Elongation-Competent Pauses Govern the Fidelity of a Viral RNA-Dependent RNA Polymerase

•The RNAP elongation dynamic of dsRNA virus is captured by magnetic tweezers•Large sets of high-resolution data allow quantification of polymerase fidelity•Maximum likelihood estimation allows extraction of critical model parameters•Errors are predominantly incorporated via new error-prone catalytic pathway

SummaryRNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage Φ6 RNA-dependent RNA polymerase. We extract large datasets fingerprinting real-time polymerase dynamics over four magnitudes in time, in the presence of nucleotide analogs, and under varying NTP and divalent cation concentrations and fork stability. Quantitative analysis reveals a new pause state that modulates polymerase fidelity and so ties viral polymerase pausing to the biological function of optimizing virulence. Adjusting the frequency of such pauses offers a target for therapeutics and may also reflect an evolutionary strategy for virus populations to track the gradual evolution of their hosts.

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