Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies

Abstract

The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the mean fitness reduction caused by single mutations is remarkably constant (0.10-0.13), whereas the fraction of lethals varies only modestly (0.20-0.41). Other summary statistics are provided. These generalizations about the distribution of mutational fitness effects can help us to better understand the evolution of RNA and single-stranded DNA viruses.

Figure 1.

Diagram showing different steps of the site-directed mutagenesis protocol for RNA or ssDNA viruses. (a) The viral genome can be amplified using adjacent divergent primers (PCR) or complementary primers (non-PCR, linear amplification). The mutated residue is indicated with a white dot. Cloning and purification, eventually followed by in vitro transcription, are required for eukaryotic viruses, whereas for phages the mutagenesis product can be directly used to transfect bacteria. Sequencing is needed at some point (preferably after virus recovery) to verify the presence of the mutation. (b) Typical lethality tests for phage ΦX174. Left picture: agarose gel electrophoresis of several PCR mutagenesis reactions. The upper band corresponds to the viral genome in linear form. The genome position and nucleotide substitution of each mutant are shown above. The two numbers below each mutation label correspond to the number of plaques obtained in transfection assays for the real mutagenesis (left) and a control reaction using non-mutagenic but otherwise identical primers (right). In this particular test, all mutations expect A402T were confirmed to be lethal. Right picture: typical aspect of E. coli lawns transfected with the real versus control mutagenesis reactions.

Figure 2.

Observed MFE frequency histograms for four different RNA or ssDNA viruses. (a) VSV; (b) TEV; (c) Qβ; (d) ΦX174.