A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Lability and Confer Resistance to Acid-Dependent Uncoating Inhibition

Abstract

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 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

“…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). Consistently, blockage of endosome acidification using the lysosomotropic weak base ammonium chloride (NH 4 Cl), which acts as a proton sink within the endosomes (19,20), inhibited FMDV infection by impairing virus uncoating without affecting virus binding or entry (10,13,21). We previously reported that under the conditions of impaired acidification of endosomes in cells treated with NH 4 Cl, FMDV variants with enhanced acid sensitivity could be selected (10).…”

“…In contrast, the most acid-labile viruses were those carrying amino acid replacements VP3 A116V and VP3 A118V (pH 50 s, 6.87 and 6.93 [10], respectively). The VP3 A116V ϩ VP1 N17D double mutant (pH 50 , 6.67) displayed an intermediate phenotype between acid-resistant and acid-labile viruses, with an inactivation profile similar to that of C-S8c1 virus (pH 50 , 6.65 [10,11]). Therefore, these results indicated that the introduction of replacement VP1 N17D into a highly acid-labile capsid carrying replacement VP3 A116V increased virion acid resistance to a level similar to that of the parental C-S8c1 virus, with minor effects on viral growth.…”

“…The C-S8c1 isolate is a biological clone from type C FMDV (26). Mutants m6 and c2 are two C-S8c1 variants with increased acid resistance and enhanced acid sensitivity, respectively (10,11). O1K is a type O FMDV recovered from the infectious clone pO1K (27).…”

“…Consistently, blockage of endosome acidification using the lysosomotropic weak base ammonium chloride (NH 4 Cl), which acts as a proton sink within the endosomes (19,20), inhibited FMDV infection by impairing virus uncoating without affecting virus binding or entry (10,13,21). We previously reported that under the conditions of impaired acidification of endosomes in cells treated with NH 4 Cl, FMDV variants with enhanced acid sensitivity could be selected (10). 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).…”

“…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).…”