Coevolutionary constraints of Zika virus nonstructural protein 5 replication and interferon antagonism activities

The flavivirus nonstructural protein 5 performs multiple functions during infection, including RNA replication and type I interferon signaling antagonism. Although flavivirus NS5 proteins inhibit IFN signaling through distinct mechanisms, which suggests evolutionary flexibility, the evolutionary constraints for these activities to coexist within a single protein remain to be determined. Here, we map the Zika virus NS5 STAT2 antagonism determinants and compare them with replication constraints defined by deep mutational scanning. Antagonism and replication determinants extensively overlap, and no single amino acid substitution eliminates antagonism without impairing replication. Resolving these fitness landscapes in parallel identifies specific combinations of partially functional substitutions that retain replication capacity while markedly reducing antagonism. These viruses are profoundly attenuated in human STAT2 knock-in mice. Our results uncover a fundamental evolutionary constraint linking replication and immune evasion activities in NS5, highlight that STAT2 antagonism is essential for ZIKV pathogenesis and provide new avenues for attenuated ZIKV vaccines.