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Structure and assembly of the Ebola virus nucleocapsid

Nature volume 551, pages 394–397 (2017)Cite this article

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Abstract

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever1. Filoviruses are within the order Mononegavirales2, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein–nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein3,4,5,6, and cryo-electron microscopy of nucleocapsid7,8,9,10,11,12 or nucleocapsid-like structures11,12,13. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.

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Figure 1: Structure of Ebola NP 1–450.
Figure 2: Structure of Ebola NP–VP24–VP35–VP40 VLPs.
Figure 3: Structure of Ebola and Marburg NCs from intact viruses.

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Acknowledgements

The Briggs laboratory acknowledges financial support from the European Molecular Biology Laboratory and from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-CoG-648432 MEMBRANEFUSION). The Becker group was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 1021) and by the German Center for Infection Research (DZIF). This work was supported by an EMBO long-term fellowship, ALTF 748-2014, awarded to W.W. We thank Y. Kawaoka for support during this collaborative study and W. J. H. Hagen for assistance during tomographic data collection.

Author information

Author notes

  1. William Wan

    Present address: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany

Authors and Affiliations

  1. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, Heidelberg, 69117, Germany

    William Wan, Mairi Clarke & John A. G. Briggs

  2. Institut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Straße 2, Marburg, 35043, Germany

    Larissa Kolesnikova, Alexander Koehler & Stephan Becker

  3. Laboratory of Ultrastructural Virology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan

    Takeshi Noda

  4. PRESTO, Japan Science and Technology Agency, Saitama, 332-0012, Japan

    Takeshi Noda

  5. Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    John A. G. Briggs

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Contributions

W.W., S.B., and J.A.G.B. designed and interpreted experiments. L.K., M.C., A.K., and T.N. prepared specimens. W.W. collected data and performed image processing. W.W. and J.A.G.B. analysed data and wrote the manuscript with support from all authors.

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Correspondence to John A. G. Briggs.

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Reviewer Information Nature thanks I. Gutsche, M. Luo and E. Saphire for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Representative orthoslices of tomograms and subtomogram averages.

Top row, orthoslices through the middle of representative tomograms of the NC and NC-like assemblies. Bottom row, orthoslices taken through the structures determined by subtomogram averaging. The inside of the NC helix is on the left. Scale bars, 20 nm.

Extended Data Figure 2 Local resolution maps and FSCs of NC and NC-like assemblies.

In each panel, the small inset shows the full subtomogram averaging structure of part of the helix with a central subunit coloured. Subtomogram averaging maps are locally filtered and sharpened. The isolated central subunits are shown zoomed in. The surface is coloured according to local resolution in ångströms, as defined by the colour map on the right. FSC curves are calculated using cylindrical masks, with a circular cross-section of approximately one viral subunit (two NPs and outer protrusions) and height centred at each protein layer; green is for NP core, yellow is for VP24 layer, and red is for outer unassigned density layer. In general, the NP core has high, homogeneous resolutions while outer subunits have decreasing resolution with respect to their distance from the NP core.

Extended Data Figure 3 Model of NP from subtomogram averaging compared with NP crystal structure and with modelled RNA.

a, Different views of the subtomogram averaging derived NP model (cyan) and the NP crystal structure17 (pink, PDB accession number 4YPI). Left is a view from inside the NC helix, centre is a cross-sectional view of the NC, and right is a view from outside the NC. b, The NP model in the EM density from our NP 1–450 subtomogram averaging structure. Left shows a cross-sectional view of NC. In the RNA density (yellow) is a rigid-body fit of the six-nucleotide RNA segment from measles virus NP9 (PDB accession number 4UFT). Centre and right, three NP–RNA models from the left panel are fitted as rigid bodies. Centre is a view from the outside of the NC, and right is a focused view of the RNA density. Scale bars, 20 Å.

Extended Data Figure 4 Sequence and secondary structure of NP model.

Our NP model contains NP residues 16–405. Secondary structure elements are highlighted and coloured according to Fig. 1j. Secondary structure labels follow that of crystal structure 4YPI17.

Extended Data Figure 5 Comparison of mononegavirus NPs and encapsidated RNA.

Each row shows two views of each NP: left is a cross-sectional view of the NC, with the centre of the NC axis to the left; right is a view of the NP from outside the NC. Scale bar, 20 Å.

Extended Data Figure 6 Global fit of Ebola crystal structures into the subtomogram averaging structure of Ebola NP–VP24–VP35–VP40 NC-like assembly.

Left column shows histograms for random rigid-body fits. Coloured arrows identify the fits illustrated in the other columns. Second column shows the top two scoring fits within the targeted density. The last two columns show detailed views of the highest and second-highest scoring fits, respectively. a, Fits into the full outer densities; densities are shown at 1.5σ, except the detailed view of the VP24 fits, which are shown at 2.5σ. The fits of VP24 are high-scoring outliers indicating correct fits, and are those shown in Fig. 2. b, Fits into the VP24-subtracted outer protrusion densities; densities are shown at 1.2σ.

Extended Data Figure 7 Stabilization of NP helix 6 upon outer protein binding.

The top of each panel shows the cross-section of NC-like structures. In light grey are two NC subunits. The bottom of each panel shows a detailed view of the two NC subunits with molecular models fitted. Helix 6 (arrow) in NP 1–450 shows nearly no density, while in NP–VP24–VP35–VP40, binding of VP24 stabilizes this helix. Scale bars, 50 Å.

Extended Data Figure 8 Different inter-rung contacts between Ebola NP 1–450 and NP–VP24–VP35–VP40.

Top row shows part of the helix viewed from the inside. Subunits on adjacent rungs are highlighted in grey; these correspond to the subunits fitted by the NP models on the bottom rows. The relative positions of NP molecules across the inter-rung contact differ by 8.3 Å. Scale bars, 20 Å.

Extended Data Figure 9 Lattice map revealing the helical symmetry of Ebola NC.

Cyan arrows denote the positions of one asymmetric unit (two copies of NP, each of which has VP24 bound in a different orientation), as determined by subtomogram averaging. Orange arrows denote positions where a single NP subunit is present that disrupts the normal alternating arrangement of VP24 binding position. Single NP subunits often appear on consecutive rungs along higher-order helical symmetries, forming ‘seams’ along the NC helix. This suggests that VP24 binding is not only influenced by the neighbouring subunits along the NP helix, but also by neighbouring subunits on adjacent rungs.

Extended Data Table 1 Summary of tomographic data acquisition and image processing
Full size table

Supplementary information

Life Sciences Reporting Summary (PDF 68 kb)

A tour of the NC structures presented in this manuscript

A 3D visualization of the structures presented in Figures 1-3. (MP4 27390 kb)

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Wan, W., Kolesnikova, L., Clarke, M. et al. Structure and assembly of the Ebola virus nucleocapsid. Nature 551, 394–397 (2017). https://doi.org/10.1038/nature24490

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