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Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

ABSTRACT: Infection by viruses depends on a balance between capsid stability and dynamics. This study investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability and between thermostability and infectivity. In the FMDV capsid, a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essential role for virus infection of most of the 8 tested charged groups is not related to substantial changes in capsid protein expression or processing or in capsid assembly or stability against a thermally induced dissociation into pentamers. However, the positively charged side chains of R2018 and H3141, located at the interpentamer interfaces close to the capsid 2-fold symmetry axes, were found to be critical both for virus infectivity and for keeping the capsid in a state of weak thermostability. A charge-restoring substitution (N2019H) that was repeatedly fixed during amplification of viral genomes carrying deleterious mutations reverted both the lethal and capsid-stabilizing effects of the substitution H3141A, leading to a double mutant virus with close to normal infectivity and thermolability. H3141A and other thermostabilizing substitutions had no detectable effect on capsid resistance to acid-induced dissociation into pentamers. The results suggest that FMDV infectivity requires limited local stability around the 2-fold axes at the interpentamer interfaces of the capsid. The implications for the mechanism of genome uncoating in FMDV and the development of thermostabilized vaccines against foot-and-mouth disease are discussed.IMPORTANCE This study provides novel insights into the little-known structural determinants of the balance between thermal stability and instability in the capsid of foot-and-mouth disease virus and into the relationship between capsid stability and virus infectivity. The results provide new guidelines for the development of thermostabilized empty capsid-based recombinant vaccines against foot-and-mouth disease, one of the economically most important animal diseases worldwide.

SUBMITTER: Lopez-Arguello S

PROVIDER: S-EPMC6498042 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

López-Argüello Silvia S Rincón Verónica V Rodríguez-Huete Alicia A Martínez-Salas Encarnación E Belsham Graham J GJ Valbuena Alejandro A Mateu Mauricio G MG

Journal of virology 20190501 10

Infection by viruses depends on a balance between capsid stability and dynamics. This study investigated biologically and biotechnologically relevant aspects of the relationship in foot-and-mouth disease virus (FMDV) between capsid structure and thermostability and between thermostability and infectivity. In the FMDV capsid, a substantial number of amino acid side chains at the interfaces between pentameric subunits are charged at neutral pH. Here a mutational analysis revealed that the essentia ...[more]

PMID: 30867300

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Project description:Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.

Project description:Viral RNA-dependent RNA polymerases (RdRps) are major determinants of high mutation rates and generation of mutant spectra that mediate RNA virus adaptability. The RdRp of the picornavirus foot-and-mouth disease virus (FMDV), termed 3D, is a multifunctional protein that includes a nuclear localization signal (NLS) in its N-terminal region. Previous studies documented that some amino acid substitutions within the NLS altered nucleotide recognition and enhanced the incorporation of the mutagenic purine analogue ribavirin in viral RNA, but the mutants tested were not viable and their response to lethal mutagenesis could not be studied. Here we demonstrate that NLS amino acid substitution M16A of FMDV serotype C does not affect infectious virus production but accelerates ribavirin-mediated virus extinction. The mutant 3D displays polymerase activity, RNA binding, and copying processivity that are similar to those of the wild-type enzyme but shows increased ribavirin-triphosphate incorporation. Crystal structures of the mutant 3D in the apo and RNA-bound forms reveal an expansion of the template entry channel due to the replacement of the bulky Met by Ala. This is a major difference with other 3D mutants with altered nucleotide analogue recognition. Remarkably, two distinct loop ?9-?11 conformations distinguish 3Ds that exhibit higher or lower ribavirin incorporation than the wild-type enzyme. This difference identifies a specific molecular determinant of ribavirin sensitivity of FMDV. Comparison of several polymerase mutants indicates that different domains of the molecule can modify nucleotide recognition and response to lethal mutagenesis. The connection of this observation with current views on quasispecies adaptability is discussed.IMPORTANCE The nuclear localization signal (NLS) of the foot-and-mouth disease virus (FMDV) polymerase includes residues that modulate the sensitivity to mutagenic agents. Here we have described a viable NLS mutant with an amino acid replacement that facilitates virus extinction by ribavirin. The corresponding polymerase shows increased incorporation of ribavirin triphosphate and local structural modifications that implicate the template entry channel. Specifically, comparison of the structures of ribavirin-sensitive and ribavirin-resistant FMDV polymerases has identified loop ?9-?11 conformation as a determinant of sensitivity to ribavirin mutagenesis.

Dataset Information

Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

Publications

Thermostability of the Foot-and-Mouth Disease Virus Capsid Is Modulated by Lethal and Viability-Restoring Compensatory Amino Acid Substitutions.

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