Equine Rhinitis A Virus Mutants with Altered Acid Resistance Unveil a Key Role of VP3 and Intrasubunit Interactions in the Control of the pH Stability of the Aphthovirus Capsid

Abstract

Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and is genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intraprotomer interface between VP3 and VP2 or between VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV. IMPORTANCEThe viral capsid constitutes a sort of dynamic nanomachine that protects the viral genome against environmental assaults while accomplishing important functions such as receptor attachment for viral entry or genome release. We have explored the molecular determinants of aphthovirus capsid stability by isolating and characterizing a panel of equine rhinitis A virus mutants that differed on their acid sensitivity. All the mutations were located within a specific region of the capsid, the intraprotomer interface among capsid proteins, thus providing new insights into the regions that control the acid stability of aphthovirus capsid. These findings could positively contribute to the development of antiviral approaches targeting aphthovirus uncoating or the refinement of vaccine strategies based on capsid stabilization. E quine rhinitis A virus (ERAV) is a picornavirus associated with febrile respiratory disease in horses (1, 2). Due to its physical properties and its identification as a respiratory pathogen, ERAV was historically classified into the genus Rhinovirus (former Equine rhinovirus-1) of the family Picornaviridae (1). However, because of its resemblance at the nucleotide sequence level and at the genomic structure to foot-and-mouth disease virus (FMDV), ERAV has been reclassified into the Aphthovirus genus (3). Consistently, ERAV is now considered the phylogenetically most closely related virus to FMDV, which actually constitutes the prototype aphthovirus (http://www.ictvonline.org/virusTaxonomy .asp). Since FMDV is an extremely contagious and pathogenic aphthovirus that can only be manipulated in high-containment biosafety level 3 facilities, the utilization o...

A Heat-Induced Mutation on VP1 of Foot-and-Mouth Disease Virus Serotype O Enhanced Capsid Stability and Immunogenicity

“…Mature virions are ultimately formed, with the genome’s entry into capsids and VP0 autocatalytic cleavage into VP2 and VP4 ( 12 ). FMDV is more highly sensitive to acids and heat than other picornaviruses ( 10 ), which is conducive to virus infection and replication ( 13 , 14 ). During the normal virus purification process, nearly 10% of the intact particles (146S) dissociates into 12S pentamers ( 15 ).…”

Adaptive value of foot-and-mouth disease virus capsid substitutions with opposite effects on particle acid stability

“…1 C). These substitutions included: (1) bulkier residues in VP3 located close to the pentameric interface that destabilize inter pentameric interactions, destabilize viral capsid and increase its acid lability (A116V, A116T, and A118V) 11 , 13 ; (2) amino acid replacements located in the N terminus of VP1 (N17D, T22N, T12A, and V11I) 12 , 13 that regulate acid stability by a not well characterized mechanism in which only the relevance of the electrostatic interaction with negatively charged RNA has been identified for VP1 N17D mutant 35 ; (3) the amino acid replacement VP2 H145Y located near the intra protomeric interface, region key for Aphthovirus stability 36 , 37 , which has been also shown to increase acid resistance in several FMDV serotypes 17 , 18 . As expected, the yield of acid-resistant viruses was more inhibited by NH 4 Cl than that of the WT.…”