Codon usage determines the mutational robustness, evolutionary capacity, and virulence of an RNA virus.

RNA viruses exist as dynamic and diverse populations shaped by constant mutation and selection. Yet little is known about how the mutant spectrum contributes to virus evolvability and pathogenesis. Because several codon choices are available for a given amino acid, a central question concerns whether viral sequences have evolved to optimize not only the protein coding consensus, but also the DNA/RNA sequences accessible through mutation. Here we directly test this hypothesis by comparing wild-type poliovirus to synthetic viruses carrying re-engineered capsid sequences with hundreds of synonymous mutations. Strikingly, such rewiring of the population's mutant network reduced its robustness and attenuated the virus in an animal model of infection. We conclude that the position of a virus in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenicity. This organizing principle for RNA virus populations confers tolerance to mutations and facilitates replication and spread within the dynamic host environment.