Cryomicroscopy provides structural snapshots of influenza virus membrane fusion

Abstract

The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy.

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Figure 1: Atomic models of influenza hemagglutinin at neutral and low pH.
Figure 2: Time course of fusion, studied by cryomicroscopy.
Figure 3: Tomogram section at the 30-min, pH 5–incubation time point.
Figure 4: 3D contact zones.
Figure 5: Tomogram sections showing HA and membrane architecture during fusion.
Figure 6: Summary of structural steps in membrane fusion.

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Acknowledgements

We are grateful to S. Wharton for advice on experiments, M. Maiorca for assistance in building segmentation models of the membranes, and J. Skehel and J. Molloy for discussions. Research was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council (program code U117581334 to P.B.R.), and the Wellcome Trust.

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L.J.C. and P.B.R. designed experiments and wrote the manuscript. L.J.C. performed experiments and analyses.

Corresponding author

Correspondence to Peter B Rosenthal.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 2D cryomicroscopy images of low-pH time course after trypsin treatment (pH 7; pH 5, 1 min; pH 5, 5 min; pH 5, 30 min).

Images are unfiltered.(a) pH7 images show no contact between virus and liposomes, which are content-less, sometimes multi-lamellar and heterogeneous in size.(b) pH5, 1 min. Viral particles retain capsular shape but are in contact with many liposomes.(c) pH5, 5 mins. Viral particles are less ordered and contacting many liposomes. Some particles have fused with liposomes and viral contents can be observed in some liposomes.(d) pH5, 30 mins. Large liposomes contain viral protein structures, similar to those seen in low pH virions. Glycoproteins are widely distributed around the membrane and contacts with other liposomes occur.

Supplementary Figure 2 Tomogram section showing virus interactions with liposomes at the pH 5, 1-min time point.

Images are slices of tomograms (projections of a 22 Å slab) showing the morphology of the viral particles with intact M1 matrix layer, NA patch, and closely packed RNP segments labeled.

Supplementary Figure 3 Gallery of 2D images of the HA extended intermediate.

Images in every second row are identical to row above but indicate membranes (red lines) bridged by extended HA intermediate (green lines). images are filtered as described in methods

Supplementary Figure 4 Gallery of 3D tomogram sections of the HA extended intermediate.

Images are slices of tomograms (projections of 22 Å slab) showing additional examples to those in Figure 5a. Images in every second row are identical to row above but indicate membranes (red lines) bridged by extended HA intermediate (green lines).

Supplementary Figure 5 Histograms of lengths of different HA structures observed and measured from tomograms.

(a) Length measurements for pH7 HA and HA extended intermediate. (b) Length measurements for bars, lines in star arrangements, and HA2. Model projections (right column) have been calculated from crystal structures described in Figure 1.

Supplementary Figure 6 Tomogram sections of a 3D contact zone between virus and liposome (1-min time point).

Images are tomogram slices (projections of a 13 Å slab) at intervals of 43 Å. Images in every second row are identical to row above but indicate membranes (red lines) bridged by extended HA intermediate (green lines). Last panel: shows membranes and extended HA intermediate for all tomogram sections. Corresponds to contact zones in Figure 4a-d.

Supplementary Figure 7 Tomogram sections of a 3D contact zone between virus and liposome (1-min time point).

Images are tomogram slices (projections of a 13 Å slab) at intervals of 43 Å. Images in every second row are identical to row above but indicating membranes (red lines) bridged by extended HA intermediate (green lines). Last panel: shows membranes and extended HA intermediate for all tomogram sections.

Supplementary Figure 8 Gallery of bar images.

Shows additional examples to those in Figure 5c,d of dense bars at membrane contacts (yellow arrows). First two panels of first row are tomogram sections. Remaining images are 2D images filtered as described in methods. Black spots densities are gold fiducial markers.

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Calder, L., Rosenthal, P. Cryomicroscopy provides structural snapshots of influenza virus membrane fusion. Nat Struct Mol Biol 23, 853–858 (2016). https://doi.org/10.1038/nsmb.3271

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