Influenza Hemagglutinin Protein Stability, Activation, and Pandemic Risk

Abstract

For decades, hemagglutinin (HA) protein structure and its refolding mechanism have served as a paradigm for understanding protein-mediated membrane fusion. HA trimers are in a high-energy state and are functionally activated by low pH. Over the past decade, HA stability (or the pH at which irreversible conformational changes are triggered) has emerged as an important determinant in influenza virus host range, infectivity, transmissibility, and human pandemic potential. Here, we review HA protein structure, assays to measure its stability, measured HA stability values, residues and mutations that regulate its stability, the effect of HA stability on interspecies adaptation and transmissibility, and mechanistic insights into this process. Most importantly, HA stabilization appears to be necessary for adapting emerging influenza viruses to humans.

Keywords: fusion glycoprotein; influenza A virus; interspecies adaptation; pandemic; virus entry; virus transmission.

Figure 1. Model of influenza A virus transmission

Endemic circulation of representative subtypes is shown within circles. Major pathways of transmission between species are denoted by straight arrows. Diverse influenza A viruses of 16 known HA and 9 known NA subtypes circulate in a reservoir of wild aquatic birds (dark blue), occasionally transmitting to other wild and domestic species. Bats host two recently identified subtypes, H17N10 and H18N11. A major pathway for the transfer of genetic diversity occurs from wild birds to domestic poultry (light blue). Infections in domestic poultry sporadically spread to farm animals and humans. Influenza viruses frequently transmit between swine (dark orange) and humans (orange). Examples of endemic strains circulating within a species are described within circles. Examples of sporadic (non-endemic) transmission of influenza viruses between species have been omitted for simplicity including recent human outbreaks such as H5N1 and H7N9.

Figure 2. Influenza virus replication cycle and properties influencing adaptation to humans and ferrets

Major steps during replication are denoted in yellow boxes. These include receptor binding, endocytosis, low-pH-induced membrane fusion, uncoating, nuclear import, transcription, mRNA export, viral protein translation, viral protein import into the nucleus, viral genome replication, viral ribonucleoprotein (vRNP) export, vRNP transport to the plasma membrane, virus assembly, virus budding, and virus release. Properties identified in interspecies adaptation are denoted by blue boxes. Extracellular adaptive properties include the stability of virions and the HA protein, virion morphology, balance of HA binding and NA receptor-destroying activities, and NA stalk length. Intracellular adaptive properties include receptor-binding specificity by the HA protein, HA stability, polymerase efficiency, and interferon antagonism.

Figure 3. Prefusion structure of the HA protein and residues known to affect its stability

(A) HA domain structure. HA1 domains include the fusion (F1, blue), vestigial esterase (VE, yellow), and receptor-binding domain (RBD, green). HA2 includes a stalk domain (orange), transmembrane region (TM, white), and cytoplasmic tail (CT, white). Solid circles identify residues to which stabilizing or destabilizing mutations have been identified in review articles [21, 22] and primary manuscripts [, –, , , –87]. (B) Domain insertion in the HA protein, adapted from [116]. (C) Prefusion structure of one HA monomer. (D) Prefusion structure of an HA trimer. Residues regulating stability (black balls) are located throughout the trimer in the receptor-binding pocket (R.B.P.), between HA1 heads (head-head), between the HA1 head the stalk (head-stalk), in the B loop and adjacent helix C (B loop area), in the core of the coiled coil (core), in the HA1 stalk, between helix A and the coiled coil (helix A), in and around the fusion peptide pocket (fus. pep. & pocket), and in the membrane-proximal region (M.P.R.). In panel D, two protomers are colored gray. Structures were generated using MacPYMOL using A/California/4/2009 (H1N1) protein data bank structure 3UBE [117].

Figure 4. HA activation pH values for influenza A viruses

(A) Highest pH values of overlaid media that trigger virus-infected cells to form syncytia, or fuse, grouped by host species. Isolates from wild birds include H1N1 and H5Nx (H5N2 and H5N8) [54, 68]. Isolates from poultry include H5Nx, H5N1, and H9N2 [57, 58, 68, 72, 73, 98]. Swine isolates include H1N1, H1N2, and H3N2 [54, 66]. Human isolates include zoonotic infections (H5N1, H7N7, H7N9, and H9N2) and 2009 pandemic H1N1 (pH1N1) viruses [54]. Mean values for each group are shown by horizontal bars. All data was collected using the same methods in the same laboratory. Statistical significance between the human pH1N1 group and the other groups was determined by one-way ANOVA analysis followed by a Tukey post-hoc test: *P < 0.05, **P < 0.01, ***P < 0.001. (B) Relationship between HA activation pH and transmissibility between mallards. A/chicken/Vietnam/C58/2004 (C58) (H5N1) wild-type (WT, closed circle) and HA1-H18Q (closed square) were transmissible between mallards, while CH58 HA2-K58I (open circle) was loss-of-function for transmissibility [73]. (C) Relationship between HA activation pH and transmissibility between ferrets by the airborne route. Viruses incapable of ferret airborne transmission included A/Tennessee/560-1/2009 (H1N1, closed circle) containing an HA1-Y17H mutation [54], A/Indonesia/5/2005 (H5N1, open square) wild-type [60, 95], and an H5N1 reassortant virus containing the wild-type HA protein from A/Vietnam/1203/2004 (VN1203, gray triangle) [96]. Airborne transmissible gain-of-function viruses had stabilized HA proteins including pH1N1 HA2-R106K, H5N1 HA1-H110Y, and H5N1 HA1-T318I. H3 numbering is used.

Figure 5. Respiratory tract pH values

Compilation of reported respiratory pH values. Values for healthy humans include those from the nasal cavity [106, 108], nasopharynx [118], soft palate [119], oropharynx [120], trachea [121, 122], and lungs [–125]. Limited data is available for non-human species. Reported values for animals include those for the ferret trachea [126], swine trachea [–129], and mouse nasal cavity [123] and trachea [130, 131]. Values for co-morbidities include those for cystic fibrosis patients [107, 132, 133], healthy children [107, 119, 134, 135], and respiratory infected [135, 136]. Reported values include average values (bold type) and the range of data in parentheses. For illustrative purposes, reported values are shaded using a spectral color scheme (bottom left).

Figure 6. Intracellular pH values

pH values for various subcellular compartments in a prototypic mammalian cell [137, 138]. For illustrative purposes, pH values are shaded using the same spectral color scheme (bottom) as in Figure 5.