Acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology

Abstract

Membrane fusion is not spontaneous. Therefore, enveloped viruses have evolved membrane-fusion mediating glycoproteins that, once activated, refold, and release energy that fuses viral and cellular membranes. The influenza A virus hemagglutinin (HA) protein is a prototypic structural class I viral fusion glycoprotein that, once primed by proteolytic cleavage, is activated by endosomal low pH to form a fusogenic "leash-in-grooves" hairpin structure. Low-pH induced HA protein refolding is an irreversible process, so acid exposure in the absence of a target membrane leads to virus inactivation. The HA proteins of diverse influenza virus subtypes isolated from a variety of species differ in their acid stabilities, or pH values at which irreversible HA protein conformational changes are triggered. Recently, efficient replication of highly pathogenic avian influenza (HPAI) viruses such as H5N1 in avian species has been associated with a relatively high HA activation pH. In contrast, a decrease in H5N1 HA activation pH has been shown to enhance replication and airborne transmission in mammals. Mutations that alter the acid stabilities of H1 and H3 HA proteins have also been discovered that influence the amantadine susceptibilities, replication rates, and pathogenicities of human influenza viruses. An understanding of the role of HA acid stability in influenza virus biology is expected to aid in identifying emerging viruses with increased pandemic potential and assist in developing live attenuated virus vaccines. Acid-induced HA protein activation, which has provided a paradigm for protein-mediated membrane fusion, is now identified as a novel determinant of influenza virus biology.

Fig. 1

Alignment of HA protein sequences with key features identified. Residues are identified by H3 numbering above the alignment and real numbering (starting with methionine 1) to the right of the sequences. Alpha helical—(cylinders) and beta strand—(arrows) secondary structures are included above the alignments. Both prefusion and postfusion secondary structures are shown in HA2 (they are equivalent in HA1). Fusion (red), esterase (yellow), and receptor-binding (blue) subdomains are color-coded in the secondary structures. Signal peptide, fusion peptide, transmembrane (TM), and cytoplasmic tail (CT) regions are also identified. Residues governing HA acid stability (Table 1) are identified in the primary sequence by yellow boxes. Sequences are from A/Aichi/2/68/X-31 (H3N2), A/Shanghai/02/2013 (H7N9), A/California/04/2009 (H1N1), and A/Vietnam/1203/04 (H5N1)

Fig. 2

Structural changes in the HA protein after low-pH induced activation. a Prefusion HA1/HA2 trimer with HA1 shown in magenta and HA2 secondary structural elements shown in multiple colors. The receptor-binding pocket (R.B.P.) is located in the membrane-distal head domain and the metastable fusion domain is located in the membrane-proximal stalk. b Prefusion HA2 trimer shown without HA1 residues. c Postfusion HA2 trimer showing changes in secondary and tertiary structure after acid-induced irreversible protein refolding. In each panel, two protomers are colored gray. Figure adapted from (Bullough et al. 1994) using A/Aichi/2/68/X-31 (H3N2) protein data bank structures 1HGF and 1QUI

Fig. 3

Locations of HA acid stability mutations in the prefusion conformation. a RBD residues near or in the receptor-binding pocket (R.B.P.) or at the interface of HA1-HA1 protomers. b Esterase sub-domain residues at the interface of the receptor-binding subdomain and the stalk region. c HA1 and HA2 residues in the spring-loaded coiled coil region, in and around the fusion peptide pocket, and in the membrane-proximal region. In panels A and B, the receptor-binding subdomain is colored blue and the esterase subdomain is colored yellow. In panel C, HA1 residues in the stalk are colored magenta and HA2 secondary structural elements are colored as in Fig. 2. Residues governing HA acid stability are identified using H3 numbering on the A/Aichi/2/68/X-31 (H3N2) protein data bank structure 1HGF. Acid stability mutations are described in detail in Table 1