Structure-Guided Functional Annotation of the Influenza A Virus NS1 Protein Reveals Dynamic Evolution of the p85β-Binding Site during Circulation in Humans

Rational characterization of virulence and host-adaptive markers in the multifunctional influenza A virus NS1 protein is hindered by a lack of comprehensive knowledge about NS1-host protein protein interfaces. Here, we surveyed the impact of amino acid variation in NS1 at its structurally defined binding site for host p85β, a regulator of phosphoinositide 3-kinase (PI3K) signaling. Structure-guided alanine scanning of all viral residues at this interface defined 10 positions contributing to the interaction, with residues 89, 95, 98, 133, 145, and 162 being the most important. A bioinformatic study of >24,000 publicly available NS1 sequences derived from viruses infecting different hosts highlighted several prevalent amino acid variants at the p85β interface that either enhanced (I95) or weakened (N135, T145, L161, Y161, S164) p85β binding. Interestingly, analysis of viruses circulating in humans since the 1918 pandemic revealed the temporal acquisition of functionally relevant variants at this interface. I95 (which enhanced p85β binding) quickly became prevalent in the 1940s and experimentally conferred a fitness advantage to a recombinant 1930s-based H1N1 virus in human lung epithelial cells. Surprisingly, H1N1 and H3N2 viruses recently acquired T145 or N135, respectively, which diminished p85β binding but apparently not the overall fitness in the human population. Evolutionary analyses revealed covariation of the NS1-p85β binding phenotype in humans with functional changes at multiple residues in other viral proteins, suggesting an unexplored compensatory or synergistic interplay between phenotypes in vivo. Overall, our data provide a resource to understand the consequences of the NS1-p85β binding spectrum of different influenza viruses and highlight the dynamic evolution of this property in viruses circulating in humans. IMPORTANCE In humans, influenza A viruses are responsible for causing seasonal epidemics and occasional pandemics. These viruses also circulate and evolve in other animal species, creating a reservoir from which novel viruses with distinct properties can emerge. The viral nonstructural protein NS1 is an important host range determinant and virulence factor that exhibits strain-specific interactions with several host factors, although few have been characterized extensively. In the study described here, we comprehensively surveyed the impact of natural and unnatural NS1 variations on the binding of NS1 to host p85β, a subunit of phosphoinositide 3-kinase that regulates intracellular metabolism and contributes to virus replication and virulence. We define the p85β-binding site on NS1 and provide a predictive resource to assess this ability of NS1 in viruses from different hosts. Strikingly, we uncover a spectrum of p85β binding by different NS1 proteins and reveal that viruses evolving in humans have undergone dynamic changes in this NS1 function over the last century.