Implications of localized charge for human influenza A H1N1 hemagglutinin evolution: Insights from deep mutational scans

Abstract

Seasonal influenza A viruses of humans evolve rapidly due to strong selection pressures from host immune responses, principally on the hemagglutinin (HA) viral surface protein. Based on mouse transmission experiments, a proposed mechanism for immune evasion consists of increased avidity to host cellular receptors, mediated by electrostatic charge interactions with negatively charged cell surfaces. In support of this, the HA charge of the globally circulating H3N2 has increased over time since its pandemic. However, the same trend was not seen in H1N1 HA sequences. This is counter-intuitive, since immune escape due to increased avidity (due itself to an increase in charge) was determined experimentally. Here, we explore whether patterns of local charge of H1N1 HA can explain this discrepancy and thus further associate electrostatic charge with immune escape and viral evolutionary dynamics. Measures of site-wise functional selection and expected charge computed from deep mutational scan data on an early H1N1 HA yield a striking division of residues into three groups, separated by charge. We then explored evolutionary dynamics of these groups from 1918 to 2008. In particular, one group increases in net charge over time and consists of sites that are evolving the fastest, that are closest to the receptor binding site (RBS), and that are exposed to solvent (i.e., on the surface). By contrast, another group decreases in net charge and consists of sites that are further away from the RBS and evolving slower, but also exposed to solvent. The last group consists of those sites in the HA core, with no change in net charge and that evolve very slowly. Thus, there is a group of residues that follows the same trend as seen for the entire H3N2 HA. It is possible that the H1N1 HA is under other biophysical constraints that result in compensatory decreases in charge elsewhere on the protein. Our results implicate localized charge in HA interactions with host cells, and highlight how deep mutational scan data can inform evolutionary hypotheses. The hemagglutinin (HA) surface protein of influenza A viruses evolves rapidly to evade host immunity, leading to sizable yearly epidemics in human populations. Previous transmission experiments with H1N1 in mice have tied immune escape to an increase in HA avidity for cellular receptors, mediated by electrostatic charge. Furthermore, retrospective sequence analyses from a previous study confirmed that the HA of circulating global H3N2 has increased in net charge, yet surprisingly, that of H1N1 has not varied significantly. How is a stable net charge related to local patterns of H1N1 HA charge in response to selection? To elucidate the role of local electrostatic charge in host-virus interactions, we investigate characteristics of local charge on the H1N1 HA using functional data from deep mutational scan experiments. Combining measures of functional selection and expected charge at each site on DMS data from a 1933 H1N1 HA yields a striking visual pattern that identifies three groups of sites that have different biophysical properties and that prove to have distinct evolutionary patterns in natural human sequences. Essentially, we find evidence for an increase in charge near the receptor binding site and for compensatory changes elsewhere on the protein. Thus, our findings may reconcile disparate results from transmission experiments and natural sequence analyses, and highlight the importance of local properties of the HA protein. Overall, our findings further support the hypothesis of immune escape due to increased HA avidity to cellular receptors mediated by electrostatic charge. More generally, our work illustrates a novel method of leveraging deep mutational scans in conjunction with natural sequences to shed light on virus-host interactions.