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Residues within the Foot-and-Mouth Disease Virus 3D^pol Nuclear Localization Signal Affect Polymerase Fidelity.

ABSTRACT: 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. 3D^pol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3D^pol 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 3D^pol We identify two residues of 3D^pol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3D^pol 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 3D^pol 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 3D^pol 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 3D^pol, 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 3D^pol 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 3D^pol, 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 3D^pol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3D^pol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

SUBMITTER: Kloc A

PROVIDER: S-EPMC7431809 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:UnlabelledThe N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop β9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues.ImportanceThe study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms.

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Residues within the Foot-and-Mouth Disease Virus 3D^pol Nuclear Localization Signal Affect Polymerase Fidelity.

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Residues within the Foot-and-Mouth Disease Virus 3Dpol Nuclear Localization Signal Affect Polymerase Fidelity.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Residues within the Foot-and-Mouth Disease Virus 3D^pol Nuclear Localization Signal Affect Polymerase Fidelity.