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Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation.

ABSTRACT: Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al., 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3Dpol fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substitution of 3Dpol in different serotypes of FMDV strains had different effects on viral fidelity. In addition, nucleoside analogues were used to select high-fidelity mutagen-resistant type O FMDV variants. The rescued mutagen-resistant type O FMDV high-fidelity variants exhibited significantly attenuated fitness and a reduced virulence phenotype. These results have important implications for understanding the molecular mechanism of FMDV evolution and pathogenicity, especially in developing a safer modified live-attenuated vaccine against FMDV.

SUBMITTER: Li C

PROVIDER: S-EPMC5911202 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation.

Li Chen C Wang Haiwei H Yuan Tiangang T Woodman Andrew A Yang Decheng D Zhou Guohui G Cameron Craig E CE Yu Li L

Virology 20180220

Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al., 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3D<sup>pol</sup> fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substit ...[more]

PMID: 29455065

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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.

Project description:The low fidelity of foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase allows FMDV to exhibit high genetic diversity. Previously, we showed that the genetic diversity of FMDV plays an important role in virulence in suckling mice. Here, we mutated the amino acid residue Phe257, located in the finger domain of FMDV polymerase and conserved across FMDV serotypes, to a cysteine (F257C) to study the relationship between viral genetic diversity, virulence, and transmissibility in natural hosts. The single amino acid substitution in FMDV polymerase resulted in a high-fidelity virus variant, rF257C, with growth kinetics indistinguishable from those of wild-type (WT) virus in cell culture, but it displayed smaller plaques and impaired fitness in direct competition assays. Furthermore, we found that rF257C was attenuated in vivo in both suckling mice and pigs (one of its natural hosts). Importantly, contact exposure experiments showed that the rF257C virus exhibited reduced transmissibility compared to that of wild-type FMDV in the porcine model. This study provides evidence that FMDV genetic diversity is important for viral virulence and transmissibility in susceptible animals. Given that type O FMDV exhibits the highest genetic diversity among all seven serotypes of FMDV, we propose that the lower polymerase fidelity of the type O FMDV could contribute to its dominance worldwide.IMPORTANCE Among the seven serotypes of FMDV, serotype O FMDV have the broadest distribution worldwide, which could be due to their high virulence and transmissibility induced by high genetic diversity. In this paper, we generated a single amino acid substitution FMDV variant with a high-fidelity polymerase associated with viral fitness, virulence, and transmissibility in a natural host. The results highlight that maintenance of viral population diversity is essential for interhost viral spread. This study provides evidence that higher genetic diversity of type O FMDV could increase both virulence and transmissibility, thus leading to their dominance in the global epidemic.

Project description:Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Project description:BACKGROUND: Putative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity. RESULTS: To derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations. CONCLUSION: These values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies.

Project description:The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

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Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation.

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Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation.

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