Uncovering Structural Plasticity of Enterovirus A through Deep Insertional and Deletional Scanning

Insertions and deletions (InDels) are essential sources of novelty in protein evolution. In RNAviruses, InDels cause dramatic phenotypic changes contributing to the emergence of viruseswith altered immune profiles and host engagement. This work aimed to expand our currentunderstanding 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). Usingnewly described deep InDel scanning approaches, we engineered approximately 45,000insertions and 6,000 deletions at every site across the viral proteome, quantifying their effectson viral fitness. As a general trend, most InDels were lethal to the virus. However, our screenreproducibly identified a set of InDel-tolerant regions, demonstrating our ability tocomprehensively map tolerance to these mutations. Tolerant sites highlighted structurallyflexible and mutationally plastic regions of viral proteins that avoid core structural andfunctional elements. Phylogenetic analysis on EV-A species infecting diverse mammalian hostsrevealed that the experimentally-identified hotspots overlapped with sites of InDels across theEV-A species, suggesting structural plasticity at these sites is an important function for InDelsin EV speciation. Our work reveals the fitness effects of InDels across EV-A71, identifyingregions of evolutionary capacity that require further monitoring, which could guide thedevelopment of Enterovirus vaccines.