Energy landscape reshaped by strain-specific mutations underlies epistasis in NS1 evolution of influenza A virus

Elucidating how individual mutations affect the protein energy landscape is crucial for understanding how proteins evolve. However, predicting mutational effects remains challenging because of epistasis—the nonadditive interactions between mutations. Here, we investigate the biophysical mechanism of strain-specific epistasis in the nonstructural protein 1 (NS1) of influenza A viruses (IAVs). We integrate structural, kinetic, thermodynamic, and conformational dynamics analyses of four NS1s of influenza strains that emerged between 1918 and 2004. Although functionally near-neutral, strain-specific NS1 mutations exhibit long-range epistatic interactions with residues at the p85β-binding interface. We reveal that strain-specific mutations reshaped the NS1 energy landscape during evolution. Using NMR spin dynamics, we find that the strain-specific mutations altered the conformational dynamics of the hidden network of tightly packed residues, underlying the evolution of long-range epistasis. This work shows how near-neutral mutations silently alter the biophysical energy landscapes, resulting in diverse background effects during molecular evolution.