Insertions and deletions (InDels) are essential sources of novelty in protein evolution. In RNA
viruses, InDels cause dramatic phenotypic changes contributing to the emergence of viruses
with altered immune profiles and host engagement. This work aimed to expand our current
understanding of viral evolution and explore the mutational tolerance of RNA viruses to InDels,
focusing on Enterovirus A71 (EV-A71) as a prototype for Enterovirus A species (EV-A). Using
newly described deep InDel scanning approaches, we engineered approximately 45,000
insertions and 6,000 deletions at every site across the viral proteome, quantifying their effects
on viral fitness. As a general trend, most InDels were lethal to the virus. However, our screen
reproducibly identified a set of InDel-tolerant regions, demonstrating our ability to
comprehensively map tolerance to these mutations. Tolerant sites highlighted structurally
flexible and mutationally plastic regions of viral proteins that avoid core structural and
functional elements. Phylogenetic analysis on EV-A species infecting diverse mammalian hosts
revealed that the experimentally-identified hotspots overlapped with sites of InDels across the
EV-A species, suggesting structural plasticity at these sites is an important function for InDels
in EV speciation. Our work reveals the fitness effects of InDels across EV-A71, identifying
regions of evolutionary capacity that require further monitoring, which could guide the
development of Enterovirus vaccines. Less...