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Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2013 Nov 08; Vol. 288 (45), pp. 32753-32765. Date of Electronic Publication: 2013 Sep 30. - Publication Year :
- 2013
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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.
- Subjects :
- Amino Acid Motifs
Amino Acid Substitution
Humans
Nuclear Magnetic Resonance, Biomolecular
Poliovirus genetics
Poliovirus pathogenicity
Poliovirus Vaccines genetics
Poliovirus Vaccines metabolism
RNA-Dependent RNA Polymerase genetics
RNA-Dependent RNA Polymerase metabolism
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Ribavirin chemistry
Viral Proteins genetics
Viral Proteins metabolism
Virulence genetics
Molecular Dynamics Simulation
Mutation, Missense
Poliovirus enzymology
Poliovirus Vaccines chemistry
RNA-Dependent RNA Polymerase chemistry
Viral Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 288
- Issue :
- 45
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 24085299
- Full Text :
- https://doi.org/10.1074/jbc.M113.484428