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Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV, genus aphthovirus), one of the most notorious pathogens in global livestock industry, needs to navigate antivirals host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I IFN response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV Lpro or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2, but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved amongst aphthoviruses. Neither FMDV or ERAV Lpro interfered with phosphorylation of PKR or eIF2α, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response.IMPORTANCE The picornavirus foot and mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on global life stock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral dsRNA is detected which results in activation of the PKR-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here we show that FMDV can suppress SG formation via its leader protease (Lpro). Simultaneously we observed that Lpro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of FMDV's antiviral host response evasion strategies may help to develop countermeasures to control FMDV infections in the future.

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (IFN) response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV leader protease (L(pro)) or 3C(pro), we demonstrate an essential role for L(pro) in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking L(pro) resulted in SG formation. Additionally, we show that L(pro) cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) L(pro) also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV L(pro) interfered with phosphorylation of RNA-dependent protein kinase (PKR) or eIF2α, indicating that L(pro) does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response. IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (L(pro)). Simultaneously, we observed that L(pro) can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future.

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-Å resolution. 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.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH4Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations., Work at the F.S. laboratory was supported by grants from Ministerio de Ciencia e Innovación (MICINN) BIO2008-0447-C03-01 and CSD2006-0007; work at the M.G.M. laboratory was supported by grants from MICINN (BIO2009-10092) and Comunidad de Madrid (S-2009/MAT/1467). An institutional grant from Fundación Ramón Areces is also acknowledged.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8 T cells in FMDVvaccinated and -infected cattle. In the current study, we aimed to identify CD8 T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-)producing CD8 T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8 T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN- release, proliferation, and target cell killing by CD8 T cells, but not CD4 T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8 T cells from FMDV serotype O-vaccinated A31 cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8 T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes. Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae, genus Aphthovirus. The FMDV particle consists of a positive-strand RNA molecule of approximately 8,500 nucleotides, enclosed within an icosahedral capsid. The genome encodes a unique polyprotein from which four structural proteins (P1A, P1B, P1C, and P1D; also referred to as VP4, VP2, VP3, and VP1, respectively) and nine nonstructural proteins are cleaved by viral proteases (48). FMDV shows a high genetic and antigenic variability, which is reflected in the seven serotypes and multiple subtypes reported so far (13). The virus causes a highly contagious infection in cloven-hoofed animals which is characterized by the formation of vesicles on the mouth, tongue, nose, and feet. In addition, most infected animals develop viremia. The virus elicits a rapid humoral response in both infected and vaccinated animals (26). Virus-specific antibodies protect animals in a serotype-specific manner against reinfection or against infection in the case of vaccination, and protection is generally correlated with high levels of neutralizing antibodies (38). Control of the disease is achieved by vaccination with a chemically inactivated whole-virus vaccine emulsified with adjuvant; however, this provides only short-term, serotype-specific protection (2). The introduction of this vaccine has been very successful in areas of the world where the disease is enzootic. However, one of the major difficulties in implementing vaccination is the inability to distinguish vaccinated animals from infected/recovered animals, which may still be shedding virus. Currently, a number of assays specifically developed for

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo . FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin αvβ6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-β) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-β mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-α mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo . In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH 4 Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH 4 Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH 4 Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.

The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed inEscherichia colias myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin αvβ6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cproin vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (alphavbeta1, alphavbeta3, alphavbeta6, and alphavbeta8) as receptors to initiate infection. Infection mediated by alphavbeta6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express alphavbeta8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by alphavbeta8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent ( approximately 35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE.

Foot-and-mouth disease virus (FMDV) is the causative agent of an acute vesicular disease affecting wild and domesticated animals. Despite the economic burden of the disease and all efforts to eradicate it, FMD outbreaks continue to emerge unexpectedly in developed and developing countries. Correlation of the mutational dynamics of the virus with its epidemiology remains unexplored. Analysis of 103 complete genomes representing the seven serotypes shows the important role that selection plays in the genomic evolution of viral isolates for serotypes. We identified selection and relaxed constraints due to genetic drift through analyses of synonymous sites. Finally, we investigated interactions between mutations that showed coevolving patterns and analyzed, based on protein structures, slightly deleterious and compensatory mutational dynamics. Specifically, we demonstrate that structurally exposed capsid proteins present a greater number of adaptive mutations and relaxed selection than nonstructural proteins. Such events have been magnified during the evolution of the southern African virus types (SATs). These shifts in selection-drift balance have generated the great antigenic and genetic diversity observed for SAT serotypes and that are responsible for epizootics on the continent of Africa. The high number of slightly deleterious and compensatory mutations in SAT serotypes in structural proteins is testament to such balance plasticity. The significant accumulation of these coevolving mutations in African serotypes supports their contribution in generating adaptive immune-escaping mutants and in establishing persistent infections. The reverse of this pattern in nonstructural proteins reveals the neutral fixation of mutations in the more widely spread and commonly studied Euro-Asiatic serotypes.

The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) self-processes inefficiently at the Lpro/VP4 cleavage site LysLeuLys*GlyAlaGly (* indicates cleaved peptide bond) when the leucine at position P2 is replaced by phenylalanine. Molecular modeling and energy minimization identified the Lproresidue L143 as being responsible for this discrimination. The variant LproL143A self-processed efficiently at the Lpro/VP4 cleavage site containing P2 phenylalanine, whereas the L143M variant did not. LproL143A self-processing at the eIF4GII sequence AspPheGly*ArgGlnThr was improved but showed more-extensive aberrant processing. Residue 143 in Lprois occupied only by leucine and methionine in all sequenced FMDV serotypes, implying that these bulky side chains are one determinant of the restricted specificity of Lpro.

The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin αvβ6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to αvβ6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin αvβ6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.

Foot-and-mouth disease virus (FMDV), like other RNA viruses, exhibits high mutation rates during replication that have been suggested to be of adaptive value. However, even though genetic variation in RNA viruses and, more specifically, FMDV has been extensively examined during virus replication in a wide variety of in vitro cell cultures, very little is known regarding the generation and effects of genetic variability of virus replication in the natural host under experimental conditions and no genetic data are available regarding the effects of serial passage in natural hosts. Here, we present the results of 20 serial contact transmissions of the highly pathogenic, pig-adapted O Taiwan 97 (O Tw97) isolate of FMDV in swine. We examined the virus genomic consensus sequences for a total of 37 full-length viral genomes recovered from 20 in vivo passages. The characteristics and distributions of changes in the sequences during the series of pig infections were analyzed in comparison to the O Tw97 genomes recovered from serially infected BHK-21 cell cultures. Unexpectedly, a significant reduction of virulence upon pig passages was observed, and finally, interruption of the viral transmission chain occurred after the14th pig passage (T14). Virus was, however, isolated from the tonsils and nasal swabs of the asymptomatic T15 pigs at 26 days postcontact, consistent with a natural establishment of the carrier state previously described only for ruminants. Surprisingly, the region encoding the capsid protein VP1 (1D) did not show amino acid changes during in vivo passages. These data demonstrate that contact transmission of FMDV O Tw97 in pigs mimics the fitness loss induced by the bottleneck effect, which was previously observed by others during plaque-to-plaque FMDV passage in vitro, suggesting that unknown mechanisms of virulence recovery might be necessary during the evolution and perpetuation of FMDV in nature.

The nucleoside analogue ribavirin (R) is mutagenic for foot-and-mouth disease virus (FMDV). Passage of FMDV in the presence of increasing concentrations of R resulted in the selection of FMDV with the amino acid substitution M296I in the viral polymerase (3D). Measurements of progeny production and viral fitness with chimeric viruses in the presence and absence of R documented that the 3D substitution M296I conferred on FMDV a selective replicative advantage in the presence of R but not in the absence of R. In polymerization assays, a purified mutant polymerase with I296 showed a decreased capacity to use ribavirin triphosphate as a substrate in the place of GTP and ATP, compared with the wild-type enzyme. The results suggest that M296I has been selected because it attenuates the mutagenic activity of R with FMDV. Replacement M296I is located within a highly conserved stretch in picornaviral polymerases which includes residues that interact with the template-primer complex and probably also with the incoming nucleotide, according to the three-dimensional structure of FMDV 3D. Given that a 3D substitution, distant from M296I, was associated with resistance to R in poliovirus, the results indicate that picornaviral polymerases include different domains that can alter the interaction of the enzyme with mutagenic nucleoside analogues. Implications for lethal mutagenesis are discussed.

The 3C protease (3C pro ) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C pro , a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a β-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This β-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the β-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C pro with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the β-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C pro inhibitors.

Picornaviridae are a large virus family causing widespread, often pathogenic infections in humans and other mammals. Picornaviruses are genetically and antigenically highly diverse, with evidence for complex evolutionary histories in which recombination plays a major part. To investigate the nature of recombination and selection processes underlying the evolution of serotypes within different picornavirus genera, large-scale analysis of recombination frequencies and sites, segregation by serotype within each genus, and sequence selection and composition was performed, and results were compared with those for other nonenveloped positive-stranded viruses (astroviruses and human noroviruses) and with flavivirus and alphavirus control groups. Enteroviruses, aphthoviruses, and teschoviruses showed phylogenetic segregation by serotype only in the structural region; lack of segregation elsewhere was attributable to extensive interserotype recombination. Nonsegregating viruses also showed several characteristic sequence divergence and composition differences between genome regions that were absent from segregating virus control groups, such as much greater amino acid sequence divergence in the structural region, markedly elevated ratios of nonsynonymous-to-synonymous substitutions, and differences in codon usage. These properties were shared with other picornavirus genera, such as the parechoviruses and erboviruses. The nonenveloped astroviruses and noroviruses similarly showed high frequencies of recombination, evidence for positive selection, and differential codon use in the capsid region, implying similar underlying evolutionary mechanisms and pressures driving serotype differentiation. This process was distinct from more-recent sequence evolution generating diversity within picornavirus serotypes, in which neutral or purifying selection was prominent. Overall, this study identifies common themes in the diversification process generating picornavirus serotypes that contribute to understanding of their evolution and pathogenicity.

The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 × 10 −5 per site per day (95% confidence interval [CI], 1.75 × 10 −5 to 2.80 × 10 −5 ) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10 6 PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID 50 ) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 × 10 9 PFU each and challenged 24 h later with 100 ID 50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.

We have previously shown that the leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) blocks cap-dependent mRNA translation and that a genetically engineered FMDV lacking the leader proteinase coding region (A12-LLV2) is attenuated in cell culture and susceptible animals. The attenuated phenotype apparently is a consequence of the inability of A12-LLV2 to block the expression of type I interferon (IFN-α/β) protein, resulting in IFN-induced inhibition of FMDV replication. Here we show that in addition to preventing IFN-α/β protein synthesis, Lproreduces the level of immediate-early induction of IFN-β mRNA and IFN-stimulated gene products such as double-stranded RNA-dependent protein kinase R (PKR), 2′,5′-oligoadenylate synthetase, and Mx1 mRNAs in swine cells. Down-regulation of cellular PKR by RNA interference did not affect wild-type virus yield but resulted in a higher yield of A12-LLV2, indicating a direct role of PKR in controlling FMDV replication in the natural host. The observation that Lprocontrols the transcription of genes involved in innate immunity reveals a novel role of this protein in antagonizing the cellular response to viral infection.

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the α V subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the βG-βH loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the α V β 3 and α V β 6 integrins, virus adsorbed to the cells at 4°C appears to colocalize almost exclusively with the α V β 6 integrin. Upon shifting the infected cells to 37°C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.

The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D pol . To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D pol in the presence of the 3CD precursor plus an internal RNA sequence termed a c is-acting r eplication e lement ( cre ). The FMDV cre has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3 B - u ridylylation s ite, or bus ) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5′ and 3′ untranslated regions (UTR), as was tolerance for insertions/deletions in the 5′ UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L pro , 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.

RNA interference (RNAi) is a powerful tool to silence gene expression posttranscriptionally. In this study, we evaluated the antiviral potential of small interfering RNA (siRNA) targeting VP1 of foot-and-mouth disease virus (FMDV), which is essential during the life cycle of the virus and plays a key role in virus attachment to susceptible cells. We investigated in vivo the inhibitory effect of VP1-specific siRNAs on FMDV replication in BHK-21 cells and suckling mice, a commonly used small animal model. The results showed that transfection of siRNA-expressing plasmids gave an 80 to 90% reduction in the expression of FMDV VP1 in BHK-21 cells. Moreover, BHK-21 cells transiently transfected with siRNA-expressing plasmids were specifically resistant to FMDV infection when exposed to 100 50% tissue culture infective doses of virus, and the antiviral effects extended to almost 48 h postinfection. Furthermore, subcutaneous injection of siRNA-expressing plasmids in the neck made suckling mice significantly less susceptible to FMDV. In conclusion, our data suggests that RNAi may provide a viable therapeutic approach to treat FMDV infection.

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three alphav integrins, alphavbeta1, alphavbeta3, and alphavbeta6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human beta8 cDNA and expression of alphavbeta8 at the cell surface. The involvement of alphavbeta8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the alphavbeta8 heterodimer or the alphav chain. Similar results were obtained with a chimeric alphavbeta8 including the beta6 cytodomain (alphavbeta8/6), showing that the beta6 cytodomain can substitute efficiently for the corresponding region of beta8. In contrast, virus binding to alphavbeta6 including the beta8 cytodomain (alphavbeta6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the alphavbeta6 chimera was recognized poorly by antibodies specific for the ectodomain of alphavbeta6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the beta6 cytodomain is important for maintaining alphavbeta6 in a conformation required for productive infection by FMDV.

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 A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus , the only non- Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S -transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections.

In 1997, an epizootic in Taiwan, Province of China, was caused by a type O foot-and-mouth disease virus which infected pigs but not cattle. The virus had an altered 3A protein, which harbored a 10-amino-acid deletion and a series of substitutions. Here we show that this deletion is present in the earliest type O virus examined from the region (from 1970), whereas substitutions surrounding the deletion accumulated over the last 29 years. Analyses of the growth of these viruses in bovine cells suggest that changes in the genome in addition to the deletion, per se, are responsible for the porcinophilic properties of current Asian viruses in this lineage.

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5′ nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 10 6 -fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin α v β 3 as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin α v β 3 and have compared the two receptors for utilization by FMDV. Both the α v and β 3 subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human α v subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the β 3 subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with α v and β 3 subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine α v β 3 subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine α v subunit and a human β 3 subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human α v and the bovine β 3 subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine β 3 subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human β 3 subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine β 3 subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143→Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin α v β 3 . In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin alphavbeta3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, alphavbeta6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin beta6 subunit and expression of alphavbeta6 at the cell surface. Integrin alphavbeta6 is the major site for virus attachment on the beta6-transfected cells, and binding to alphavbeta6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the beta6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes alphavbeta6. These studies establish a role for alphavbeta6 as a cellular receptor for FMDV.

Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3C-mut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.

Empty capsids of foot-and-mouth disease virus (FMDV) type A22 Iraq 24/64, whose structure has been solved by X-ray crystallography, are unusual for picornaviruses since they contain VP2 and VP4, the cleavage products of the protein precursor VP0. Both the N terminus of VP1 and the C terminus of VP4, which pack together close to the icosahedral threefold symmetry axis where three pentamers associate, are more disordered in the empty capsid than they are in the RNA-containing virus. The ordering of these termini in the presence of RNA strengthens interactions within a single protomer and between protomers belonging to different pentamers. The disorder in the FMDV empty capsid forms a subset of that seen in the poliovirus empty capsid, which has VP0 intact. Thus, VP0 cleavage confers stability on the picornavirus capsid over and above that attributable to RNA encapsidation. In both FMDV and poliovirus empty capsids, the internal disordering uncovers a conserved histidine which has been proposed to be involved in the cleavage of VP0. A comparison of the putative active sites in FMDV and poliovirus suggests a structural explanation for the sequence specificity of the cleavage reaction.

DNA vaccine candidates for foot-and-mouth disease (FMD) were engineered to produce FMD virus (FMDV) particles that were noninfectious in cell culture or animals. The prototype plasmid, pWRM, contains a cytomegalovirus immediate-early promoter-driven genome-length type A12 cDNA followed by the bovine growth hormone polyadenylation site. BHK cells transfected with this plasmid produced virus, but the specific infectivity of pWRM was much lower than that achieved with in vitro-generated RNA genomes. To improve the infectivity of the plasmid, a cDNA encoding the hepatitis delta virus ribozyme was added to the 3' end of the FMDV cDNA. The resulting plasmid, pWRMH, exhibited slightly increased infectivity in cell culture and produced virus when inoculated into suckling mice. A third plasmid, pWRMHX, was created by removal of the sequences encoding the cell binding site found in capsid protein VP1 of pWRMH. Although cells transfected with pWRMHX produced viral capsids, this plasmid was not lethal in suckling mice, indicating that particles lacking the cell binding site were not able to initiate secondary infectious cycles. Swine inoculated with pWRMHX did not show any signs of disease and produced neutralizing antibodies to FMDV, and 20% of the vaccinated animals were protected from challenge. A derivative of pWRMHX, pWRMHX-pol-, harboring a mutation designed to inactivate the viral polymerase was much less immunogenic, indicating that immunogenicity of pWRMHX resulted, in part, from amplification of the viral genome in the animal.

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.

A comparison of picornavirus internal ribosome entry site (IRES) secondary structures revealed the existence of conserved motifs located on loops. We have carried out a mutational analysis to test their requirement for IRES-driven translation. The GUAA sequence, located in the aphthovirus 3A loop, did not tolerate substitutions that disrupt the GNRA motif. Interestingly, this motif was found at similar positions in all picornavirus IRESs, suggesting that it may form part of a tertiary-structure element. The RAAA tetranucleotide located in the 3B loop was conserved only in cardiovirus and aphthovirus. A mutational analysis of the RAAA motif revealed that activities of 3B loop mutants correlated with both the presence of a sequence close to CAAA at the new 3B loop and the absence of reorganization of the 3B and 3C stem-loops. In support of this conclusion, insertion of a large number of nucleotides close to the 3B loop, which was predicted to reorganize the 3B-3C stem-loop structure, led to defective IRES elements. We conclude that the aphthovirus IRES loops located at the most distal part of domain 3, which carries GNRA and RAAA motifs, are essential for IRES function.

The RNA genomes from the cardioviruses, hepatoviruses, and aphthoviruses encode two to five tandem pseudoknots within their 5' untranslated regions. These pseudoknots lie adjacent to a pyrimidine-rich sequence, which in cardio- and aphthoviruses takes the form of a homopolymeric poly(C) tract. Seven deletion mutations within mengovirus pseudoknots PK(B) and PK(C) were created and characterized. tested in tissue culture, mengovirus genomes with alterations in PK(C) were viable but had small plaque phenotypes. Larger plaque revertants were isolated and partially characterized, and each proved to be a second-site pseudorevertant with (unmapped) changes elsewhere in the genome. The infectious PK(C) mutant viruses were highly lethal to mice, and deletions in this motif did not affect mengovirus virulence in the same manner as deletions in the adjacent poly(C) tract. In contrast, deletions in PK(B), or deletions which spanned PK(B) + PK(C), produced nonviable genomes. Cell-free translations directed by any of the altered PK sequences gave normal polyprotein amounts relative to wild-type mengovirus. But viral RNA accumulation during HeLa cell infection was dramatically impaired, even with the least disruptive of the PK(C) changes, suggesting the pseudoknots play an essential though undefined role in RNA synthesis and moreover that an intact PK(B) structure is critical to this function.

A population replacement experiment has been devised to test the ability of a challenge virus to replace the resident virus in a persistently infected cell culture. BHK-21 cells persistently infected with foot-and-mouth disease virus of serotype C (clone C-S8c1) were challenged with a large excess of either the parental foot-and-mouth disease virus C-S8c1, genetically marked variants differing in their degree of virulence, or a mutant rescued after prolonged persistence in BHK-21 cells. After challenge, the composition of the resident virus population in the carrier culture was analyzed by reverse transcription-PCR amplification and nucleotide sequencing. The dominance of the initial persisting virus was seen in all cases, except when virulent viruses were used in the challenge. The experiments document that, paradoxically, virulence can be a positive factor in the reestablishment of a virus population in a persistently infected cell culture. A model based on the selection of virus-resistant cell variants during persistence is proposed to interpret these observations. Implications about the persistence of viruses in their host cells and organisms are discussed.

Binding of foot-and-mouth disease virus (FMDV) to cells requires an arginine-glycine-aspartic acid (RGD) sequence in the capsid protein VP1. We have genetically engineered an FMDV in which these three amino acids have been deleted, producing a virus particle which is unable to bind to cells. Cattle vaccinated with these receptor binding site-deleted virions were protected from disease when challenged with a virulent virus, demonstrating that these RGD-deleted viruses could serve as the basis for foot-and-mouth disease vaccines safer than those currently in use. This strategy may prove useful in the development of vaccines for other viral diseases.

The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and in the cleavage of translation initiation factor p220, a subunit of the cap-binding protein complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this information, we have investigated the active-site residues by introducing specific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cis activity at the L/VP4 cleavage site, trans activity on a truncated L-P1 substrate, and p220 cleavage activity. Mutations of Cys-23 to ser or His-110 to Leu resulted in enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-length FMDV cDNA, and RNA transcripts derived from these cDNAs were translated in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within P1, and virus obtained from transfected cells reverted to wild type. The H110L mutant cleaved the L/P1 junction almost as well as the wild-type enzyme, and virus recovered from transfected cells retained the mutation and displayed wild-type viral protein synthesis and host shut-off kinetics.

The effect of deletion of each of the two authentic polyprotein translation initiation sites of foot-and-mouth disease virus on viral protein synthesis and replication was analyzed. Deletion of either the first or the second initiation site led to the expression of only one form of the leader protein, L or L', respectively, but in vitro processing of the viral polyprotein and cleavage of eIF-4 gamma were not affected by either deletion. Whereas RNA in which the first translation initiation site had been deleted led to the production of viruses in transfected BHK cells, deletion of the second translation initiation site abolished virus replication.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.

We recently have shown that binding of foot-and-mouth disease virus (FMDV) to cells in culture requires an arginine-glycine-aspartic acid (RGD) sequence in the G-H loop of the capsid protein VP1 (P. W. Mason, E. Rieder, and B. Baxt, Proc. Natl. Acad. Sci. USA 91:1932-1936, 1994). In this report, we show that FMDV type A12 viruses found in infected bovine tongue tissue (BTT) differ from their tissue culture-grown derivatives at amino acid residues near the RGD. Viruses genetically engineered to contain VP1 sequences found in animal tissue (BTT viruses) were antigenically different from their tissue culture derivatives and bound to BHK cells more poorly than did the tissue culture-adapted viruses. Passage of the genetically engineered BTT viruses in BHK cells resulted in the rapid selection of variants with cell-binding properties, antigenic characteristics, and sequences typical of tissue culture-adapted viruses. These data indicate that residues near the RGD are critical for cell binding and that interpretations of antigenic variation of FMDV can be affected by virus cultivation in vitro.

A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.

A region of about 435 bases from the 5' noncoding region of foot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive secondary structure. Precise deletion of five predicted secondary structure features has been performed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of the modified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intact foot-and-mouth disease virus IRES complemented four of these defective elements to an efficiency of up to 80% of wild-type activity. No complementation was observed with the structurally analogous element from encephalomyocarditis virus. The role of RNA-RNA interactions in the function of the picornavirus IRES is discussed.

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae . For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.

Synthetic peptides based on the VP1 proteins of two serotypes of foot-and-mouth disease virus (FMDV) and having the general formula C-C-(200-213)-P-P-S-(141-158)-P-C-G induce heterologous as well as homologous protection against challenge. Substitution of the sequence consisting of residues 200 to 213 (200-213 sequence) with a second copy of the homologous 141-158 sequence (i.e., homodimers) resulted in failure of either serotype peptide to protect heterologously. The antiviral and antipeptide titers of sera from guinea pigs immunized with the homodimeric 141-158 peptides showed serotype specificity and, with the data from the heterodimeric peptide vaccines, suggested that the C-terminal 141-158 sequence was more effectively recognized by the immune system than the N-terminal sequence. Whereas heterologous antiviral titers as measured by enzyme-linked immunosorbent assay and virus neutralization tests have not been observed with sera from cross-protected animals, epitope-mapping studies established that there was heterologous recognition of an octapeptide within the 200-213 sequence. That the 200-213 sequence was required for the induction of heterologous protection was also confirmed with a number of peptides, including hybrids based on the 200-213 sequence of one virus and the 141-158 sequence of a second virus. Thus, peptides of the general formula given above induce serotype-specific and serotype-cross-reactive protective antibodies and are unique in their induction of significant levels of heterologous protection, a property which has never been reported for whole FMDV vaccines.