Influenza HA Subtypes Demonstrate Divergent Phenotypes for Cleavage Activation and pH of Fusion: Implications for Host Range and Adaptation

Abstract

The influenza A virus (IAV) HA protein must be activated by host cells proteases in order to prime the molecule for fusion. Consequently, the availability of activating proteases and the susceptibility of HA to protease activity represents key factors in facilitating virus infection. As such, understanding the intricacies of HA cleavage by various proteases is necessary to derive insights into the emergence of pandemic viruses. To examine these properties, we generated a panel of HAs that are representative of the 16 HA subtypes that circulate in aquatic birds, as well as HAs representative of the subtypes that have infected the human population over the last century. We examined the susceptibility of the panel of HA proteins to trypsin, as well as human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2). Additionally, we examined the pH at which these HAs mediated membrane fusion, as this property is related to the stability of the HA molecule and influences the capacity of influenza viruses to remain infectious in natural environments. Our results show that cleavage efficiency can vary significantly for individual HAs, depending on the protease, and that some HA subtypes display stringent selectivity for specific proteases as activators of fusion function. Additionally, we found that the pH of fusion varies by 0.7 pH units among the subtypes, and notably, we observed that the pH of fusion for most HAs from human isolates was lower than that observed from avian isolates of the same subtype. Overall, these data provide the first broad-spectrum analysis of cleavage-activation and membrane fusion characteristics for all of the IAV HA subtypes, and also show that there are substantial differences between the subtypes that may influence transmission among hosts and establishment in new species. IAV is associated with significant morbidity and mortality, and represents a challenging public health threat that affects social and economic welfare each year, particularly during IAV pandemics. Although we know that all human strains derive, either directly or via intermediate hosts, from avian viral sources, we know very little about the phenotypic characteristics of the 16 HA subtypes that circulate in aquatic birds and have potential to infect mammals. HA membrane fusion properties, in conjunction with the characteristics for protease activation of HA, a requirement for fusion, are critical factors involved in the ecology and transmission of IAVs, and need to be understood if we are to derive explanations for how pandemic viruses emerge in humans. We examined the cleavage-activation and membrane fusion characteristics for the 16 HA subtypes by transiently expressing HA proteins in cells. Our findings show that the cleavability of the HAs vary considerably between subtypes and depending on the protease. Additionally, analysis of the pH of fusion for each subtype showed that HA stability varied significantly among the subtypes, as well as within subtypes from viruses isolated from different species. Overall, these data have implications for host range, potential for adaptation, and persistence in natural environments.