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• W2034699053 abstract "All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.Background: The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysis and fidelity.Results: A vaccine-derived mutation in motif D decreases RdRp fidelity by changing motif D conformational dynamics.Conclusion: Non-conserved residues of motif D can alter RdRp function.Significance: Motif D of the RdRp may be a universal target permitting creation of enzymes with perturbed fidelity and viruses with reduced virulence. All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose. Background: The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysis and fidelity. Results: A vaccine-derived mutation in motif D decreases RdRp fidelity by changing motif D conformational dynamics. Conclusion: Non-conserved residues of motif D can alter RdRp function. Significance: Motif D of the RdRp may be a universal target permitting creation of enzymes with perturbed fidelity and viruses with reduced virulence. Positive-strand RNA viruses cause a number of acute and chronic diseases, including the common cold, myocarditis, encephalitis, hepatitis, and paralytic poliomyelitis (1.Hayden F.G. Update on influenza and rhinovirus infections.Adv. Exp. Med. Biol. 1999; 458: 55-67Crossref PubMed Scopus (12) Google Scholar, 2.Hayden F.G. Antivirals for influenza. Historical perspectives and lessons learned.Antiviral Res. 2006; 71: 372-378Crossref PubMed Scopus (81) Google Scholar, 3.Hayden F.G. Respiratory viral threats.Curr. Opin. Infect. Dis. 2006; 19: 169-178Crossref PubMed Scopus (55) Google Scholar, 4.Kim W.R. The burden of hepatitis C in the United States.Hepatology. 2002; 36: S30-S34Crossref PubMed Google Scholar, 5.Weiss S.R. Navas-Martin S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus.Microbiol. Mol. Biol. Rev. 2005; 69: 635-664Crossref PubMed Scopus (749) Google Scholar, 6.Racaniello V.R. One hundred years of poliovirus pathogenesis.Virology. 2006; 344: 9-16Crossref PubMed Scopus (213) Google Scholar, 7.Howard R.S. Poliomyelitis and the postpolio syndrome.BMJ. 2005; 330: 1314-1318Crossref PubMed Scopus (90) Google Scholar). The enzyme responsible for RNA genome replication is the virally encoded RNA-dependent RNA polymerase (RdRp). 3The abbreviations used are: RdRp, RNA-dependent RNA polymerase; cPVR, poliovirus receptor; HSQC, heteronuclear single quantum correlation; IACUC, Institutional Animal Care and Use Committee; MD, molecular dynamics; PV, poliovirus; SDKIE, solvent deuterium kinetic isotope effect; sym/sub, symmetrical primer-template substrate; HRV16, human rhinovirus serotype 16. RdRps belong to a superfamily of template-directed nucleic acid polymerases, including DNA-dependent DNA polymerases, DNA-dependent RNA polymerase, and RNA/DNA-dependent DNA polymerases (i.e. reverse transcriptases) (8.Svarovskaia E.S. Cheslock S.R. Zhang W.H. Hu W.S. Pathak V.K. Retroviral mutation rates and reverse transcriptase fidelity.Front. Biosci. 2003; 8: d117-d134Crossref PubMed Google Scholar, 9.Joyce C.M. Benkovic S.J. DNA polymerase fidelity. Kinetics, structure, and checkpoints.Biochemistry. 2004; 43: 14317-14324Crossref PubMed Scopus (281) Google Scholar, 10.Berdis A.J. Mechanisms of DNA polymerases.Chem. Rev. 2009; 109: 2862-2879Crossref PubMed Scopus (75) Google Scholar, 11.Ng K.K. Arnold J.J. Cameron C.E. Structure-function relationships among RNA-dependent RNA polymerases.Curr. Top. Microbiol. Immunol. 2008; 320: 137-156PubMed Google Scholar). A strategy to rationally design live, attenuated vaccine strains has been proposed based on modifying RdRps to change their nucleotide incorporation fidelity (12.Vignuzzi M. Stone J.K. Arnold J.J. Cameron C.E. Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.Nature. 2006; 439: 344-348Crossref PubMed Scopus (804) Google Scholar, 13.Vignuzzi M. Wendt E. Andino R. Engineering attenuated virus vaccines by controlling replication fidelity.Nat. Med. 2008; 14: 154-161Crossref PubMed Scopus (225) Google Scholar); viral strains encoding RdRps with altered fidelity (either higher or lower fidelity) have been shown to lead to virus incapable of causing disease but capable of acting as vaccine strains to protect mice from a lethal challenge from wild-type virus (12.Vignuzzi M. Stone J.K. Arnold J.J. Cameron C.E. Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.Nature. 2006; 439: 344-348Crossref PubMed Scopus (804) Google Scholar, 13.Vignuzzi M. Wendt E. Andino R. Engineering attenuated virus vaccines by controlling replication fidelity.Nat. Med. 2008; 14: 154-161Crossref PubMed Scopus (225) Google Scholar). A better understanding of the fidelity determinants of RdRps would thus provide a framework for rational vaccine design. The structure of viral RdRps has been described as a “cupped right hand” with fingers, thumb, and palm subdomains (11.Ng K.K. Arnold J.J. Cameron C.E. Structure-function relationships among RNA-dependent RNA polymerases.Curr. Top. Microbiol. Immunol. 2008; 320: 137-156PubMed Google Scholar) (see Fig. 1A). Most of the catalytic machinery resides in the palm region, which can be further divided into five major structural motifs (A–E). Motifs A and C contain absolutely conserved Asp residues critical for binding Mg2+ ions required for RdRp function, and conserved residues in motif B are important for interacting with the sugar of the incoming nucleoside triphosphate (NTP) (15.Poch O. Sauvaget I. Delarue M. Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.EMBO J. 1989; 8: 3867-3874Crossref PubMed Scopus (975) Google Scholar, 16.O'Reilly E.K. Kao C.C. Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.Virology. 1998; 252: 287-303Crossref PubMed Scopus (262) Google Scholar, 17.Lang D.M. Zemla A.T. Zhou C.L. Highly similar structural frames link the template tunnel and NTP entry tunnel to the exterior surface in RNA-dependent RNA polymerases.Nucleic Acids Res. 2013; 41: 1464-1482Crossref PubMed Scopus (25) Google Scholar, 18.Zemla A.T. Lang D.M. Kostova T. Andino R. Ecale Zhou C.L. StralSV. Assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase.BMC Bioinformatics. 2011; 12: 226Crossref PubMed Scopus (10) Google Scholar, 19.Gohara D.W. Arnold J.J. Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3Dpol). Kinetic, thermodynamic, and structural analysis of ribonucleotide selection.Biochemistry. 2004; 43: 5149-5158Crossref PubMed Scopus (63) Google Scholar, 20.Gohara D.W. Crotty S. Arnold J.J. Yoder J.D. Andino R. Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3Dpol). Structural, biochemical, and biological analysis of conserved structural motifs A and B.J. Biol. Chem. 2000; 275: 25523-25532Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21.Korneeva V.S. Cameron C.E. Structure-function relationships of the viral RNA-dependent RNA polymerase. Fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket.J. Biol. Chem. 2007; 282: 16135-16145Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 22.Garriga D. Ferrer-Orta C. Querol-Audí J. Oliva B. Verdaguer N. Role of motif B loop in allosteric regulation of RNA-dependent RNA polymerization activity.J. Mol. Biol. 2013; 425: 2279-2287Crossref PubMed Scopus (47) Google Scholar). Structures of RdRps bound with RNA and/or nucleotide have highlighted structural rearrangements within these palm motifs that are necessary before nucleotide incorporation (23.Gong P. Peersen O.B. Structural basis for active site closure by the poliovirus RNA-dependent RNA polymerase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 22505-22510Crossref PubMed Scopus (228) Google Scholar, 24.Thompson A.A. Albertini R.A. Peersen O.B. Stabilization of poliovirus polymerase by NTP binding and fingers-thumb interactions.J. Mol. Biol. 2007; 366: 1459-1474Crossref PubMed Scopus (53) Google Scholar, 25.Thompson A.A. Peersen O.B. Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase.EMBO J. 2004; 23: 3462-3471Crossref PubMed Scopus (196) Google Scholar, 26.Ferrer-Orta C. Arias A. Agudo R. Pérez-Luque R. Escarmís C. Domingo E. Verdaguer N. The structure of a protein primer-polymerase complex in the initiation of genome replication.EMBO J. 2006; 25: 880-888Crossref PubMed Scopus (103) Google Scholar, 27.Zamyatkin D.F. Parra F. Machín A. Grochulski P. Ng K.K. Binding of 2′-amino-2′-deoxycytidine-5′-triphosphate to norovirus polymerase induces rearrangement of the active site.J. Mol. Biol. 2009; 390: 10-16Crossref PubMed Scopus (46) Google Scholar). Our recent kinetic and solution-state nuclear magnetic resonance (NMR) studies have also identified motif D as an important determinant of RdRp fidelity. Motif D contains an absolutely conserved Lys (Lys-359 in PV RdRp) that protonates the pyrophosphate leaving group in the nucleotide addition reaction (28.Castro C. Smidansky E. Maksimchuk K.R. Arnold J.J. Korneeva V.S. Götte M. Konigsberg W. Cameron C.E. Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 4267-4272Crossref PubMed Scopus (125) Google Scholar, 29.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.Nat. Struct. Mol. Biol. 2009; 16: 212-218Crossref PubMed Scopus (169) Google Scholar). NMR experiments have indicated that there are important structural rearrangements in motif D when correct, but not incorrect, nucleotide binds (30.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.Structure. 2012; 20: 1519-1527Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 31.Yang X. Welch J.L. Arnold J.J. Boehr D.D. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.Biochemistry. 2010; 49: 9361-9371Crossref PubMed Scopus (42) Google Scholar), which might be responsible for repositioning Lys-359 for catalysis. Computational studies are also consistent with the motif D Lys acting as a general acid (32.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?.PLoS Comput. Biol. 2012; 8: e1002851Crossref PubMed Scopus (48) Google Scholar). Amino acid substitutions at Lys-359 (e.g. K359R) lead up to a 50-fold decrease in the maximum turnover rate constant (kpol), a 5-fold increase in RdRp fidelity (as determined by kpol,correct/kpol,incorrect), and lead to the severe attenuation of the virus (29.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.Nat. Struct. Mol. Biol. 2009; 16: 212-218Crossref PubMed Scopus (169) Google Scholar, 30.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.Structure. 2012; 20: 1519-1527Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 33.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.J. Biol. Chem. 2012; 287: 31618-31622Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We have suggested that the role of motif D in RdRp and reverse transcriptase fidelity is analogous to the function of the O/P helices in A/B family DNA polymerases (30.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.Structure. 2012; 20: 1519-1527Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Amino acid substitutions in the O/P helices of DNA polymerases appear to be able to tune polymerase fidelity (34.Bell J.B. Eckert K.A. Joyce C.M. Kunkel T.A. Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain.J. Biol. Chem. 1997; 272: 7345-7351Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 35.Kaushik N. Pandey V.N. Modak M.J. Significance of the O-helix residues of Escherichia coli DNA polymerase I in DNA synthesis. Dynamics of the dNTP binding pocket.Biochemistry. 1996; 35: 7256-7266Crossref PubMed Scopus (43) Google Scholar, 36.Ogawa M. Tosaka A. Ito Y. Yoshida S. Suzuki M. Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I.Mutat. Res. 2001; 485: 197-207Crossref PubMed Scopus (16) Google Scholar, 37.Suzuki M. Avicola A.K. Hood L. Loeb L.A. Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I.J. Biol. Chem. 1997; 272: 11228-11235Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 38.Suzuki M. Yoshida S. Adman E.T. Blank A. Loeb L.A. Thermus aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-helix.J. Biol. Chem. 2000; 275: 32728-32735Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Amino acid changes in motif D may likewise alter PV RdRp fidelity. One particularly noteworthy substitution is the T362I substitution that is encoded by the RdRp from the Sabin type I strain of PV. The T362I substitution may interfere with the structural dynamics of the motif D loop to disrupt RdRp catalytic function and may play a role in the attenuation of the Sabin vaccine. In this article we demonstrate that the T362I substitution lowers RdRp fidelity by altering the conformational dynamics of motif D and other active-site regions for more efficient nucleotide misincorporation. The decrease in RdRp fidelity induced by the T362I substitution likely contributes to the reduced virulence of the strain carrying this genetic change. These studies highlight motif D as a potential, rational target for modifying viral RdRps to produce attenuated viruses for use as vaccine candidates. [γ-32P]ATP (>7000 Ci/mmol) was from VWR-MP Biomedical; nucleoside 5′-triphosphates and 2′-deoxynucleoside 5′-triphosphates (all nucleotides were ultrapure solutions) were from GE Healthcare; 3′-deoxyadenosine 5′-triphosphate (cordycepin) was from Trilink Biotechnologies. All RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, CO); T4 polynucleotide kinase was from New England Biolabs, Inc; [methyl-13C]methionine was from Cambridge Isotope Laboratories. All other reagents were of the highest grade available from Sigma or Fisher. The T362I variant was generated using the QuikChange method (Stratagene) and appropriate primers. The mutation was confirmed by DNA sequencing (Nucleic Acid Facility, The Pennsylvania State University). It should be noted that wild-type (WT) and T362I RdRp used in the kinetic and NMR studies also contain two interface I amino acid substitutions (L446D and R455D) to prevent polymerase dimerization. Overexpression and purification of PV RdRp were performed as described previously (31.Yang X. Welch J.L. Arnold J.J. Boehr D.D. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.Biochemistry. 2010; 49: 9361-9371Crossref PubMed Scopus (42) Google Scholar, 39.Arnold J.J. Bernal A. Uche U. Sterner D.E. Butt T.R. Cameron C.E. Mattern M.R. Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity.Anal. Biochem. 2006; 350: 214-221Crossref PubMed Scopus (23) Google Scholar, 40.Arnold J.J. Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub).J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 41.Gohara D.W. Ha C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Production of “authentic” poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli.Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). Both kinetic and NMR experiments involved protein samples labeled by [methyl-13C]Met (31.Yang X. Welch J.L. Arnold J.J. Boehr D.D. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.Biochemistry. 2010; 49: 9361-9371Crossref PubMed Scopus (42) Google Scholar). The incorporation of this isotope did not change RdRp kinetics (data not shown). Purifying, 5′-32P end labeling and reannealing of RNA templates 10-mer sym/subU (5′-GCAUGGGCCC-3′) and 11-mer sym/subUA (5′-GCAUGGGCCCA-3′) were carried out as described previously (31.Yang X. Welch J.L. Arnold J.J. Boehr D.D. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.Biochemistry. 2010; 49: 9361-9371Crossref PubMed Scopus (42) Google Scholar, 40.Arnold J.J. Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub).J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Enzyme assays were conducted at 30 °C in reaction buffer consisting of 50 mm HEPES, pH 7.5, 10 mm 2-mercaptoethanol, 5 mm MgCl2, and 60 μm ZnCl2. Immediately before the reaction, RNA oligonucleotides were reannealed, and concentrated RdRps were diluted to the required concentration by reaction buffer (50 mm HEPES, pH 7.5, 10 mm 2-mercaptoethanol, 20% glycerol, and 60 μm ZnCl2). The volume of enzyme added into any reactions was never above 110 of the total volume. For stopped flow experiments, the RdRp-RNA binary complexes were formed by incubating RdRp (0.5 μm) with sym/sub RNA (0.25 μm duplex) for 2 min at room temperature. Experiments were initiated by rapidly mixing RdRp-RNA binary complexes in 50 mm HEPES, pH 7.5, 10 mm 2-mercaptoethanol, 5 mm MgCl2, and 60 μm ZnCl2 with the equal volume of NTP solution in the same buffer. The utilized RNA templates sym/subU and sym/subUA were substituted with 2-aminopurine and pyrrolo-cytosine at the 5′ side of the template RNA respectively. These fluorescent bases create fluorescence changes to monitor the chain elongation reaction. For 2-aminopurine-modified sym/sub-U RNA, excitation and emission wavelengths were 313 and 370 nm, respectively. For pyrrolo-cytosine-modified sym/sub-UA RNA, the excitation and emission wavelengths were 350 and 450 nm, respectively. For benchtop assays, the RdRp-RNA binary complexes were formed through incubating RdRp (0.5 μm) with RNA (0.25 μm duplex) for 3 min at room temperature followed by 2 min at 30 °C. The RNA template was 32P end-labeled. After initiation, the reaction was quenched by adding an equal volume of quench buffer (50 mm EDTA, 85% formamide, 0.025% bromphenol blue, and 0.025% xylene cyanol). The quenched samples were analyzed by denaturing PAGE, and products were quantitated by phosphorimaging as previously described (40.Arnold J.J. Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub).J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). NMR sample preparation followed procedures previously described using [methyl-13C]Met-labeled PV RdRp (30.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.Structure. 2012; 20: 1519-1527Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 31.Yang X. Welch J.L. Arnold J.J. Boehr D.D. Long-range interaction networks in the function and fidelity of poliovirus RNA-dependent RNA polymerase studied by nuclear magnetic resonance.Biochemistry. 2010; 49: 9361-9371Crossref PubMed Scopus (42) Google Scholar). 13C,1H HSQC (heteronuclear single quantum correlation) NMR spectra were collected at 293 K on a Bruker Avance III 600 MHz spectrometer equipped with a 5-mm “inverse detection” triple-resonance (13C,1H,15N) single axis gradient TCI for the following samples: free RdRp, RdRp-RNA complex, RdRp-RNA complex, and excess 3′-dATP, RdRp-RNA-3′-blocked complex after passage over the desalting column (RdRp-RNA binary complex), RdRp-RNA-binary complex and excess of second nucleotide (i.e. RdRp-RNA-NTP ternary complex), and RdRp-RNA-NTP ternary complex after passage over a second de-salting column. Atomistic molecular dynamics (MD) simulation for T362I PV RdRp (20 ns) was performed following the same method used in our previous MD simulations of picornaviral RdRps (42.Moustafa I.M. Shen H. Morton B. Colina C.M. Cameron C.E. Molecular dynamics simulations of viral RNA-dependent RNA polymerases link conserved and correlated motions of functional elements to fidelity.J. Mol. Biol. 2011; 410: 159-181Crossref PubMed Scopus (71) Google Scholar). Because there is no structure available for the T362I variant, the initial structural model of T362I RdRp was constructed based on the WT PV RdRp (Protein Data Bank code 1RA6) by substituting Thr-362 with an Ile residue using the TLEAP module of the AMBER package (43.Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6520) Google Scholar). The protein was immersed in a truncated octahedral box filled with TIP3P (44.Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. Comparison of simple potential functions for simulating liquid water.J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (29895) Google Scholar) water molecules, with a distance of at least 20 Å between any protein atom and the edge of the solvent box. MD simulation was carried out using SANDER/PMEMD in AMBER10 package (43.Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6520) Google Scholar) using the AMBER99SB force field. Initial minimization of the modeled structure was done in multiple steps using SANDER; all subsequent steps of the MD simulation were carried out using PMEMD. The solvated system was heated stepwise from 0 to 300 K with an increment of 50 K over a period of 10 ps under constant volume and temperature conditions (NVT) followed by 200 ps of NVT simulation. Finally, the simulation was switched to constant pressure and temperature dynamics for the remainder of the MD simulation. An integration time step of 1 fs was used, and the cutoff distance for nonbonded interactions was set to 9 Å. The neighbor pair list was updated every 10 steps. The Berendsen thermostat (45.Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNola A. Haak J.R. Molecular dynamics with coupling to an external bath.J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23462) Google Scholar) was applied with temperature and pressure coupling constants of 1 ps (weak coupling). Electrostatic interactions were calculated with the Particle Mesh Ewald method (46.Darden T. York D. Pedersen L. Particle mesh Ewald. An N [center-dot] log(N) method for Ewald sums in large systems.J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (20832) Google Scholar), and all bond lengths involving hydrogen atoms were constrained using SHAKE algorithm (47.Ryckaert J.-P. Ciccotti G. Berendsen H.J.C. Numerical integration of the cartesian equations of motion of a system with constraints. Molecular dynamics of n-alkanes.J. Comput. Phys. 1977; 23: 327-341Crossref Scopus (16908) Google Scholar). 50 plaque forming units of WT, G64S, or T362I PV were used to infect HeLa cell monolayers pretreated with the various concentrations of ribavirin and then washed and overlaid with agarose media containing the same concentration of ribavirin. Cells were incubated for 3–4 days at 37 °C before being stained with crystal violet (48.Arnold J.J. Vignuzzi M. Stone J.K. Andino R. Cameron C.E. Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase.J. Biol. Chem. 2005; 280: 25706-25716Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 49.Deleted in proof.Google Scholar, 50.Arnold J.J. Cameron C.E. Poliovirus RNA-dependent RNA polymerase (3Dpol). Pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+.Biochemistry. 2004; 43: 5126-5137Crossref PubMed Scopus (121) Google Scholar). Mice were bred and housed in standard ventilated caging for all experiments. Protocols for animal studies were approved by The Pennsylvania State University Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance to guidelines and regulations overseen by the IACUC. Viral stocks were generated in serum-free media and harvested, and genome copies were quantitated by quantitative PCR performed by the Genomics Core Facility of The Pennsylvania State University. 4–6-Week-old outbred (ICR) mice transgenic for the PV receptor (cPVR) were infected with PV at the indicated genome copy by intraperitoneal injection in 3 ml of serum-free media. Mice were observed for 10 days for signs of disease and were euthanized upon showing dual limb paralysis or paralysis such that their ability to obtain food and water was compromised; this was in accordance with approval by the IACUC at The Pennsylvania State University. The T362I substitution as derived from the PV Sabin I vaccine strain is located in the motif D loop of the palm subdomain (Fig. 1A). Motif D also contains the highly conserved Lys (for PV RdRp, Lys-359), which has been shown to behave as a general acid to protonate the pyrophosphate leaving group in the nucleotide incorporation reaction (28.Castro C. Smidansky E. Maksimchuk K.R. Arnold J.J. Korneeva V.S. Götte M. Konigsberg W. Cameron C.E. Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases.Proc. Natl. A" @default.
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• W2034699053 date "2013-11-01" @default.
• W2034699053 modified "2023-09-28" @default.
• W2034699053 title "Vaccine-derived Mutation in Motif D of Poliovirus RNA-dependent RNA Polymerase Lowers Nucleotide Incorporation Fidelity" @default.
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• W2034699053 doi "https://doi.org/10.1074/jbc.m113.484428" @default.
• W2034699053 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3820909" @default.
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