Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C pro to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C pro , using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C pro at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

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confidence: 99%

Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

Gullberg

Polacek

Bøtner

et al. 2013

“…The outer capsid surfaces are formed by VP1, which surrounds the five-fold symmetry axis, and VP2 and VP3, which alternate around the three-fold axis (5). VP4 is myristoylated and located inside the capsid and is thought to play an essential role in the final stage of assembly and in endosomal membrane penetration by the viral RNA (6,7). In vivo, FMDV has a strong tropism for epithelial cells, which is in part due to the epithelial cell-restricted expression of integrin ␣v␤6, which is the principal receptor used by field viruses to initiate infection (8)(9)(10)(11)(12).…”

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confidence: 99%

Positively Charged Residues at the Five-Fold Symmetry Axis of Cell Culture-Adapted Foot-and-Mouth Disease Virus Permit Novel Receptor Interactions

Berryman

Clark

Kakker

et al. 2013

Field isolates of foot-and-mouth disease virus (FMDVF oot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most widespread, epizootic transboundary animal diseases, affecting many species of wildlife and livestock, such as cattle, sheep, goats, and pigs. The significant economic losses that result from FMD are due to the high morbidity of infected animals and stringent trade restrictions imposed on affected countries (1). Vaccination plays a major role in controlling FMD, either to lessen the effects of an outbreak in FMDfree countries or for control and eradication in regions where it is endemic. The etiological agent of FMD, foot-and-mouth disease virus (FMDV), exists as seven distinct serotypes (O, A, C, Asia-1, and the Southern African Territories [SAT] serotypes SAT-1, SAT-2, and SAT-3). Within each serotype, a large number of antigenic variants exist (2). Intraserotype diversity is driven by a high mutation rate during replication that is caused by an error-prone viral RNA-dependent RNA polymerase (3) and thus complicates efforts to control disease by vaccination due to incomplete protection between some antigenic variants (4). Hence, the most effective vaccines closely match the outbreak virus, which can necessitate the development of new vaccine strains. The current vaccines are inactivated virus preparations grown in large-scale cell culture. Therefore, the production of a new vaccine is critically dependent upon adaptation of viruses from the field for growth in cell culture, which can prove problematical for some viruses.Foot-and-mouth disease virus is the type species of the Aphthovirus genus of the Picornaviridae, a family of nonenveloped, single-stranded positive-sense RNA viruses. The viral capsid is formed by 60 copies each of four structural proteins (VP1 to VP4) arranged in icosahedral symmetry. The outer capsid surfaces are formed by VP1, which surrounds the five-fold symmetry axis, and VP2 and VP3, which alternate around the three-fold axis (5). VP4 is myristoylated and located inside the capsid and is thought to play an essential role in the final stage of assembly and in endosomal membrane penetration by the viral RNA (6, 7). In vivo, FMDV has a strong tropism for epithelial cells, which is in part due to the epithelial cell-restricted expression of integrin ␣v␤6, which is the principal receptor used by field viruses to initiate infection (8-12). Integrin binding is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) motif located at the apex of a structurally disordered loop (the GH loop of VP1). The integrin specificity of FMDV has been the subject of several studies, and three other RGD-dependent integrins (␣v␤1, ␣v␤3, and ␣v␤8) have also been reported to be receptors for field strains of the virus (13-15); however, the role of these integrins in pathogenesis is unclear, and we have found that ␣v␤3 is a poor receptor for FMDV in vitro (16). Furthermore, despite recognizing their ligands via the RGD motif, two other RGD-dependent integrins ...

“…Human picornaviruses produce symptoms ranging from mild respiratory illness to hemorrhagic conjunctivitis, myocarditis, acute flaccid paralysis, and neonatal organ failure (19,27,28,33,40,41). Veterinary picornaviruses, such as foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), and porcine teschovirus (PTV), can have devastating effects on livestock (5,8,21).Although picornaviruses such as poliovirus (PV) are known to evolve more rapidly than other viruses with single-stranded RNA (ssRNA) genomes (9, 29), little research has been conducted to investigate how they evolve more rapidly than other viruses with similarly error-prone RNA-dependent RNA polymerases (29, 57) or if certain picornaviruses evolve more rapidly than others. Understanding the evolutionary potentials and constraints of these important pathogens is imperative for the development of durable vaccines and effective treatment plans for individual pathogens (42).…”

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confidence: 99%

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confidence: 99%

Genus-Specific Substitution Rate Variability among Picornaviruses

Hicks

Duffy

2011

Picornaviruses have some of the highest nucleotide substitution rates among viruses, but there have been no comparisons of evolutionary rates within this broad family. We combined our own Bayesian coalescent analyses of VP1 regions from four picornaviruses with 22 published VP1 rates to produce the first within-family meta-analysis of viral evolutionary rates. Similarly, we compared our rate estimates for the RNA polymerase 3D pol gene from five viruses to four published 3D pol rates. Both a structural and a nonstructural gene show that enteroviruses are evolving, on average, a half order of magnitude faster than members of other genera within the Picornaviridae family.Members of the Picornaviridae family are the most common cause of human viral infections in developed countries (28,39). Human picornaviruses produce symptoms ranging from mild respiratory illness to hemorrhagic conjunctivitis, myocarditis, acute flaccid paralysis, and neonatal organ failure (19,27,28,33,40,41). Veterinary picornaviruses, such as foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), and porcine teschovirus (PTV), can have devastating effects on livestock (5,8,21).Although picornaviruses such as poliovirus (PV) are known to evolve more rapidly than other viruses with single-stranded RNA (ssRNA) genomes (9, 29), little research has been conducted to investigate how they evolve more rapidly than other viruses with similarly error-prone RNA-dependent RNA polymerases (29, 57) or if certain picornaviruses evolve more rapidly than others. Understanding the evolutionary potentials and constraints of these important pathogens is imperative for the development of durable vaccines and effective treatment plans for individual pathogens (42). As even small, 3-fold differences in RNA virus mutation rates can have dramatic consequences, such as driving a population into lethal mutagenesis (7), similar differences in long-term evolutionary rates could indicate significantly dissimilar evolutionary potentials.While viral evolutionary rates were previously calculated only by linear regression, modern simulation software such as BEAST (11) allows for the estimation of more complex models of viral evolution. These Bayesian coalescent programs can produce both estimated mean rates of evolution and 95% credibility intervals (CIs) that provide a measure of the variability around mean rates. Instead of comparing single-point estimates, now nonoverlapping CIs provide the strongest evidence that genes or organisms are evolving at different rates (11). Many substitution rate estimates have been published for picornaviruses, especially for the antigenically significant VP1 gene, which encodes the most external of the picornavirus structural proteins and interacts with cellular receptors (37,49). Based on sequence availability in GenBank, four novel analyses were conducted, measuring the rate of evolution of the VP1 gene for two enteroviruses and producing the first rate estimates for the type species of the genera Cardiovirus and Teschoviru...