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Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo*

Open AccessPublished:May 02, 2016DOI:https://doi.org/10.1074/jbc.M116.726596
      Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg2+ ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe364 and Pro357, which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe364 to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.

      Introduction

      The RNA-dependent RNA polymerases (RdRPs)
      The abbreviations used are: RdRP
      RNA-dependent RNA polymerase
      3Dpol
      picornaviral RNA-dependent-RNA polymerase
      CV
      coxsackievirus
      TCID50
      50% tissue culture infective dose
      2AP
      2-aminopurine.
      from positive strand RNA viruses close their active sites for catalysis via a subtle NTP-induced conformational change within conserved motifs A and C (
      • Gong P.
      • Peersen O.B.
      Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase.
      ). This palm domain-based closure mechanism differs from what is observed in other classes of replicative polymerases where the palm is fully structured prior to NTP binding, and the nascent base pair is delivered into the active site for catalysis via molecular motions originating in the finger domain (
      • Kornberg R.D.
      The molecular basis of eukaryotic transcription.
      ,
      • Steitz T.A.
      The structural changes of T7 RNA polymerase from transcription initiation to elongation.
      ). Despite these different molecular motions, the structural end points are essentially equivalent, and the RdRPs retain the highly conserved polymerase active site geometry with aspartate residues and a magnesium-catalyzed two-metal reaction mechanism (
      • Steitz T.A.
      A mechanism for all polymerases.
      ,
      • Ferrer-Orta C.
      • Ferrero D.
      • Verdaguer N.
      RNA-dependent RNA polymerases of picornaviruses: from the structure to regulatory mechanisms.
      ). The origin of the palm-based movement in the viral RdRPs is likely the conserved molecular contact between the finger and thumb domains that stabilizes the protein structure at the expense of reducing finger domain flexibility (
      • Thompson A.A.
      • Albertini R.A.
      • Peersen O.B.
      Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.
      ).
      One key characteristic of the viral RdRPs is their relatively low replication fidelity with mutation frequencies of 10−4–10−5 that result in a heterogeneous virus population often referred to as a quasispecies (
      • Andino R.
      • Domingo E.
      Viral quasispecies.
      ,
      • Lauring A.S.
      • Frydman J.
      • Andino R.
      The role of mutational robustness in RNA virus evolution.
      ). The population consensus sequence defines a particular virus and strain, but closer inspection of individual genomes reveals that they each contain a few random point mutations relative to the consensus. This pool of continually generated genetic diversity allows RNA viruses to rapidly adapt to different environments, enabling efficient replication in multiple cell types when infecting a host organism. The quasispecies population diversity is critically important for pathogenesis and virus growth, which can be attenuated in vivo by either decreasing diversity with high fidelity polymerase or increasing diversity with low fidelity polymerases (
      • Gnädig N.F.
      • Beaucourt S.
      • Campagnola G.
      • Bordería A.V.
      • Sanz-Ramos M.
      • Gong P.
      • Blanc H.
      • Peersen O.B.
      • Vignuzzi M.
      Coxsackievirus B3 mutator strains are attenuated in vivo.
      ,
      • Korboukh V.K.
      • Lee C.A.
      • Acevedo A.
      • Vignuzzi M.
      • Xiao Y.
      • Arnold J.J.
      • Hemperly S.
      • Graci J.D.
      • August A.
      • Andino R.
      • Cameron C.E.
      RNA virus population diversity, an optimum for maximal fitness and virulence.
      ,
      • Pfeiffer J.K.
      • Kirkegaard K.
      Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice.
      ,
      • Stapleford K.A.
      • Rozen-Gagnon K.
      • Das P.K.
      • Saul S.
      • Poirier E.Z.
      • Blanc H.
      • Vidalain P.O.
      • Merits A.
      • Vignuzzi M.
      Viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide depletion.
      ).
      The molecular interactions involved in the palm domain-based active site closure step led us to previously carry out mutagenesis studies showing that the fidelity of coxsackievirus B3 (CVB3) and poliovirus polymerases can be drastically altered by mutations within motif A (
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ). These structurally homologous enzymes differ 3–4-fold in processive elongation rates and nucleotide selectivity. Interestingly, mutations at structurally identical positions tend to increase the fidelity of the lower fidelity poliovirus polymerase but decrease the fidelity of the inherently higher fidelity CVB3 polymerase. Those data demonstrated that the RdRP active site closure mechanism provides a platform for the evolutionary fine-tuning of virus replication fidelity and suggested that protein engineering approaches to alter fidelity may be an avenue for developing live-attenuated virus vaccines.
      Multiple lines of experimental data have also identified motif D as an important regulator of RdRP function and as a reporter for active site conformational states. Motif D forms the outer rim of the NTP entry channel and is located immediately exterior to motif A (Fig. 1). NMR dynamics measurements using sparse 13C labeling show significant changes in local motion within motif D upon nucleotide binding (
      • Yang X.
      • Smidansky E.D.
      • Maksimchuk K.R.
      • Lum D.
      • Welch J.L.
      • Arnold J.J.
      • Cameron C.E.
      • Boehr D.D.
      Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition.
      ), molecular dynamics trajectories suggest a role in NTP transport into the active site (
      • Moustafa I.M.
      • Shen H.
      • Morton B.
      • Colina C.M.
      • Cameron C.E.
      Molecular dynamics simulations of viral RNA polymerases link conserved and correlated motions of functional elements to fidelity.
      ,
      • Moustafa I.M.
      • Korboukh V.K.
      • Arnold J.J.
      • Smidansky E.D.
      • Marcotte L.L.
      • Gohara D.W.
      • Yang X.
      • Sánchez-Farrán M.A.
      • Filman D.
      • Maranas J.K.
      • Boehr D.D.
      • Hogle J.M.
      • Colina C.M.
      • Cameron C.E.
      Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.
      ), and kinetic isotope effects indicate that a conserved lysine in motif D is a proton donor during catalysis (
      • Castro C.
      • Smidansky E.D.
      • Arnold J.J.
      • Maksimchuk K.R.
      • Moustafa I.
      • Uchida A.
      • Götte M.
      • Konigsberg W.
      • Cameron C.E.
      Nucleic acid polymerases use a general acid for nucleotidyl transfer.
      ). Comparisons of open and closed active site conformations in RdRP crystal structures suggest an essentially rigid body movement of motif D that is tightly coupled to the movement of the adjacent motif A; there are only minor internal differences in how the motif is packed against the rest of the polymerase structure in the two states (Fig. 1B). However, Phe363 of poliovirus 3Dpol appears to undergo a sliding motion atop the motif D α-helix when comparing the open versus closed active site conformations (Fig. 1B). Structures of CVB3 polymerase have shown that certain motif A mutations that affect active site closure result in large conformational changes within motif D, including the displacement of Phe364, the structural equivalent of poliovirus 3Dpol Phe363, from its binding pocket (
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ). CVB3 elongation complex structures also show density for a polymerase-bound “metal A” Mg2+ ion that must be transported ≈5 Å into the catalytic center during active site closure where it would join the metal B ion that is delivered as part of the NTP-Mg2+ complex (
      • Gong P.
      • Peersen O.B.
      Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase.
      ,
      • Gong P.
      • Kortus M.G.
      • Nix J.C.
      • Davis R.E.
      • Peersen O.B.
      Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.
      ). The extended hydration shell of the metal A ion and its likely path into the active site involve motif D, suggesting that the motif may play a role in controlling the dynamics of delivering Mg2+ for catalysis.
      Figure thumbnail gr1
      FIGURE 1Structural overview of picornaviral RdRP active site movements and motif D conservation. A, coxsackievirus B3 3Dpol elongation complex structure (Protein Data Bank code 4K4X) highlighting the location of motif D (green) within the palm domain and residues targeted for mutation (yellow spheres). B, close-up view of two conformations of the poliovirus 3Dpol elongation complex active site (Protein Data Bank codes 3OL6 and 3OL7) showing the concerted movements of motifs A and D that close the active site and reposition Asp233 and the prebound metal A Mg2+ ion (green sphere) to yield the two-metal ion (magenta) coordination geometry required for catalysis. Note the sliding movement of Phe364 atop Ala341 and Ala345 that are located on the motif D helix (CVB3 numbering used). C, sequence and structure alignment of the motif D loop from multiple picornaviruses showing the conservation of residues Pro357 and Phe364 (CVB3 numbering) that likely stabilize the loop conformation. The maximum likelihood structural superpositioning (
      • Theobald D.L.
      • Wuttke D.S.
      THESEUS: maximum likelihood superpositioning and analysis of macromolecular structures.
      ,
      • Theobald D.L.
      • Wuttke D.S.
      Accurate structural correlations from maximum likelihood superpositions.
      ) emphasizes how the proline-phenylalanine interaction stabilizes the conserved architecture of the motif D loop. D, close-up view of the open conformation CVB3 3Dpol active site (Protein Data Bank 4K4Z) showing the hydration shell around the prebound Mg2+ ion (green). Mg2+ coordination by waters (red spheres) and Asp330 is indicated by black dashes; Asp233 hydrogen bonds to two of the coordinating waters are shown as yellow dashes. Residues framing this hydration network that were targeted for mutagenesis are shown as yellow sticks. PV, poliovirus; FMDV, foot-and-mouth disease virus; EMCV, encephalomyocarditis virus; HRV, human rhinovirus; EV, enterovirus.
      To further assess the role of motif D in controlling CVB3 polymerase rate and replication fidelity, we have carried out a biochemistry and virology study of CVB3 polymerase mutations that target two groups of structural interactions. First, mutations of highly conserved Phe364 (Fig. 1C) and its binding pocket slow processive elongation up to 7-fold in vitro and increase virus replication fidelity 2-fold in vivo, giving rise to the first genetically stable high fidelity CVB3 variant viruses. Second, mutations that disrupt the hydration network surrounding the bound metal A Mg2+ ion (Fig. 1D) also slow the polymerase and give rise to stable progeny viruses, but their effects on both rate and fidelity are fairly minor. These data provide further insights into the molecular mechanisms underlying viral RdRP active site closure and provide additional control points for engineering viral polymerase fidelity.

      Discussion

      Building upon our previous work identifying structure-function relationships in engineered fidelity variant picornaviral polymerases (
      • Gnädig N.F.
      • Beaucourt S.
      • Campagnola G.
      • Bordería A.V.
      • Sanz-Ramos M.
      • Gong P.
      • Blanc H.
      • Peersen O.B.
      • Vignuzzi M.
      Coxsackievirus B3 mutator strains are attenuated in vivo.
      ,
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ), here we focused our attention on the conformational flexibility of motif D and its impact on CVB3 3Dpol kinetics, fidelity, and structure. The initial structures of poliovirus 3Dpol elongation complexes showed that RdRP active site closure involves a concerted rigid body movement of motif A and the loop portion of motif D that establishes antiparallel β-sheet backbone hydrogen bonding between motifs A and C. This process fully structures the canonical polymerase palm domain active site by repositioning the essential Asp233 in motif A to coordinate both divalent metals needed for catalysis (
      • Gong P.
      • Peersen O.B.
      Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase.
      ). Point mutations within motif A can have profound effects on polymerase elongation rate and replication fidelity, which can be either increased or decreased depending on the specific viral polymerase being studied (
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ). An alternate conformation of the loop portion of motif D was then captured in a crystal structure of the low fidelity CVB3 3Dpol F232L variant (
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ). This alternate motif D loop conformation involved Phe364 moving from its binding pocket under Pro357 to instead bind near the base of the thumb domain, and it showed that the motif D loop could be restructured independently of motif A. This is consistent with the conformational heterogeneity within motif D that has been observed by NMR (
      • Yang X.
      • Smidansky E.D.
      • Maksimchuk K.R.
      • Lum D.
      • Welch J.L.
      • Arnold J.J.
      • Cameron C.E.
      • Boehr D.D.
      Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition.
      ) and in molecular dynamics simulations (
      • Moustafa I.M.
      • Shen H.
      • Morton B.
      • Colina C.M.
      • Cameron C.E.
      Molecular dynamics simulations of viral RNA polymerases link conserved and correlated motions of functional elements to fidelity.
      ) where data indicate that motions in this region of the polymerase are sensitive to NTP binding. Sequence and structure alignments of picornaviral polymerases further show that the original conformation is highly conserved and seemingly stabilized by a stacking interaction between Phe364 and Pro357 (Fig. 1C), suggesting that the interactions of these two residues may be important for RdRP function by controlling the motif D loop conformation.
      In this study, we examined whether perturbations of this interaction could impact loop flexibility and reveal aspects of the functional role motif D plays during active site closure. We targeted residue Phe364 with mutagenesis to small and β-branched hydrophobic residues (Ala, Val, Leu, and Ile) and with more conservative changes to tyrosine and tryptophan, the other two planar aromatic amino acids. We also mutated Ala341 and Ala345, two residues that form a relatively flat floor at the bottom of the hydrophobic pocket into which Phe364 is inserted. In a second set of mutations, we targeted the active site metal A ion by changes to residues Ala231, Ala358, and Val367 that may impact the transport of magnesium into the active site for catalysis by virtue of being indirectly involved in the extended ion hydration shell (
      • Gong P.
      • Kortus M.G.
      • Nix J.C.
      • Davis R.E.
      • Peersen O.B.
      Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.
      ). Crystallography, stopped-flow kinetics, rapid quench reactions, and infectious virus studies were used to characterize the effects of the mutants on 3Dpol structure and biochemistry and on coxsackievirus B3 growth and pathogenesis.
      The data point to Phe364 being a key control point for both processive replication rate and fidelity. Mutations of this residue slow the polymerase elongation rate (kpol) with a continuum of effects ranging from 1.2- to 7-fold, whereas the Km for NTP remains largely unchanged, suggesting that residue 364 plays a role in the dynamics of nucleotide reorientation and active site closure that occurs after the initial NTP binding event. The crystal structures show that a planar amino acid (Phe, Tyr, or Trp) is needed at residue 364 to maintain the active site in an open conformation. Mutations to Ile, Ala, Val, and Leu all show Pro357 dropping down toward the Phe364 binding pocket to result in either a partially or a fully closed active site. Furthermore, Tyr and Trp were the only Phe364 mutations that supported virus growth, indicating that stabilizing the loop conformation and/or the default open active site is essential for proper 3Dpol function during infection. Sequencing of the progeny virus population further revealed that F364Y does not affect replication fidelity, but F364W increased fidelity 2.3-fold, resulting in a very strong high fidelity variant making only 1.8 mutations per 10 kb synthesized. This is the first isolation of a genetically stable high fidelity variant of CVB3, an enzyme that already has fairly high fidelity in comparison with the poliovirus RdRP. Combined with our previous studies of motif A mutations that result in low fidelity variants (
      • Gnädig N.F.
      • Beaucourt S.
      • Campagnola G.
      • Bordería A.V.
      • Sanz-Ramos M.
      • Gong P.
      • Blanc H.
      • Peersen O.B.
      • Vignuzzi M.
      Coxsackievirus B3 mutator strains are attenuated in vivo.
      ,
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ), we have now recovered viable CVB3 isolates with 3Dpol variants whose mutation frequencies vary over a 6-fold range from 1.8 to 11.2 mutations per 10 kb of RNA synthesized.
      The details of the 3Dpol catalytic cycle have been studied in great detail to reveal five major steps: NTP binding, a precatalysis conformational change that reorients the NTP into the active site, catalysis, a postcatalysis conformational change, and finally translocation of the RNA to reset the active site (
      • Ng K.K.
      • Arnold J.J.
      • Cameron C.E.
      Structure-function relationships among RNA-dependent RNA polymerases.
      ). The precatalysis reorientation step is rate-limiting and a major fidelity checkpoint, resulting in a strong correlation between elongation rate and replication fidelity. The structures of picornaviral elongation complexes show that the molecular rearrangement that takes place during the active site closure step is the inward movement of motif A to fully fold the core palm domain β-sheet and create a canonical replicative polymerase active site (Fig. 1B) (
      • Gong P.
      • Peersen O.B.
      Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase.
      ,
      • Gong P.
      • Kortus M.G.
      • Nix J.C.
      • Davis R.E.
      • Peersen O.B.
      Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.
      ). Our prior studies of 3Dpol motif A mutations showed a strong correlation between replication fidelity obtained from deep sequencing and the presence of an NTP 2′-hydroxyl group based on the CTP-versus-dCTP discrimination assay (
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ). Those findings suggested that proper hydroxyl positioning and the resulting hydrogen bonding network emanating from the 2′-OH play a major role in driving active site closure for catalysis. However, such a 2′-OH correlation does not appear to hold for the set of motif D mutants described in this work. For example, the F364Y and F364W mutants both have slightly elevated discrimination factors, but only the tryptophan has a significant fidelity effect in an infectious virus context. Among the mutants targeting the metal A site, the biochemical data would predict lowered fidelity, but the virus-based observation is one of slightly higher fidelity variants. These new data do retain the in vitro biochemical observation that faster polymerases have reduced 2′-OH discrimination (Fig. 5B), but for motif D mutants these effects are no longer clearly predictive of change to in vivo virus mutation frequencies.
      The kinetic data also indicate that the cycling of the active site and motif D from an open to closed conformation is important for the first round of catalysis, supporting molecular dynamics data arguing that motif D plays a role in nucleotide transport into the active site (
      • Shen H.
      • Sun H.
      • Li G.
      What is the role of motif D in the nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus?.
      ). Wild type 3Dpol and the F364Y mutant have open active sites and comparable rates for the first nucleotide incorporation event based on the observed disappearance of starting material, but the F364A mutant is significantly slower despite having a preclosed active site that might be expected to accelerate incorporation of the first nucleotide (Fig. 6C). Thus, we conclude that the active site must be cycled to the open state to enable initial NTP binding, and the NTP is then repositioned to trigger active site closure and catalysis. Subsequent incorporation steps, i.e. processive elongation, are slowed in both F364Y and F364A mutants because they stabilize the open or the closed state, respectively, and either effect will reduce the rate at which the polymerase progresses through the full catalytic cycle. The molecular origin of the lower in vivo fidelity observed for F364W variant virus is not clear from the structures or the biochemical data, and the in vitro biochemical behavior of this mutant 3Dpol is very similar to that of the non-fidelity variant F364Y (Fig. 4). The fidelity effect is likely due to differences in motif D dynamics that alter the efficiency of NTP delivery into the active site or the coupling between motifs A and D, and elucidating the fine details of these motions will likely require atomic level dynamics data from NMR and computational approaches.
      The emergence of the F364W mutations as a genetically stable high fidelity variant is attributed to two distinct effects. First, the sole tryptophan codon is UGG, and any single base mutation of this codon will result in small or charged amino acids (i.e. Gly, Ser, Cys, Leu, and Arg) that likely cannot support virus replication based on our virus growth results. Thus, the Trp364 variant virus effectively includes a genetic poison pill that minimizes its reversion potential. Second, at the level of a functional polymerase, the tryptophan creates a larger surface for the sliding motion that takes place relative to the motif D helix (Fig. 1B), but it does so by nonspecific hydrophobic interactions that do not strongly favor either the open or the closed conformation of the active site. Consequently, it has a small effect on the elongation rate, reducing it from 20 to 17 nucleotides/s, and remains fast enough to support virus growth. This is in stark contrast to two known high fidelity variants that were first identified in poliovirus but are not functional in coxsackievirus (
      • Gnädig N.F.
      • Beaucourt S.
      • Campagnola G.
      • Bordería A.V.
      • Sanz-Ramos M.
      • Gong P.
      • Blanc H.
      • Peersen O.B.
      • Vignuzzi M.
      Coxsackievirus B3 mutator strains are attenuated in vivo.
      ,
      • Campagnola G.
      • McDonald S.
      • Beaucourt S.
      • Vignuzzi M.
      • Peersen O.B.
      Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.
      ): 3Dpol G64S was originally selected as a ribavirin-resistant poliovirus (
      • Pfeiffer J.K.
      • Kirkegaard K.
      A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity.
      ), and in CVB3 it is slightly higher fidelity than wild type based on in vitro polymerase assays, but the mutation reduced virus replication by almost 100-fold, and it reverted to wild type after only three passages. Similarly, K359R is a viable high fidelity motif D mutant in poliovirus (
      • Weeks S.A.
      • Lee C.A.
      • Zhao Y.
      • Smidansky E.D.
      • August A.
      • Arnold J.J.
      • Cameron C.E.
      A Polymerase mechanism-based strategy for viral attenuation and vaccine development.
      ), but the structurally equivalent K360R slows the CVB3 3Dpol elongation rate by ≈35-fold, and it does not support virus growth.
      A long term goal of understanding structure-function relationships in the viral RdRPs is to use such information to attenuate in vivo virus growth in ways that can lead to suitable vaccine strains. Prior studies with poliovirus and coxsackievirus have shown that either increasing or decreasing RdRP fidelity can attenuate growth in vivo. One potential advantage of doing this via a protein engineering approach is the identification of specific mutations that can retain function but are unlikely to arise by virus-based adaptation pathways. Such mutations may also be less likely to revert by the same pathways. In this study, we used a combination of structure-based protein engineering and virology to identify 3Dpol F364W as a new high fidelity variant of CVB3 that significantly attenuates virus growth in mice. The parental phenylalanine residue is highly conserved among picornaviruses, and a tryptophan is never observed, suggesting that the nuances of codon usage have prevented this fully functional polymerase variant from appearing in any enterovirus, and likewise this limits the reversion potential of the variant. Our findings show that motif D and the conserved structural interactions with Phe364 provide a powerful control point for engineering polymerase fidelity as a tool for attenuating virus growth.

      Author Contributions

      S. M. conducted the biochemistry and structural biology experiments, analyzed the results, and wrote the relevant parts of the paper with O. B. P. A. B. conducted the experiments relating to the metal A binding site. S. B. and G. M. conducted the virology studies and wrote those sections of the paper together with V. M. S. M. and O. B. P. conceived the idea for the project.

      Acknowledgments

      We thank G. Campagnola for helpful discussions and experiment assistance and J. Nix for beamline support.

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