Host Switching Mutations in H5N1 Influenza Hemagglutinin Suppress Site-specific Activation Dynamics

Increase in the occurrence of human H5N1 spillover infections resulting from dissemination of highly pathogenic avian influenza (HPAI) virus into bird and mammal populations raises concerns about HPAI adapting to become human transmissible. Studies identified hemagglutinin (HA) acid stability and receptor preference as essential traits that shape host tropism. Mutations that increase HA stability and affinity for α-2,6-linked sialic acids have been shown to confer airborne transmissibility in a ferret model, however mechanisms of activation of H5 subtype HA have not been probed and the effect of adaptive mutations on HA function has been largely inferred from static structures. Here, we use hydrogen/deuterium-exchange mass spectrometry to dissect activation dynamics for two ancestral HPAI H5 HA, their matched HA with adaptive mutations, and a contemporary H5 HA. By measuring dynamics, we identify variation in active site flexibility among the HA and demonstrate that adaptive mutations result in suppression of fusion peptide dynamics and stabilization of a key subunit interface involved in activation. The contemporary H5 isolated from a recent human spillover case exhibits a relatively protected fusion peptide and moderately depressed pH of activation compared to the HAs examined in this study. Our studies of activation dynamics in the H5 HAs in conjunction with prior analysis of H1 and H3 HA reveal subtype-specific patterns that correlate with adaptive mutation sites and indicate underlying physical constraints on influenza HA adaptation.