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

Nature Microbiology volume 1, Article number: 16050 (2016) Cite this article

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Abstract

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.

To characterize the VLP-based fusion system, we first compared the liposomal fusion products of intact influenza virus (X31) formed at low pH to those of WT VLPs. Virus or VLPs were incubated at neutral pH with liposomes (66 mol% phosphatidylcholine (PC), 13 mol% phosphatidylethanolamine (PE), 16 mol% cholesterol and 5 mol% gangliosides as receptor) and then exposed to low pH for 2 min at 37 °C before vitrification and cryo-electron microscopy (cEM). Observed events were classified as binding, fusion intermediate and complete fusion (see Methods). In the binding class, the liposomes bound to VLPs retained their spherical shape or were compressed by adhesive forces to HA glycoproteins. The binding areas between the liposomes and VLPs were typically between 100 and 600 nm2, corresponding to occupancies of one to seven glycoproteins, respectively (Fig. 1b,c). In contrast, the fusion intermediate class showed liposomes with teardrop shapes pointing towards the VLPs where HA glycoproteins were disorganized. WT VLPs had similar numbers of binding and fusion intermediates and lower numbers of complete fusion events compared to X31 virus (Supplementary Fig. 2). For G1S VLP–liposome mixtures, binding and fusion intermediates were observed, but not complete fusion, as expected from this mutation's phenotype in cell–cell fusion (Supplementary Fig. 1). G1S HA supports lipid mixing but not fusion pore opening or content mixing, conditions necessary for observable hemifusion (Supplementary Fig. 1)5. Structurally, the influenza virus and VLPs form similar fusion intermediates with liposomes. However, unlike virus, we can produce VLPs carrying mutations in the HA fusion peptide that prevent viral replication.

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Figure 1: VLP structural features are resolved using VPP and indicate that influenza VLPs carrying G1S mutated HA engage one to seven HA glycoproteins on binding to liposomes.
Figure 2: Defocus–phase contrast cET and VPP–cET of liposomal membrane, Y-shaped lipidic junctions and hemifusion diaphragms.
Figure 3: HA spikes in close proximity to lipidic junctions and ruptured membranes.
Figure 4: Two independent, cholesterol concentration-dependent pathways lead to HDs of different diameter.

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Acknowledgements

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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Authors and Affiliations

  1. Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, 20892, Maryland, USA

    Petr Chlanda, Elena Mekhedov, Hang Waters, Paul S. Blank & Joshua Zimmerberg

  2. Rocky Mountain Laboratories, Electron Microscopy Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton 59840, Montana, USA

    Cindi L. Schwartz & Elizabeth R. Fischer

  3. Department of Mathematics, Fordham University, Bronx, 10458, New York, USA

    Rolf J. Ryham

  4. Department of Molecular Biophysics and Physiology, Rush University, Chicago, 60612, Illinois, USA

    Fredric S. Cohen

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Contributions

The project was planned by P.C., P.S.B. and J.Z. Experimental work was performed by P.C. and E.M. pCAGGS-M1 and pCAGGS-M2 constructs were generated by H.W. Cryo-electron microscopy and tomography was done by P.C., C.L.S. and E.R.F. Statistical analysis and image processing was performed by P.C. and P.S.B. Continuum analysis was carried out by R.J.R. and F.S.C. The manuscript was written by P.C., R.J.R., F.S.C., P.S.B. and J.Z. All authors assisted in editing the manuscript and contributed to data analysis.

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Correspondence to Petr Chlanda or Joshua Zimmerberg.

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Supplementary information

Supplementary Information

Supplementary Results, Figures 1–10, Tables 1–4, Video legends and References. (PDF 14180 kb)

Supplementary Video 1

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)

Supplementary Video 2

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)

Supplementary Video 3

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)

Supplementary Video 4

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)

Supplementary Video 5

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)

Supplementary Video 6

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)

Supplementary Video 7

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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