A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus

William Fitzsimmons; Robert J. Woods; John T. McCrone; Andrew Woodman; Jamie J. Arnold; Madhumita Yennawar; Richard Evans; Craig E. Cameron; Adam S. Lauring

No explicit power analyses were used in designing the experiments. In most cases we used 5 552 biological replicates. In a few cases, we used fewer (3) or more (7) The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/309880 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/309880 doi: bioRxiv preprint Figure 2 . R-selection leads to increased mutation rates. (A) A point mutant of 3DG64S (GGTgly to AGTser) was introduced into a poliovirus genome that is marked with a nearby point mutation that ablates an AccI restriction site. Viruses were serially passaged every 4.5 hours (r-selected) or every 24 hours (control) for 15 passages. Chromatograms show the codon for position 64 (either GGTgly or AGTser). Gel image of AccI restriction digest of all passage 15 populations showing that the reversion occurred in the parental backbone and was not due to contamination with WT virus, which retains the AccI site. (B) WT and a "locked in" version of 3DG64S (GGTgly to UCAser) were subjected to r-selection (3.5-4 hour and 4-4.5 hour, respectively) or control (24 hour) passages for 50 passages as described in the text. Heatmap shows all mutations identified at >0.025 frequency in ≥2 out of the 20 total lineages, colored by log frequency. Diagram at left shows regions of the poliovirus genome. (C) Fitness of indicated variants relative to WT as determined by competition assay. Each symbol is a replicate competition assay and exact p-values for the key comparisons are provided in the main text. (D) Mutation rate of indicated variants in mutations per nucleotide per strand copied as determined by Luria Delbruck fluctuation test. Each symbol is a replicate fluctuation test and exact p-values for the key comparisons are provided in the main text. (E) In vitro kinetics of purified RdRp. Purified RdRp (2µM), primer-template (1µM) and ATP were incubated, and samples were quenched at the indicated time points (schematic). The kinetics of complex assembly and single-base incorporation are expressed as µM extended template (y-axis) over time (x-axis). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/309880 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/309880 doi: bioRxiv preprint . Each simulation represents 27 mice and each mouse has a bottleneck size drawn from a zero-truncated Poisson with an average lambda of 2.43 (blue) or 10 (magenta). Line is actual data from reference 48, the shaded regions represent the area occupied by 95% of the simulations, and the dark shaded regions representing the interquartile range of the simulations. (B) Survival curves showing mice with paralysis free survival over time for groups (n=18 per virus) infected intramuscularly with 10^5 pfu (left), 10^6 pfu (center), and 10^7 pfu (right) of WT (black) or 3DG64S (dashed blue). * p<0.05, *** p<0.001 by Log rank test. (C) Viral titer in brain and spinal cord 5 days post intravenous inoculation with 10^7 pfu of WT (filled circles) or 3DG64S (open circles). * p<0.05, **p<0.005 by Mann Whitney U test, n=7 mice in each group (out of 8 that were infected, one mouse in each group had titers below the limit of detection, dotted line). One mouse in each group had titers bel