Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography

Abstract

Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoprotein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoprotein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoprotein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the viral matrix protein VP40 to the C terminus of the nucleoprotein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoprotein. Four proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of viral proteins in its structure and assembly.

Fig. 1.

CryoEM of EBOV. (A) Low-magnification cryoEM images of purified EBOV. Protein density is black. Filamentous particles of varying lengths, spherical particles, and other irregularly shaped particles are observed. (B) CryoEM image of a filamentous EBOV virion. White arrow, EBOV virion with an NC. Black arrow, a thin particle without an internalized NC. (C) Histograms of virion length (Left) and diameter (Right) for filamentous EBOV virions containing an NC. (D) Corresponding histograms for MARV. More details in Fig. S1 and SI Materials and Methods.

Fig. 2.

CryoET and 3D reconstruction of the EBOV NC from subtomogram averaging. (A) A slice through a reconstructed, filtered tomogram of EBOV. Protein density is black. White arrowhead indicates the rod-like NC within the virion. (B) Reconstruction of the EBOV NC from cryoET and subtomogram averaging (Left) compared with the MARV NC reconstruction (Right) (16). Isosurfaces have been contoured at 1.5 σ away from the mean, and the helical axis is vertical in the plane of the paper. (C) The same reconstructions as B, viewed along the helical axis.

Fig. 3.

Minimum assembly component of the EBOV NC. (A) CryoEM image of purified full-length EBOV NP. Protein density is black. (B) Image of purified NP(1-451). Inset: 2D average of extracted helical segments. Width of box 720 nm, protein density white. (C) Corresponding images of the NC helix purified from NP+VP40 VLPs. (D) Comparison of proportion of condensed helices (green) and loose coils (yellow) observed in the three samples. Data values are in Table S1.

Fig. 4.

Protein recruitment and formation of a rigid NC. (A) Detection of viral proteins in respective VLPs. Purified VLPs were collected, and Western blot analysis using rabbit anti-NP, -40, -35, and -24 antibodies was performed. (B) A tomographic slice through an empty VLP. Protein density is black. (C) Slice through a VLP with a broken NC. Points of breakages in the NC helix have been highlighted with white arrows. (D) A VLP with a rigid NC. (E) Proportion of particles observed with a rigid NC (dark green), with an overall broken NC (orange), and without an NC (gray) in different samples. Data values are in Table S2.

Fig. 5.

Location of viral proteins in the EBOV NC. (A) 2D class averages of the NC from NP+VP40 VLPs. (B) 2D class averages of the NC from NP+VP24+VP35+VP40 VLPs. (C) 2D class averages of the NC from EBOV virions. Black arrows indicate protrusions. (D) Subtomogram averaging reconstruction of the NC helix from NP+VP24+VP35+VP40 VLPs. Isosurfaces have been contoured at 1.5 σ away from the mean, and the helical axis is vertical in the plane of the paper. (E) The same reconstruction viewed along the helical axis.

Fig. 6.

Steps involved in EBOV NC assembly. A schematic illustration of the samples described in this study and their assembly properties. Assembly of a virus particle is indicated by the thick arrow. Initial condensation of the NP-RNA complex can be achieved in vitro by removal of the disordered C-terminal, or in cells by coexpression with VP40 (thin arrows). The condensed helix can be converted into a rigid NC-like helix inside VLPs only if all NP, VP24, VP35, and VP40 are expressed. The resulting NC helix is indistinguishable from that in EBOV virions.