Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion1,2. For viral fusion, the hemifusion structures are not determined3. Here, influenza virus-like particles4 carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S5 and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a ‘lipidic junction’. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture–insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.
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The authors thank V. Nair for assistance with the Krios transmission electron microscope at Rocky Mountain Laboratories Microscopy Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The authors also thank L.-A. Carlson, L. Chernomordik, I. Morales and T. Reese for critical reading of the manuscript. This work was supported by the Division of Intramural Research of the Intramural Program of the National Institutes of Health.
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Supplementary Results, Figures 1–10, Tables 1–4, Video legends and References. (PDF 14180 kb)
Tomogram showing ‘Y’ (lipidic) junctions mediated by G1S VLP corresponding to Fig. 2b. Tomogram was acquired at defocus –5 μm without VPP and denoised using nonlinear anisotropic diffusion (NAD) filter with k value 10 and 10 iterations. Scale bar: 50 nm. (MOV 2721 kb)
Tomogram showing ‘Y’ (lipidic) junctions mediated by G1S VLP corresponding to Fig. 2c. Tomogram was acquired at defocus –1 μm with VPP and denoised using NAD filter with k value 10 and 10 iterations. Scale bar: 50 nm. (MOV 6384 kb)
Tomogram showing liposome and complete HD connected to the membrane of the G1S filamentous VLP from top (corresponding to Fig. 2e). Tomogram was acquired at defocus –1 μm with VPP. (MOV 907 kb)
Animated visualization of the isosurface of a spherical VLP and several liposomes at low pH shown in Figs 2c and 3a–d. Liposome marked by A corresponds to the liposome in Figs (MOV 6651 kb)
Tomogram showing ruptured liposomes and lipidic junctions with G1S filamentous VLP (corresponding to Fig. 3e). Tomogram was acquired at defocus –1 μm with VPP and denoised using the NAD filter with a k value of 1 for 10 iterations. (MOV 2543 kb)
Animated visualization of the isosurface of ruptured liposomes and lipidic junctions with G1S filamentous VLP (corresponding to Fig. 3e). Scale bar: 50 nm. (MOV 2901 kb)
Tomogram showing WT VLP and liposomes fusion product (FP) after complete fusion characteristic of areas of membrane free of influenza glycoproteins. Red arrows and marks sparsely distributed influenza glycoproteins and red line highlights the membrane harbouring influenza glycoproteins. The unmarked area is free of influenza glycoproteins. Scale bar: 50 nm. (MOV 11311 kb)
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Chlanda, P., Mekhedov, E., Waters, H. et al. The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes. Nat Microbiol 1, 16050 (2016). https://doi.org/10.1038/nmicrobiol.2016.50
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