Globally Defining the Effects of Amino Acid Mutations across a Picornavirus Capsid ^†

RNA viruses are characterized by their extreme mutation rates, which play key roles in their biology and give them the ability to rapidly adapt to new environments. However, non-synonymous mutations tend to be largely deleterious to protein function, raising the question of how the proteins of RNA viruses maintain functionality in the face of high mutation rates. This is of particular relevance to the capsids of non-enveloped RNA viruses, which form highly complex protein structures that assemble from numerous subunits, interact with cellular host factors to mediate entry and uncoating, and are under strong immune selection. To better understand how viral capsids accommodate mutations, we generated viral populations harboring a large fraction of all possible single amino acid mutations in a picornavirus capsid. We then used high-fidelity next-generation sequencing to derive the relative fitness of these mutations compared to the wildtype sequence. Combining our results with available structural, genetic, and phenotypic data, we are able to provide a comprehensive understanding of the ability of a viral capsid to accommodate mutations.