Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

Abstract

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...

H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion

“…Limited available data on fusion pH and stability of influenza viruses circulating in natural host species seem to indicate that human viruses fuse at a lower pH than avian and swine viruses (21,(26)(27)(28)(29)(30). However, different viral strains and distinctive assays were used by different authors, and pronounced subtype-and staindependent variation in the viral fusion activity was observed in these studies hampering solid conclusions.…”

Influenza A viruses serially passaged in different MDCK cell lines exhibit limited sequence variation across their genomes, with the exception of the hemagglutinin gene

“…For both pH1N1 and wyoH3N2 human IAV strains, most amino acid mutations altered residues within the stalk region of the HA gene, with some increasing to high frequency between 40% and 99%. Similar mutations altering the pH stability of the HA influence host tropism and stability during transmission (53,54). These mutations may therefore be due to selection for HA stability at different pHs, or for more efficient protease cleavage or fusion in MDCK cells under the culture conditions of our studies.…”

The role of fusion activity of influenza A viruses in their biological properties

“…Th e substitution of amino acid lysine in position 58 of HA2 gp, highly conserved among all subtypes (except H11, which has arginine in this position), to isoleucine led to a decrease of pH optimum of in vitro fusion by about 0.7 units (Byrd-Leotis et al, 2015). Th is decrease was associated with a better stability of HA in the acidic pH.…”

“…Th is decrease was associated with a better stability of HA in the acidic pH. On the other hand, substitution D to G on HA2 gp (D112 2 G) increased pH optimum of fusion in all subtypes (except H5N1 A/ VietNam/1204/2004 virus) and was the reason of a lower HA stability at the acidic pH (Daniels et al, 1985;Weis et al, 1990;Byrd-Leotis et al, 2015).…”