Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity. 2013

Xinran Liu, and Xiaorong Yang, and Cheri A Lee, and Ibrahim M Moustafa, and Eric D Smidansky, and David Lum, and Jamie J Arnold, and Craig E Cameron, and David D Boehr
From the Department of Chemistry.

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.

UI MeSH Term Description Entries
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012254 Ribavirin A nucleoside antimetabolite antiviral agent that blocks nucleic acid synthesis and is used against both RNA and DNA viruses. Ribovirin,Tribavirin,ICN-1229,Rebetol,Ribamide,Ribamidil,Ribamidyl,Ribasphere,Vilona,Viramide,Virazide,Virazole,ICN 1229,ICN1229
D012324 RNA-Dependent RNA Polymerase An enzyme that catalyses RNA-template-directed extension of the 3'- end of an RNA strand by one nucleotide at a time, and can initiate a chain de novo. (Enzyme Nomenclature, 1992, p293) Nucleoside-Triphosphate:RNA Nucleotidyltransferase (RNA-directed),RNA Replicase,RNA-Dependent RNA Replicase,RNA-Directed RNA Polymerase,RNA Dependent RNA Polymerase,RNA Dependent RNA Replicase,RNA Directed RNA Polymerase,RNA Polymerase, RNA-Dependent,RNA Polymerase, RNA-Directed,RNA Replicase, RNA-Dependent,Replicase, RNA,Replicase, RNA-Dependent RNA
D014764 Viral Proteins Proteins found in any species of virus. Gene Products, Viral,Viral Gene Products,Viral Gene Proteins,Viral Protein,Protein, Viral,Proteins, Viral
D014774 Virulence The degree of pathogenicity within a group or species of microorganisms or viruses as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenic capacity of an organism is determined by its VIRULENCE FACTORS. Pathogenicity
D056004 Molecular Dynamics Simulation A computer simulation developed to study the motion of molecules over a period of time. Molecular Dynamics Simulations,Molecular Dynamics,Dynamic, Molecular,Dynamics Simulation, Molecular,Dynamics Simulations, Molecular,Dynamics, Molecular,Molecular Dynamic,Simulation, Molecular Dynamics,Simulations, Molecular Dynamics
D017955 Poliovirus A species of ENTEROVIRUS which is the causal agent of POLIOMYELITIS in humans. Three serotypes (strains) exist. Transmission is by the fecal-oral route, pharyngeal secretions, or mechanical vector (flies). Vaccines with both inactivated and live attenuated virus have proven effective in immunizing against the infection. Brunhilde Virus,Human poliovirus 1,Human poliovirus 2,Human poliovirus 3,Lansing Virus,Leon Virus,Poliovirus Type 1,Poliovirus Type 2,Poliovirus Type 3,Polioviruses
D019906 Nuclear Magnetic Resonance, Biomolecular NMR spectroscopy on small- to medium-size biological macromolecules. This is often used for structural investigation of proteins and nucleic acids, and often involves more than one isotope. Biomolecular Nuclear Magnetic Resonance,Heteronuclear Nuclear Magnetic Resonance,NMR Spectroscopy, Protein,NMR, Biomolecular,NMR, Heteronuclear,NMR, Multinuclear,Nuclear Magnetic Resonance, Heteronuclear,Protein NMR Spectroscopy,Biomolecular NMR,Heteronuclear NMR,Multinuclear NMR,NMR Spectroscopies, Protein,Protein NMR Spectroscopies,Spectroscopies, Protein NMR,Spectroscopy, Protein NMR
D019943 Amino Acid Substitution The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties. Amino Acid Substitutions,Substitution, Amino Acid,Substitutions, Amino Acid

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