Nanosecond-Timescale Dynamics Of The Viral Rna-Dependent Rna Polymerase As A Determinant Of Incorporation Fidelity

The viral RNA-dependent RNA polymerase (RdRp) is required for replication of the genomes of RNA viruses. While many organisms may have evolved to make few mistakes during genome replication, RNA viruses appear to need genetic diversity for maximal fitness. This genetic diversity is created by the nucleotide misincorporation frequency of the RdRp. Perturbations in RdRp error rate therefore exhibit an antiviral effect. We have discovered a mutant poliovirus with a mutator phenotype caused by the change of His-273 of its RdRp to Arg. Kinetic experiments reveal an increase in the equilibrium constant for a conformational-change step that has been shown to be a major checkpoint for RdRp fidelity. The crystal structure of this derivative was unable to explain the biochemical observations. However, all-atom molecular dynamics (MD) simulations on the nanosecond timescale showed altered dynamics of the H273R derivative relative to the WT enzyme. By analyzing the conformational space sampled by the dihedrals of RdRp residues, we have identified RdRp residues whose dynamics correlate directly with fidelity. These residues lead to enhanced conformational flexibility of the active site of the low-fidelity H273R enzyme and diminished flexibility of the high-fidelity G64S enzyme studied previously. In general, the findings from MD simulations are supported by solution-state NMR experiments. Collectively, these experiments provide additional support for the existence of a network of RdRp residues whose dynamics control conformational changes at the active site required for incorporation fidelity. We suggest that small molecules which interfere with network dynamics should exhibit antiviral activity.