across icosahedral two-fold axes. Site-directed mutagenesis results confirmed that charge interactions dominate antibody binding and electron microscopy studies on the mAb17-IA/HRV14 complex confirmed that this neutralizing antibody binds bivalently across icosahedral two-fold axes.     

Cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type 14 uncoating

Release of the human rhinovirus (HRV) genome into the cytoplasm of the cell involves a concerted structural modification of the viral capsid. The intracellular adhesion molecule 1 (ICAM-1) cellular receptor of the major-group HRVs and the low-density lipoprotein (LDL) receptor of the minor-group HRVs have different nonoverlapping binding sites. While ICAM-1 binding catalyzes uncoating, LDL receptor binding does not. Uncoating of minor-group HRVs is initiated by the low pH of late endosomes. We have studied the conformational changes concomitant with uncoating in the major-group HRV14 and compared them with previous results for the minor-group HRV2. The structure of empty HRV14 was determined by cryoelectron microscopy, and the atomic structure of native HRV14 was used to examine the conformational changes of the capsid and its constituent viral proteins. For both HRV2 and HRV14, the transformation from full to empty capsid involves an overall 4% expansion and an iris type of movement of viral protein VP1 to open up a 10-A-diameter channel on the fivefold axis to allow exit of the RNA genome. The beta-cylinders formed by the N termini of the VP3 molecules inside the capsid on the fivefold axis all open up in HRV2, but we propose that only one opens up in HRV14. The release of VP4 is less efficient in HRV14 than in HRV2, and the N termini of VP1 may exit at different points. The N-terminal loop of VP2 is modified in both viruses, probably to detach the RNA, but it bends only inwards in HRV2.     

An externalized polypeptide partitions between two distinct sites on genome-released poliovirus particles

During cell entry, native poliovirus (160S) converts to a cell-entry intermediate (135S) particle, resulting in the externalization of capsid proteins VP4 and the amino terminus of VP1 (residues 1 to 53). Externalization of these entities is followed by release of the RNA genome (uncoating), leaving an empty (80S) particle. The antigen-binding fragment (Fab) of a monospecific peptide 1 (P1) antibody, which was raised against a peptide corresponding to amino-terminal residues 24 to 40 of VP1, was utilized to track the location of the amino terminus of VP1 in the 135S and 80S states of poliovirus particles via cryogenic electron microscopy (cryo-EM) and three-dimensional image reconstruction. On 135S, P1 Fabs bind to a prominent feature on the external surface known as the “propeller tip.” In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, at least 50 Å distant, at the icosahedral 2-fold axes. Further analysis showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips, P1 Fabs bound only at the 2-fold axes, or P1 Fabs simultaneously bound at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Hence, this portion of VP1 does not change conformation synchronously when switching from the 135S state. These conclusions are compatible with previous observations of multiple conformations of the 80S state and suggest that movement of the amino terminus of VP1 has a role in uncoating. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions.     

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.     

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T = 3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~ 350 Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~ 440 Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. &; Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA 108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.     

Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion.

Viral receptors serve both to target viruses to specific cell types and to actively promote the entry of bound virus into cells. Human rhinoviruses (HRVs) can form complexes in vitro with a truncated soluble form of the HRV cell surface receptor, ICAM-1. These complexes appear to be stoichiometric, with approximately 60 ICAM molecules bound per virion or 1 ICAM-1 molecule per icosahedral face of the capsid. The complex can have two fates, either dissociating to yield free virus and free ICAM-1 or uncoating to break down to an 80S empty capsid which has released VP4, viral RNA, and ICAM-1. This uncoating in vitro mimics the uncoating of virus during infection of cells. The stability of the virus-receptor complex is dependent on temperature and the rhinovirus serotype. HRV serotype 14 (HRV14)-ICAM-1 complexes rapidly uncoat, HRV16 forms a stable virus-ICAM complex which does not uncoat detectably at 34 degrees C, and HRV3 has an intermediate phenotype. Rhinovirus can also uncoat after exposure to mildly acidic pH. The sensitivities of individual rhinovirus serotypes to ICAM-1-mediated virus uncoating do not correlate with uncoating promoted by incubation at low pH, suggesting that these two means of virus destabilization occur by different mechanisms. Soluble ICAM-1 and low pH do not act synergistically to promote uncoating. The rate of uncoating does appear to be inversely related to virus affinity for its receptor.     

Structure of the Fab-labeled "breathing" state of native poliovirus

At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and N terminus of VP1 (residues 1 to 53) externalize reversibly. An Fab fragment of a monospecific antibody, which binds to residues 39 to 55 of VP1, was utilized to locate the N termini of VP1 in native (160S) particles in this "breathing" state. Fab and virus were mixed and imaged via cryogenic electron microscopy. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered cell entry intermediate (135S) particle, but the N terminus of VP1 is located near the 2-fold axes, instead of the "propeller tip" as in 135S particles.     

Structure of human rhinovirus serotype 2 (HRV2)

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 A resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded beta-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The betaE-betaF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.     

The three-dimensional structure of genomic RNA in bacteriophage MS2: implications for assembly

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density encompassing ∼90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.