Dataset Information

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

ABSTRACT: The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

SUBMITTER: Shi J

PROVIDER: S-EPMC6767926 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

Shi Jingjing J Perryman Jacob M JM Yang Xiaorong X Liu Xinran X Musser Derek M DM Boehr Alyson K AK Moustafa Ibrahim M IM Arnold Jamie J JJ Cameron Craig E CE Boehr David D DD

Biochemistry 20190826 36

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. ...[more]

PMID: 31424194

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Project description:BackgroundPutative high mutation rates of RNA viruses are believed to mediate undesirable phenomena, such as emergence of drug resistance. However, very little is known about biochemical fidelity rates for viral RNA-dependent RNA polymerases. Using a recently developed in vitro polymerase assay for poliovirus polymerase 3Dpol [Arnold and Cameron (2000) JBC 275:5329], we measured fidelity for each possible mismatch. Polymerase fidelity, in contrast to sequence error rate, is biochemically defined as kpol/Kd of {(correct plus incorrect) divided by incorrect} incorporations, such that a larger value connotes higher fidelity.ResultsTo derive kpol/Kd for correct base incorporation, we performed conventional pre-steady state single turnover measurements, yielding values that range from 0.62 to 9.4 microM-1 sec-1. Pre-steady state measurements for incorrect base incorporation were less straightforward: several anomalous phenomena interfered with data collection. To obtain pre-steady state kinetic data for incorrect base incorporation, three strategies were employed. (1) For some incorrect bases, a conventional approach was feasible, although care was taken to ensure that only single turnovers were being assessed. (2) Heparin or unlabeled RNA traps were used to simulate single turnover conditions. (3) Finally, for some incorrect bases, incorporation was so poor that single datapoints were used to provide kinetic estimates. Overall, we found that fidelity for poliovirus polymerase 3Dpol ranges from 1.2 x 10(4) to 1.0 x 10(6) for transition mutations and 3.2 x 10(5) to 4.3 x 10(7) for transversion mutations.ConclusionThese values are unexpectedly high showing that high RNA virus sequence variation is not due to intrinsically low polymerase fidelity. Based on unusual enzyme behavior that we observed, we speculate that RNA mismatches either directly or indirectly cause enzyme RNA dissociation. If so, high sequence variation of RNA viruses may be due to template-switch RNA recombination and/or unknown fitness/selection phenomena. These findings may lead to a mechanistic understanding of RNA virus error catastrophe and improved anti-viral strategies.

Dataset Information

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

Publications

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

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