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. 2010 Jan;184(1):221-32.
doi: 10.1534/genetics.109.108803. Epub 2009 Oct 26.

Evolution at a High Imposed Mutation Rate: Adaptation Obscures the Load in Phage T7

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Evolution at a High Imposed Mutation Rate: Adaptation Obscures the Load in Phage T7

R Springman et al. Genetics. .
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Abstract

Evolution at high mutation rates is expected to reduce population fitness deterministically by the accumulation of deleterious mutations. A high enough rate should even cause extinction (lethal mutagenesis), a principle motivating the clinical use of mutagenic drugs to treat viral infections. The impact of a high mutation rate on long-term viral fitness was tested here. A large population of the DNA bacteriophage T7 was grown with a mutagen, producing a genomic rate of 4 nonlethal mutations per generation, two to three orders of magnitude above the baseline rate. Fitness-viral growth rate in the mutagenic environment-was predicted to decline substantially; after 200 generations, fitness had increased, rejecting the model. A high mutation load was nonetheless evident from (i) many low- to moderate-frequency mutations in the population (averaging 245 per genome) and (ii) an 80% drop in average burst size. Twenty-eight mutations reached high frequency and were thus presumably adaptive, clustered mostly in DNA metabolism genes, chiefly DNA polymerase. Yet blocking DNA polymerase evolution failed to yield a fitness decrease after 100 generations. Although mutagenic drugs have caused viral extinction in vitro under some conditions, this study is the first to match theory and fitness evolution at a high mutation rate. Failure of the theory challenges the quantitative basis of lethal mutagenesis and highlights the potential for adaptive evolution at high mutation rates.

Figures

F<sc>igure</sc> 1.—
Figure 1.—
Predicted fitness equilibrium of wild-type T7 at different levels of deleterious mutation. This function is calculated for the parameters observed in the T7 mutagenic environment (10 μg/ml NG). Although there is a single value of Ud that applies in this environment, that value is estimated with uncertainty, so the graph shows the fitness equilibrium across a range of possible values of Ud. The solid circle gives the predicted fitness based on the estimated deleterious mutation rate of 2.6. This graph was calculated from Equation 3 for a viable burst size of 118, lysis time of 18 min, adsorption rate of 2.1 × 10−9, and cell density of 1 × 108/ml.
F<sc>igure</sc> 2.—
Figure 2.—
Observed number of sites in the T7 genome with mutations at different frequencies (200 generations, 95× coverage of the genome). Sequences were obtained from only short regions (200 bases), so these calculations of per genome numbers of mutations are simply products of the mutation frequency at each site summed across all sites (they thus assume linkage equilibrium among different mutations). Numbers apply to the observed genome size of 38,530. Each bar indicates the number of mutations whose frequencies were less than or equal to the label, down to the frequency of the bar to its left.
F<sc>igure</sc> 3.—
Figure 3.—
T7 genome positions of all mutations at frequencies of ≥0.75 (200 generations), distinguished by silent (top) and missense changes (bottom). Each mutation is represented by a vertical line, with the height representing the observed frequency. The position of the vertical line along the horizontal axis gives its genomic location, and horizontal bars indicate the locations of genes, identified by gene number. Genes 3, 4, 5, and 6 encode DNA metabolism genes of endonuclease, helicase/primase, DNA polymerase, and exonuclease, respectively. Genes 10 and 15 encode the major capsid protein and a virion internal core protein, respectively. The total number of high-frequency mutations is 28, with 23 in the cluster spanning genes 46 (inclusive). Some lines are too close together to be separable.
F<sc>igure</sc> 3.—
Figure 3.—
T7 genome positions of all mutations at frequencies of ≥0.75 (200 generations), distinguished by silent (top) and missense changes (bottom). Each mutation is represented by a vertical line, with the height representing the observed frequency. The position of the vertical line along the horizontal axis gives its genomic location, and horizontal bars indicate the locations of genes, identified by gene number. Genes 3, 4, 5, and 6 encode DNA metabolism genes of endonuclease, helicase/primase, DNA polymerase, and exonuclease, respectively. Genes 10 and 15 encode the major capsid protein and a virion internal core protein, respectively. The total number of high-frequency mutations is 28, with 23 in the cluster spanning genes 46 (inclusive). Some lines are too close together to be separable.
F<sc>igure</sc> 4.—
Figure 4.—
Expected and observed fractions of silent (S) and missense (M) mutations in essential (Ess) vs. nonessential (NonEss) genes at 200 generations. The expectations were generated by assigning the observed mutation spectrum throughout the genome and scaling the total number of expected mutations to equal the total observed. The total number of mutations is 7,642 and counts only those whose frequencies were ≤0.25. From left to right, expectations are 0.480, 0.237, 0.188, and 0.096.
F<sc>igure</sc> 5.—
Figure 5.—
Observed number of sites in the T7Δ5 genome with mutations at different frequencies (100 generations, 152× coverage of the population). Approximately 28 mutations per genome were unique, seen in only one sequence, so the higher number of mutations in the 0.05 category compared to that in the 200-generation population of T7+ is not due to the higher coverage identifying more singletons. Numbers apply to the observed genome size of 37,337. Otherwise as in Figure 2.
F<sc>igure</sc> 6.—
Figure 6.—
Predicted fitness equilibrium of wild-type T7 at different levels of deleterious mutation, showing both the prediction based on wild-type virus parameters (shaded lower line, from Figure 1) and the prediction based on the observed, evolved fitness at 200 generations (solid upper line). The upper line uses the lysis time and adsorption rate values estimated from wild type, as in the lower line, but uses a burst size that has been adjusted so that fitness matches the observed 21.9 when the deleterious mutation rate is 2.6. The shaded and solid circles show the predicted and observed fitnesses for a mutation rate of 2.6. The upper curve is merely parameterized with a hypothetical burst size that forces its fit to the observed fitness.

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