The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages

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 (PhiX174, G4, F1) and three ssRNA phages (Qbeta, 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 PhiX174 and 42 clones of Qbeta carrying random single-nucleotide substitutions and assayed them for fitness. In PhiX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qbeta, 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.

Figure 1. Genome structure of phages ΦX174, G4, F1, Qβ, SP, and MS2, and location of the genetic changes fixed in mutation-accumulation lines.

Genome sizes are 5.4 kb, 5.6 kb, 6.4 kb, 4.2 kb, 4.3 kb, and 3.6 kb, respectively. Protein-coding regions represent between 91% (F1) and 96% (ΦX174) of the total genome, respectively. Arrows indicate the location of each cistron and colors represent broad functional categories (blue: replication; green: particle assembly; yellow: coat; red: lysis/extrusion; grey: other/unknown). However, several genes are multifunctional; for instance A2 is involved in phage assembly and lysis. The location of each of the mutations fixed in the mutation-accumulation lines is shown within white bars, each bar corresponding to an independent line (see Table S1 for details). The three DNA phages have circular genomes but are shown in linear form for convenience. The genomes of ΦX174 and G4 are shown collinearly to indicate gene homology. However, by convention, the first position of the ΦX174 genome is assigned to the unique Pst I cleavage site (vertical dashed line).

Figure 2. Distribution of fitness effects caused by single-nucleotide substitutions in phages ΦX174 and Qβ.

Forty-five and 42 mutations, respectively, were obtained by site-directed mutagenesis and assayed for fitness. The effect of each individual mutation is provided in Table S2 and Table S3.