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Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis.

ABSTRACT: 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.

SUBMITTER: de la Higuera I

PROVIDER: S-EPMC6158410 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis.

de la Higuera Ignacio I Ferrer-Orta Cristina C Moreno Elena E de Ávila Ana Isabel AI Soria María Eugenia ME Singh Kamalendra K Caridi Flavia F Sobrino Francisco F Sarafianos Stefan G SG Perales Celia C Verdaguer Nuria N Domingo Esteban E

Journal of virology 20180926 20

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 p ...[more]

PMID: 30068642

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Project description:RNA structures can form functional elements that play crucial roles in the replication of positive-sense RNA viruses. While RNA structures in the untranslated regions (UTRs) of several picornaviruses have been functionally characterized, the roles of putative RNA structures predicted for protein coding sequences (or open reading frames [ORFs]) remain largely undefined. Here, we have undertaken a bioinformatic analysis of the foot-and-mouth disease virus (FMDV) genome to predict 53 conserved RNA structures within the ORF. Forty-six of these structures were located in the regions encoding the nonstructural proteins (nsps). To investigate whether structures located in the regions encoding the nsps are required for FMDV replication, we used a mutagenesis method, CDLR mapping, where sequential coding segments were shuffled to minimize RNA secondary structures while preserving protein coding, native dinucleotide frequencies, and codon usage. To examine the impact of these changes on replicative fitness, mutated sequences were inserted into an FMDV subgenomic replicon. We found that three of the RNA structures, all at the 3' termini of the FMDV ORF, were critical for replicon replication. In contrast, disruption of the other 43 conserved RNA structures that lie within the regions encoding the nsps had no effect on replicon replication, suggesting that these structures are not required for initiating translation or replication of viral RNA. Conserved RNA structures that are not essential for virus replication could provide ideal targets for the rational attenuation of a wide range of FMDV strains. IMPORTANCE Some RNA structures formed by the genomes of RNA viruses are critical for viral replication. Our study shows that of 46 conserved RNA structures located within the regions of the foot-and-mouth disease virus (FMDV) genome that encode the nonstructural proteins, only three are essential for replication of an FMDV subgenomic replicon. Replicon replication is dependent on RNA translation and synthesis; thus, our results suggest that the three RNA structures are critical for either initiation of viral RNA translation and/or viral RNA synthesis. Although further studies are required to identify whether the remaining 43 RNA structures have other roles in virus replication, they may provide targets for the rational large-scale attenuation of a wide range of FMDV strains. FMDV causes a highly contagious disease, posing a constant threat to global livestock industries. Such weakened FMDV strains could be investigated as live-attenuated vaccines or could enhance biosecurity of conventional inactivated vaccine production.

Project description:Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Dataset Information

Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis.

Publications

Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis.

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