Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5–6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2–5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009–2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.Wild aquatic birds are thought to be the natural reservoir of influenza A viruses (1). Influenza pandemics occur every few decades, and swine are widely believed to be a key factor in the genesis of pandemics by facilitating reassortment of the eight viral gene segments and replacing avian-like (α-2,3-linked) hemagglutinin (HA) sialic acid receptor-binding specificity with human-like (α-2,6-linked) (2). If the molecular adaptations that allow efficient human-to-human transmissibility are understood, then circulating viruses undergoing these changes (i.e., those with the greatest pandemic potential) could be identified.In 2009, pandemic (p) H1N1 emerged from swine and swiftly infected more than 60 million people, causing 12,000 US deaths in the first year (3). The pandemic strain originated by reassortment in swine, combining five genes (PB1, PB2, PA, NP, and NS) from North American triple-reassortant swine (TRS) viruses, two genes (NA and M) from Eurasian avian-like swine viruses, and an HA gene closely related to that of the classical swine lineage (4). pH1N1 viruses continue to circulate as seasonal H1N1 viruses. They retain several known pandemic traits, including α-2,6-linked sialic acid receptor-binding specificity of the HA, functional balance of HA and NA activity, and a polymerase adapted to the mammalian upper airway (5). Although these traits appear to be necessary for airborne transmissibility of influenza viruses, they do not appear to be sufficient. For example, H5N1 viruses engineered to have these traits were not air-transmissible among ferrets until a mutation increased HA thermostability and lowered the HA activation pH (6–8). The importance of HA stabilization in supporting the adaptation of influenza viruses to humans or enabling a human pandemic is not completely understood.After receptor binding and endocytosis, low pH triggers irreversible structural changes in the HA protein that fuse the viral envelope and host endosomal membrane (9). Measured HA activation pH values across all subtypes and species range from ∼5.0 to 6.0, trending higher in avian viruses (pH 5.6–6.0) and lower in human viruses (pH 5.0–5.5) (10).The goal of this study was to define the role of HA acid stability in pH1N1 pandemic capability. Our data show that HA activation pH decreased as H1N1 adapted from swine to humans. Complementary experiments in ferrets recapitulated this evolution, as we observed a loss-of-function pH1N1 virus acquired airborne transmissibility via stabilizing mutations. Overall, these studies link a fundamental molecular property, the barrier for activation of a membrane fusion protein (for influenza virus HA, its acid stability), to the interspecies adaptation of a ubiquitous respiratory virus.