Evolutionary rewiring of the dynamic network underpinning allosteric epistasis in NS1 of the influenza A virus

Viral proteins frequently mutate to evade host innate immune responses, yet the impact of these mutations on the molecular energy landscape remains unclear. Epistasis, the intramolecular communications between mutations, often renders the combined mutational effects unpredictable. Nonstructural protein 1 (NS1) is a major virulence factor of the influenza A virus (IAV) that activates host PI3K by binding to its p85f3 subunit. Here, we present a deep analysis of the impact of evolutionary mutations in NS1 that emerged between the 1918 pandemic IAV strain and its descendant PR8 strain. Our analysis reveals how the mutations rewired interresidue communications, which underlie long- range allosteric and epistatic networks in NS1. Our findings show that PR8 NS1 binds to p85f3 with approximately 10- fold greater affinity than 1918 NS1 due to allosteric mutational effects, which are further tuned by epistasis. NMR chemical shift perturbation and methyl- axis order parameter analyses revealed that the mutations induced long- range structural and dynamic changes in PR8 NS1, relative to 1918 NS1, enhancing its affinity to p85f3. Complementary molecular dynamics simulations and graph theory- based network analysis for conformational dynamics on the submicrosecond timescales uncover how these mutations rewire the dynamic network, which underlies the allosteric epistasis. Significantly, we find that conformational dynamics of residues with high betweenness centrality play a crucial role in communications between network communities and are highly conserved across influenza A virus evolution. These findings advance our mechanistic understanding of the allosteric and epistatic communications between distant residues and provide insight into their role in the molecular evolution of NS1.