Limits of structural plasticity in a picornavirus capsid revealed by a massively expanded equine rhinitis A virus particle.

UNLABELLED: The Picornaviridae family of small, nonenveloped viruses includes major pathogens of humans and animals. They have positive-sense, single-stranded RNA genomes, and the mechanism(s) by which these genomes are introduced into cells to initiate infection remains poorly understood. The structures of presumed uncoating intermediate particles of several picornaviruses show limited expansion and some increased porosity compared to the mature virions. Here, we present the cryo-electron microscopy structure of native equine rhinitis A virus (ERAV), together with the structure of a massively expanded ERAV particle, each at ∼17-Šresolution. The expanded structure has large pores on the particle 3-fold axes and has lost the RNA genome and the capsid protein VP4. The expanded structure thus illustrates both the limits of structural plasticity in such capsids and a plausible route by which genomic RNA might exit. IMPORTANCE: Picornaviruses are important animal and human pathogens that protect their genomic RNAs within a protective protein capsid. Upon infection, this genomic RNA must be able to leave the capsid to initiate a new round of infection. We describe here the structure of a unique, massively expanded state of equine rhinitis A virus that provides insight into how this exit might occur.

Virus isolate from carp: genetic characterization reveals a novel picornavirus with two aphthovirus 2A-like sequences.

Picornaviruses have been isolated from a variety of hosts, mainly mammals and birds. Here, we describe the sequence analysis of carp picornavirus 1 (CPV-1) F37/06 that was isolated from an organ pool (heart, brain, liver) of a common carp (Cyprinus carpio). This carp perished after an accidental discharge of liquid manure into a fish pond and presented without obvious clinical symptoms. Experimental intraperitoneal infection of young carp with CPV-1 revealed no clinical signs, but the virus was re-isolated from various organs. Sequence analysis of almost the complete genome (7632 nt excluding the poly-A tract) revealed a novel picornavirus clade. In phylogenetic trees, the polymerase sequence clusters with parechoviruses, duck hepatitis A virus, eel picornavirus and aquamavirus A. The ORF includes 6807 nt and encodes a polyprotein of 2269 amino acids. CPV-1 has a genome layout like that of picornaviruses except for the presence of two aphthovirus 2A-like NPGP sequence motifs: VPg+5'UTR[1AB-1C-1D-2A1(npgp)/2A2(npgp)-2B-2C(ATPase)/3A-3B(VPg)-3C(pro)-3D(pol)]3'UTR-poly-A. 2A1(npgp) and 2A2(npgp) are separated by 133 amino acids. The proteins 2A2(npgp), 2B, 3A and 3B(VPg) have no significant similarity to the corresponding proteins of other picornaviruses. Amino acid identities of the orthologous proteins P1, 2C, 3C(pro) and 3D(pol) range from 16.4 to 40.8 % in the eel picornavirus/CPV-1 comparison. 3D(pol) shows the closest similarity to eel picornavirus, with an amino acid identity of 40.8 %, followed by human parechovirus (36.5 %), duck hepatitis A virus (32.7 %) and swine pasivirus (29.3 %). Both the unique genome organization and low sequence similarity support the assignment of CPV-1 to a novel picornavirus species within a novel genus.

Crystal structure of equine rhinitis A virus in complex with its sialic acid receptor.

Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3'-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theiler's murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.

Sequence conservation and antigenic variation of the structural proteins of equine rhinitis A virus.

The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.

Equine rhinitis B virus: a new serotype.

Equine rhinovirus serotype 3 isolate P313/75 was assigned, with an unclassified genus status, to the family PICORNAVIRIDAE: The sequence from the 5' poly(C) tract to the 3' poly(A) tract of P313/75 was determined. The sequence is 8821 bases in length and contains a potential open reading frame for a polyprotein of 2583 amino acids. Sequence comparison and phylogenic analysis suggest that P313/75 is most closely related to the prototype equine rhinitis B virus (ERBV) strain P1436/71, formerly named equine rhinovirus type 2. A high degree of sequence similarity was found in the P2 and P3 regions of the two genomes. However, the deduced amino acid sequences of the P1 region of P313/75 and ERBV strain P1436/71 contained significant differences, which presumably account for the serological segregation of the two viruses. It is suggested that P313/75 can be classified as a new serotype of the genus Erbovirus, tentatively named ERBV2. Seroepidemiological data indicate that ERBV2 infection of horses may be common (24%) in Australia.

Foot-and-mouth disease virus: a long known virus, but a current threat.

Foot-and-mouth disease virus (FMDV) was the first animal virus identified. Since then, FMDV has become a model system in animal virology and a considerable amount of information on its structure, biology and vaccinology has been obtained. However, the disease that this virus produces (FMD) still constitutes one of the main animal health concerns. In this review, we have attempted to summarise the state of the knowledge in different basic and applied areas of FMDV research, with emphasis on those aspects relevant to the control of the disease.

Comparison of amino acid sequences at the amino acid 130-160 region of VP1 polypeptide of Indian field isolates of foot-and-mouth disease virus serotype Asia 1.

The nucleotide and deduced amino acid sequences in the amino acid (aa) 130-160 region of VP1 polypeptide of 65 field isolates of foot- and mouth disease virus (FMDV) serotype Asia 1 were determined and the consensus sequences were deduced. Comparison of amino acid sequences revealed conservation of NGK (130-132), TYG (134-136), RGD (142-144), and LPTSF (156-160) motifs and aa 148 (L) while variation was observed at the rest of the region (variability index (VI) of 2.06 to 16.85). Synonymous and non-synonymous mutations at the nucleotide level were well correlated with those of the corresponding amino acids. Comparison of the aa 130-160 sequence of Asia 1 serotype with those of other serotypes of FMDV revealed conservation of aa 135, 148-149, 157 and 160. Amino acids 133-138 and 148-154 were unique for Asia 1 serotype and are presumably responsible for its distinct antigenic nature. The present study revealed that the FMDV isolates of serotype Asia 1 causing outbreaks in India are very much heterogeneous in the aa 130-160 region of VP1.

A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G-H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2.3 A resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C(1). The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg-Gly-Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

[Synthetic peptide designs based on immunoactive fragments of the VP1 protein of the foot-and-mouth disease virus strain A22]. / Sinteticheskie peptidnye konstruktsii na osnove immunoaktivnykh fragmentov belka VP1 virusa iashchura shtamma A22.

Peptide constructs consisting of 44-53 aa were synthesized on the basis of sequences 135-159, 170-190 and 197-213 of VP1 from the foot-and-mouth disease A22 strain. Immunogenic and protective properties of the peptide constructs were studied in guinea pigs and mice of three lines. The constructs were shown to induce higher levels of antibodies and exhibit higher protective effects than the separate peptides. The most active among the peptides studied was the construct involving the VP1 fragments 135-160 and 170-190: it protected pigs from the experimental infection by the foot-and-mouth disease virus.

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

Foot-and-mouth disease and beyond: vaccine design, past, present and future.

The first experimental vaccines against foot-and-mouth disease were made in 1925 by Vallee, Carre and Rinjard using formaldehyde inactivation of tongue tissue from cattle infected with the virus. This method was essentially unaltered until the late 1940s when the important experiments by Frenkel in Holland showed that the quantities of virus required for vaccine production could be obtained from fragments of tongue epithelium incubated in vitro following infection with the virus. This major step made possible the comprehensive vaccination programmes which followed in Western Europe and which, in turn, resulted in the elimination of the disease from that part of the world by 1989. This spectacular success has led many to question whether other kinds of vaccine are required to control the disease worldwide. Such reservations ignore the danger to the environment associated with the growth of large amounts of virus. This can never be a zero-risk situation. Consequently, a vaccine which is not based on infectious virus as starting material has many attractions from safety considerations alone. In addition, a vaccine based on more fundamental considerations would not only be more aesthetically satisfying but could possibly provide an understanding at the molecular level of antigenic variation, still a problem in the control of the disease. The advances in our knowledge of the structure of the virus and the fragments which elicit a protective immune response now allow us to envisage a vaccine which does not require infectious virus and which protects against the multiple serotypes of the agent. Since antigenic variation is still a major problem in the control of the disease by vaccination, such a product would have important advantages over the current vaccines.

The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.

Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre-formed sulfate-binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate-binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.

Low temperature and pressure stability of picornaviruses: implications for virus uncoating.

The family Picornaviridae includes several viruses of great economic and medical importance. Poliovirus replicates in the human digestive tract, causing disease that may range in severity from a mild infection to a fatal paralysis. The human rhinovirus is the most important etiologic agent of the common cold in adults and children. Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases in cattle. These viruses have in common a capsid structure composed of 60 copies of four different proteins, VP1 to VP4, and their 3D structures show similar general features. In this study we describe the differences in stability against high pressure and cold denaturation of these viruses. Both poliovirus and rhinovirus are stable to high pressure at room temperature, because pressures up to 2.4 kbar are not enough to promote viral disassembly and inactivation. Within the same pressure range, FMDV particles are dramatically affected by pressure, with a loss of infectivity of more than 4 log units observed. The dissociation of polio and rhino viruses can be observed only under pressure (2.4 kbar) at low temperatures in the presence of subdenaturing concentrations of urea (1-2 M). The pressure and low temperature data reveal clear differences in stability among the three picornaviruses, FMDV being the most sensitive, polio being the most resistant, and rhino having intermediate stability. Whereas rhino and poliovirus differ little in stability (less than 10 kcal/mol at 0 degrees C), the difference in free energy between these two viruses and FMDV was remarkable (more than 200 kcal/mol of particle). These differences are crucial to understanding the different factors that control the assembly and disassembly of the virus particles during their life cycle. The inactivation of these viruses by pressure (combined or not with low temperature) has potential as a method for producing vaccines.

Conformational flexibility in a highly mobile protein loop of foot-and-mouth disease virus: distinct structural requirements for integrin and antibody binding.

The G-H loop of foot-and-mouth disease virus VP1 protein is a highly mobile peptide, that extends from the capsid surface and that in native virions is invisible by X-ray crystallography. In serotype C, this segment contains a hypervariable region with several continuous, overlapping, B-cell epitopes that embrace the conserved Arg-Gly-Asp (RGD) cell attachment motif. The solvent-exposed positioning of this peptide by selective insertion into different structural frameworks of E. coli beta-galactosidase, generates a spectrum of antigenic variants which react distinctively with a panel of anti-VP1 monoclonal antibodies and exhibit different efficiencies as cell ligands. The cell attachment efficiency is much less restricted by the different positioning of the viral segment at the insertion sites. A molecular model of an inserted stretch reveals a highest flexibility of the RGD tripeptide segment compared with the flanking sequences, that could allow a proper accommodation to integrin receptors even in poorly antigenic conformations. The non-converging structural requirements for RGD-mediated integrin binding and antibody recognition, explains the dynamism of the generation of neutralisation-resistant antigenic variants in the viral quasi-species, arising from a conformational space of integrin-binding competent peptides. This might be of special relevance for foot-and-moth disease virus evolution, since unlike in other picornaviruses, the cell binding motif and the major neutralising B-cell epitopes overlap in a solvent-exposed peptide accessible to the host immune system, in a virion lacking canyons and similar hiding structures.

Mutational analysis of discontinuous epitopes of foot-and-mouth disease virus using an unprocessed capsid protomer precursor.

An unprocessed capsid precursor (P1) of foot-and-mouth disease virus (FMDV) has been expressed in mammalian cells to study discontinuous epitopes involved in viral neutralization. Amino acid replacements found in virus-escape mutants were engineered in the P1 precursor by site-directed mutagenesis of the plasmid. In all cases the replacements abolished recognition of unprocessed P1 by the relevant monoclonal antibodies (MAbs), paralleling the effects of the corresponding substitutions in neutralization of infectious FMDV. Five capsid surface residues within the same discontinuous antigenic area that were never found replaced in escape mutants were also engineered in P1. None of the substitutions affected antibody recognition, suggesting that these residues were not directly involved in the interaction with the antibodies tested. The results validate site-directed mutagenesis of constructs encoding capsid precursors as an approach to probe the structure of viral discontinuous epitopes not amenable to analysis with synthetic peptides.

Characterizing sequence variation in the VP1 capsid proteins of foot and mouth disease virus (serotype 0) with respect to virion structure.

The VP1 capsid protein of foot and mouth disease virus (FMDV) is highly polymorphic and contains several of the major immunogenic sites important to effective antibody neutralization and subsequent viral clearance by the immune system. Whether this high level of polymorphism is of adaptive value to the virus remains unknown. In this study we examined sequence data from a set of 55 isolates in order to establish the nature of selective pressures acting on this gene. Using the known molecular structure of VP1, the rates and ratios of different types of nonsynonymous and synonymous changes were compared between different parts of the protein. All parts of the protein are subject to purifying selection, but this is greatest amongst those amino acid residues within beta-strands and is significantly reduced at residues exposed on the capsid surface, which include those residues demonstrated by previous mutational analyses to permit the virus to escape from monoclonal antibody binding. The ratios of nonsynonymous substitution resulting in various forms of physicochemically radical and conserved amino acid change were shown to be largely equal throughout these different parts of the protein. There was a consistently higher level of nonsynonymous and charge radical sites in those regions of the gene coding for residues exposed on the outer surface of the capsid and a marked difference in the use of amino acids between surface and nonsurface regions of the protein. However, the analysis is consistent with the hypothesis that the observed sequence variation arises where it is least likely to be disruptive to the higher-order structure of the protein and is not necessarily due to positive Darwinian selection.

Titration calculations of foot-and-mouth disease virus capsids and their stabilities as a function of pH.

Foot-and-mouth disease virus (FMDV), a non-enveloped picornavirus, is sensitive to acidic conditions. At pH values below 7 the icosahedral virus capsid, formed from 60 copies of a protomer containing four polypeptides (VP1 to 4), dissociates into 12 pentamers, releasing the viral RNA. Evidence suggests that this acid lability may assist FMDV cell entry via an endosomal pathway. Calculations of titration curves and pH-stability profiles are presented for three different strains of FMDV, O1BFS, A10(61) and A22 Iraq, and compared with experimental data for complete virions and empty capsids (which lack RNA). The finite difference Poisson-Boltzmann method was used for the calculation of electrostatic free energies with the solvent treated as a dielectric continuum. The inter-pentamer interface in the virus is formed by two protomers related by 2-fold icosahedral symmetry. As a simple model for inter-pentamer interactions, a dimer and two separate protomers were compared. The association free energy was computed by integrating the difference between the titration curves of the two species. The calculations reproduced the observed decrease in capsid stability at acidic pH but not the difference in pH sensitivities of the two type A viruses. It is shown that only residues within 15 A of the interface play a significant role in determining acid lability. For the experimentally studied pH range (5 to 7.6), histidine residues were found to dominate the pH-dependence of the stability. Two histidine residues in VP3, H142 and H145, are shown to have the greatest effect by virtue of their interactions with many polar residues across the inter-pentamer interface; the interaction of H142 with an alpha-helix in the opposite pentamer contributes only a small proportion of the destabilization energy.

Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex.

Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.

Structural comparison between retro-inverso and parent peptides: molecular basis for the biological activity of a retro-inverso analogue of the immunodominant fragment of VP1 coat protein from foot-and-mouth disease virus.

Antibodies induced against intact foot-and-mouth disease Virus (FMDV) particles bind to the retro-inverso analogue of fragment 141-159 of the viral coat protein VP1 of FMDV, variant A, equally well as to the parent peptide. A conformational investigation of this retro-inverso peptide was carried out by nmr spectroscopy and restrained molecular modeling in order to identify the structural basis for the antigenic mimicry between these retro-inverso and parent peptides. In 100% trifluoroethanol a well-defined left-handed alpha-helical region exists from residue 150 to residue 159, which is consistently present in all conformational families obtained from restrained modelling. A less-defined left-handed helical region is present in the tract 144-148, which is also consistent for all structures. Conformational flexibility exists about Gly149, which leads to two types of structures, either bent or linear. In the bent structures, a three-residue inverse tight turn is found, which can be classified as an inverse gamma-turn centered at Gly149. The overall structural features of the retro-inverso peptide are shown to be similar to those of the parent L-peptide. The two molecules, however, are roughly mirror images because they share inherently chiral secondary structure elements. By comparing these conformational conclusions with the x-ray structure of the Fab complex of a corresponding VP1 antigenic fragment, a rationale is proposed to account for the topological requirements of specific recognition that are implied by the equivalent antigenic activity of the natural and retro-inverso compounds.