The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication, but essential for the production of infectious virus.

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5' untranslated region (5' UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.

Mutagenesis Mapping of RNA Structures within the Foot-and-Mouth Disease Virus Genome Reveals Functional Elements Localized in the Polymerase (3Dpol)-Encoding Region.

RNA structures can form functional elements that play crucial roles in the replication of positive-sense RNA viruses. While RNA structures in the untranslated regions (UTRs) of several picornaviruses have been functionally characterized, the roles of putative RNA structures predicted for protein coding sequences (or open reading frames [ORFs]) remain largely undefined. Here, we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome to predict 53 conserved RNA structures within the ORF. Forty-six of these structures were located in the regions encoding the nonstructural proteins (nsps). To investigate whether structures located in the regions encoding the nsps are required for FMDV replication, we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimize RNA secondary structures while preserving protein coding, native dinucleotide frequencies, and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV subgenomic replicon. We found that three of the RNA structures, all at the 3' termini of the FMDV ORF, were critical for replicon replication. In contrast, disruption of the other 43 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. IMPORTANCE Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only three are essential for replication of an FMDV subgenomic replicon. Replicon replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify whether the remaining 43 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease, posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

Picornaviruses: A View from 3A.

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

Foot-and-mouth disease virus: Prospects for using knowledge of virus biology to improve control of this continuing global threat.

Understanding of the biology of foot-and-mouth disease virus (FMDV) has grown considerably since the nucleotide sequence of the viral RNA was determined. The ability to manipulate the intact genome and also to express specific parts of the genome individually has enabled detailed analyses of viral components, both RNA and protein. Such studies have identified the requirements for specific functional elements for virus replication and pathogenicity. Furthermore, information about the functions of individual virus proteins has enabled the rational design of cDNA cassettes to express non-infectious empty capsid particles that can induce protective immunity in the natural host animals and thus represent new vaccine candidates. Similarly, attempts to block specific virus activities using antiviral agents have also been performed. However, currently, only the well-established, chemically inactivated FMDV vaccines are commercially available and suitable for use to combat this important disease of livestock animals. These vaccines, despite certain shortcomings, have been used very successfully (e.g. in Europe) to control the disease but it still remains endemic in much of Africa, southern Asia and the Middle East. Hence there remains a significant risk of reintroduction of the disease into highly susceptible animal populations with enormous economic consequences.

A low-passage insect-cell isolate of bluetongue virus uses a macropinocytosis-like entry pathway to infect natural target cells derived from the bovine host.

Bluetongue virus (BTV) causes an economically important disease in domestic and wildlife ruminants and is transmitted by Culicoides biting midges. In ruminants, BTV has a wide cell tropism that includes endothelial cells of vascular and lymphatic vessels as important cell targets for virus replication, and several cell types of the immune system including monocytes, macrophages and dendritic cells. Thus, cell-entry represents a particular challenge for BTV as it infects many different cell types in widely diverse vertebrate and invertebrate hosts. Improved understanding of BTV cell-entry could lead to novel antiviral approaches that can block virus transmission from cell to cell between its invertebrate and vertebrate hosts. Here, we have investigated BTV cell-entry using endothelial cells derived from the natural bovine host (BFA cells) and purified whole virus particles of a low-passage, insect-cell isolate of a virulent strain of BTV-1. Our results show that the main entry pathway for infection of BFA cells is dependent on actin and dynamin, and shares certain characteristics with macropinocytosis. The ability to use a macropinocytosis-like entry route could explain the diverse cell tropism of BTV and contribute to the efficiency of transmission between vertebrate and invertebrate hosts.

Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays.

Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.

Full Genome Sequencing Reveals New Southern African Territories Genotypes Bringing Us Closer to Understanding True Variability of Foot-and-Mouth Disease Virus in Africa.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that poses a constant burden on farmers in endemic regions and threatens the livestock industries in disease-free countries. Despite the increased number of publicly available whole genome sequences, FMDV data are biased by the opportunistic nature of sampling. Since whole genomic sequences of Southern African Territories (SAT) are particularly underrepresented, this study sequenced 34 isolates from eastern and southern Africa. Phylogenetic analyses revealed two novel genotypes (that comprised 8/34 of these SAT isolates) which contained unusual 5′ untranslated and non-structural encoding regions. While recombination has occurred between these sequences, phylogeny violation analyses indicated that the high degree of sequence diversity for the novel SAT genotypes has not solely arisen from recombination events. Based on estimates of the timing of ancestral divergence, these data are interpreted as being representative of un-sampled FMDV isolates that have been subjected to geographical isolation within Africa by the effects of the Great African Rinderpest Pandemic (1887–1897), which caused a mass die-out of FMDV-susceptible hosts. These findings demonstrate that further sequencing of African FMDV isolates is likely to reveal more unusual genotypes and will allow for better understanding of natural variability and evolution of FMDV.

The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug.IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity involved for this process to occur, which could be the basis for a novel antiviral control mechanism for FMDV.

Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways.

The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from "helper" replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the "helper" replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy.

Correction: A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles.

[This corrects the article DOI: 10.1371/journal.pone.0157435.].

Truncated Bovine Integrin Alpha-v/Beta-6 as a Universal Capture Ligand for FMD Diagnosis.

Foot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV) serotype-specific antibodies in small animals (rabbits and guinea pigs) that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvß6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvß6 and FMDV empty capsids in FMD diagnostic assays.

A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles.

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvß6 (a cellular receptor for FMDV). In cattle vaccinated with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge, they were protected against disease and no FMDV RNA was detected in their sera post-challenge. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed. This prime-boost system, using reagents that can be generated outside of high-containment facilities, offers significant advantages to achieve control of FMD by vaccination.

Foot-and-mouth disease virus replicates independently of phosphatidylinositol 4-phosphate and type III phosphatidylinositol 4-kinases.

Picornaviruses form replication complexes in association with membranes in structures called replication organelles. Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol 4-kinases (PI4KIIIs) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles. For the enteroviruses, replication organelles form at Golgi membranes in a process that utilizes PI4KIIIß. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the endoplasmic reticulum and subvert PI4KIIIα to generate PI4P. Here we investigated the role of PI4KIII in foot-and-mouth disease virus (FMDV) replication. Our results showed that, in contrast to the enteroviruses and the cardioviruses, FMDV replication does not require PI4KIII (PI4KIIIα and PI4KIIIß), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at replication organelles. These results point to a unique requirement towards lipids at the FMDV replication membranes.

Application of the thermofluor PaSTRy technique for improving foot-and-mouth disease virus vaccine formulation.

Foot-and-mouth disease (FMD) has a major economic impact throughout the world and is a considerable threat to food security. Current FMD virus (FMDV) vaccines are made from chemically inactivated virus and need to contain intact viral capsids to maximize efficacy. FMDV exists as seven serotypes, each made up by a number of constantly evolving subtypes. A lack of immunological cross-reactivity between serotypes and between some strains within a serotype greatly complicates efforts to control FMD by vaccination. Thus, vaccines for one serotype do not afford protection against the others, and multiple-serotype-specific vaccines are required for effective control. The FMDV serotypes exhibit variation in their thermostability, and the capsids of inactivated preparations of the O, C and SAT serotypes are particularly susceptible to dissociation at elevated temperature. Methods to quantify capsid stability are currently limited, lack sensitivity and cannot accurately reflect differences in thermostability. Thus, new, more sensitive approaches to quantify capsid stability would be of great value for the production of more stable vaccines and to assess the effect of production conditions on vaccine preparations. Here we have investigated the application of a novel methodology (termed PaSTRy) that utilizes an RNA-binding fluorescent dye and a quantitative (q)PCR machine to monitor viral genome release and hence dissociation of the FMDV capsid during a slow incremental increase in temperature. PaSTRy was used to characterize capsid stability of all FMDV serotypes. Furthermore, we have used this approach to identify stabilizing factors for the most labile FMDV serotypes.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design.

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A - ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A - vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.

Large Cellular Inclusions Accumulate in Arabidopsis Roots Exposed to Low-Sulfur Conditions.

Sulfur is vital for primary and secondary metabolism in plant roots. To understand the molecular and morphogenetic changes associated with loss of this key macronutrient, we grew Arabidopsis (Arabidopsis thaliana) seedlings in low-sulfur conditions. These conditions induced a cascade of cellular events that converged to produce a profound intracellular phenotype defined by large cytoplasmic inclusions. The inclusions, termed low-sulfur Pox, show cell type- and developmental zone-specific localization. Transcriptome analysis suggested that low sulfur causes dysfunction of the glutathione/ascorbate cycle, which reduces flavonoids. Genetic and biochemical evidence indicated that low-sulfur Pox are the result of peroxidase-catalyzed oxidation of quercetin in roots grown under sulfur-depleted conditions.

FMDV replicons encoding green fluorescent protein are replication competent.

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.

Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.