The hemifusion structure induced by Influenza virus haemagglutinin is determined by physical properties of the target membranes

Influenza A virus hemagglutinin (HA) changes conformation and drives membrane fusion of viral and endosomal membrane at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion the hemifusion structures are not determined(3). Here, influenza virus-like particles (VLP)(4) carrying wild-type (WT) HA or HA hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate (VPP) cryo-electron tomography (cET) which improves signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm (HD) and a novel structure termed lipidic junction. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by HA. The rupture frequency and HD diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that HA induces merger between the viral and a target membrane by one of two independent pathways: rupture-insertion pathway leading to lipidic junction and hemifusion-stalk pathway leading to fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch due to its negative spontaneous curvature in the target bilayer as determined by continuum analysis.