Deep mutation, insertion and deletion scanning across the Enterovirus A proteome reveals constraints shaping viral evolution

Insertions and deletions (InDels) are essential to protein evolution. In RNA viruses, InDels contribute to the emergence of viruses with new phenotypes, including altered host engagement and tropism. However, the tolerance of viral proteins for InDels has not been extensively studied. Here, we conduct deep mutational scanning to map and quantify the mutational tolerance of a complete viral proteome to insertion, deletion and substitution. We engineered approximately 45,000 insertions, 6,000 deletions and 41,000 amino acid substitutions across the nearly 2,200 coding positions of the Enterovirus A71 proteome, quantifying their effects on viral fitness by population sequencing. The vast majority of InDels are lethal to the virus, tolerated at only a few hotspots. Some of these hotspots overlap with sites of host recognition and immune engagement, suggesting tolerance at these sites reflects the important role InDels have played in the past phenotypic diversification of Enterovirus A.