Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion

Abstract

Our previous studies showed that an angled boomerangshaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ϳ105°of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ϳ150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation.Influenza virus enters host cells by receptor-mediated endocytosis and membrane fusion as do many other enveloped viruses (1-3). When the pH in the endosome drops to about 5, membrane-anchored hemagglutinin (HA) subunit HA2 undergoes a dramatic conformational change, which ultimately drives membrane fusion (4). In an early and critical step, the fusion domain, i.e. the ϳ20 most N-terminal residues of HA2, which are often also referred to as the fusion peptide, is exposed from a hydrophobic pocket in the neutral pH HA trimer structure and is propelled toward the virus attachment site where it inserts into the endosomal membrane of the host cell (5). The structure of the endosomal membrane bilayer is perturbed by the action of the fusion domain, and an appropriately controlled perturbation of this bilayer leads to fusion between the viral and cellular membranes.To better understand the process of viral membrane fusion, it is critical to know the structure of the membrane-inserted fusion domain and to examine its interactions with the surrounding bilayer. The HA fusion domain adopts a kinked "boomerang" structure in detergent micelles and lipid bilayers as revealed by combined nuclear magnetic resonance (NMR) and site-directed spin labeling approaches (6, 7). The subtended angle of the boomerang is about 105°with a kink formed by Glu-11, Asn-12, and Gly-13. The pH 5 structure is quite compact with a hydrophobic pocket formed by several aromatic and aliphatic residues, which are all critical for shaping the boomerang and thus for fusi...

Line-Tension Controlled Mechanism for Influenza Fusion

“…Whereas the wild-type peptide has a steep kink-angle of 105 degrees, mutant G13A has a shallower kink-angle of about 150 degrees [11]. Fluorescence microscopy studies have observed that when red blood cells expressing such mutant merge, the content of the cells is released into the inter-cellular space rather than being transferred between the fusing cells [11]. Such massive leakage is readily observed more than minutes before the occurrence of lipid mixing [11].…”

“…Formation of such a pre-fusion structure is believed to involve perforation of the target membrane [6], [7], because leakage through the target membrane is detected prior to lipid mixing [6]–[8], [11], [12].…”

Structure and Mechanism of the Saposin-like Domain of a Plant Aspartic Protease

“…6C). The tryptophan within the hemagglutinin fusion peptide has been shown to induce its characteristic boomerang shape (26), which is critical to the fusogenic and membrane-disrupting activities of this fold (79). Alignment of StAP PSI with hemagglutinin fusion peptide suggested a similar role for the critical tryptophan residue (Fig.…”

The Role of Hydrophobic Residues in the Internal Fusion Loop from Ebolavirus GP2

“…The most extensive study is for the fusion peptide from Influenza HA. It was first determined that the pH dependent formation of a bent, "boomerang" conformation, and the repositioning of two charged residues out of the membrane were imperative for lipid perturbation which was further characterized through extensive structure-function mutagenesis studies [55][56][57][58].…”

“…In its fusion-active (pH 5.5) state, the hydrophobic patch at the tip of the FL is bent that the 105° angle is required for proper fusion: no fusion is observed with a point mutant that forms a single straight interfacial helix [131], and leaky fusion is observed with a mutant peptide in which the angle of the boomerang increased to ~150 o [58]. A 23-residue fusion peptide from a different subtype HA, which has two amino acid differences compared to X:31 HA, was reported to form a tighter bend at a corresponding position [59].…”