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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.

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N-and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.receptor binding | hemagglutinin | virus attachment

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The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.

To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.

Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles. IMPORTANCEThe influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components. N early every year seasonal influenza A viruses contribute to hundreds of thousands of human deaths worldwide and place an extraordinary burden on health care systems and the global economy. Of even greater concern is the unpredictable appearance of antigenically novel influenza A strains in humans, with the potential for causing devastating pandemics. A major obstacle for controlling influenza is the diversity of influenza virus A strains that circulate in the avian species, since this provides a vast genetic pool of vi...