The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

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Abstract
Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses. The fitness effects of mutations are the raw material for natural selection. It has been shown that point mutations typically have strongly deleterious effects in plant and animal RNA viruses, whereas cellular organisms are comparatively more robust. Here, we characterize the fitness effects of random mutations in DNA viruses and compare them with those found in RNA viruses, using six phage species of similar genome sizes. To achieve this goal, we introduced mutations by chemical and site-directed mutagenesis, identified the genetic changes by sequencing, and quantified their fitness effects using growth-rate assays. In all cases, mutations had a strong average impact on fitness. We conclude that mutational sensitivity is a general property of viruses with small genomes and discuss the evolutionary implications of these findings.

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