A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Resistance

Abstract

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 (NH 4 Cl 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.Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease of cloven-hoofed animals (23) that poses important restrictions for international trading (11,45,49,51). FMDV is the type species of the Aphthovirus genus within the family Picornaviridae (20). Its genome is composed of a single RNA molecule of positive polarity and about 8.5 kb in length. Like other RNA viruses, FMDV populations consist of complex and dynamic distributions of variants termed quasispecies (17) and exhibit a high potential for variation and adaptation, reflected in seven serotypes and multiple antigenic variants (18,50). FMDV RNA is protected by a capsid that comprises 60 copies of each of the four structural proteins (VP1 to VP4) arranged in an icosahedral lattice of 12 pentameric subunits, which constitute intermediates of capsid assembly and disassembly (56). After attachment to the host cell using a variety of receptors, such as different ␣ v ␤ integrins, heparan sulfate glycosaminoglycans (for some tissue culture-adapted variants), or other not-well-characterized molecules (2, 3, 5, 25-28, 46), FMDV particles are internalized by endocytosis mediated by clathrin (for viruses using integrin receptor) or caveolae (in the case of variants using heparan sulfate). In both cases, FMDV particles are delivered to early endosomes for capsid disassembly and viral genome release (7,29,33,43,44).FMDV particles display extreme acid lability, being inactivated at pH values slightly below neutrality (12,35,41,55). The pH sensitivity of FMDV is required for capsid disassembly triggered by acidification inside endosomes, allowing release of the RNA genome within infected cells (4, 9, 10). An acid-labile capsid is...

The pH Stability of Foot-and-Mouth Disease Virus Particles Is Modulated by Residues Located at the Pentameric Interface and in the N Terminus of VP1

“…An intriguing feature of the FMDV capsid is the extreme sensitivity to acidic pH, which results in virus inactivation after exposure of virions to mildly acidic pH (8). At pH slightly below neutrality, FMDV capsid disassembles into pentameric subunits (9) and there is good correlation between loss of infectivity by in vitro exposure to acidic pH and capsid dissociation (10)(11)(12). The acid sensitivity of FMDV has been related to the mechanism of virus penetration in host cells (13)(14)(15); the current model supports the hypothesis that the acidification of endosomes, where FMDV virions are sorted, triggers viral uncoating and genome release (16)(17)(18).…”

“…The analysis of these mutants revealed that the mechanism to escape the inhibitory effect of NH 4 Cl was based on the elevation of the uncoating pH (9, 13). On the other hand, point mutations in the viral capsid can enhance acid resistance by lowering the pH required for uncoating (11,12,(22)(23)(24). One such amino acid substitution, N17D, Citation Caridi F, Vázquez-Calvo A, Sobrino F, Martín-Acebes MA.…”

“…The pH stability of foot-and-mouth disease virus particles is modulated by residues located at the pentameric interface and in the N terminus of VP1. which is located at the N terminus of VP1, has been selected in three different serotypes (11,23,24).…”

“…The pH stability of foot-and-mouth disease virus particles is modulated by residues located at the pentameric interface and in the N terminus of VP1. which is located at the N terminus of VP1, has been selected in three different serotypes (11,23,24).In this study, we have further analyzed the genetic determinants of the pH stability of the FMDV capsid by isolating and characterizing a novel panel of acid-labile mutants. Amino acid replacements altering virion stability were preferentially located in two well-defined regions of the capsid: the N terminus of VP1 and the pentameric interface.…”

“…1B). In these experiments, two derivatives of C-S8c1, one with increased acid resistance (m6, which carries replacement VP1 N17D [11]) and the other with enhanced acid sensitivity (c2, which carries replacement VP3 A118V [10]), were included as controls. The infectivity of av1 was significantly reduced by treatment with pH 6.7 compared to that of C-S8c1, thus confirming that this mutant, directly isolated from an animal lesion, displayed enhanced acid sensitivity.…”