A Comprehensive Map of Evolutionary Constraints Across the Enterovirus A Genome

These data were collected by sequencing the input and output libraries from deep mutational, insertional and deletional scanning experiments. Data was processed by next-gen sequencing pipelines, in-house scripts and published software to interpret the fitness effects of mutations engineered in the EV-A71 genome. Data was visualized using R packages, inlcuding ggplot2.

Abstract: Insertions and deletions (InDels) are essential sources of novelty in protein evolution. In RNA viruses, InDels cause dramatic phenotypic changes that contribute to the emergence of viruses with altered immune profiles and host engagement. This work aims to comprehensively quantify the mutational tolerance of an RNA virus to insertion, deletion, and substitution. Using Enterovirus A71 (EV-A71) as a prototype for the Enterovirus A species (EV-A) of picornaviruses, we engineered approximately 45,000 insertions, 6,000 deletions, and 41,000 amino acid changes across the 2,193 coding positions of the EV-A71 proteome, quantifying their effects on viral fitness and comprehensively mapping evolutionary constraint across the viral proteome. In contrast with amino acid changes, the vast majority of InDels are lethal to virus growth. Most that are tolerated reside at a few hotspot regions. These tolerant sites highlight structurally flexible and mutationally plastic regions of EV-A71 proteins that avoid core structural and functional elements, but often overlap with key sites of host- and immune-recognition, suggesting a complex evolutionary role for InDels and substitutions at these sites. Phylogenetic analysis examining EV-A species isolated from diverse mammalian hosts reveals that many of the experimentally-identified hotspots also correspond to sites of natural InDel diversity across the more diverse EV-A species, suggesting these hotspots of mutational tolerance in EV-A genomes may have contributed to past phenotypic diversification of EV-A. Insights from this and future mutational scanning studies mapping viral evolutionary potential will inform better epidemiological monitoring and Enterovirus vaccine development.