4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus

Abstract

Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.

Figure 1

3D reconstruction of VEEV. (A) A typical CCD image of VEEV TC-83 strain embedded in vitreous ice. Scale bar: 50 nm. (B) Radially coloured 3D reconstruction of VEEV, showing the E1 basal triangle (green) and E2 central protrusion (blue) for each spike. Scale bar: 10 nm. (C) A slice through the 3D density map 20 pixels from the origin. The insert is the 1D radial density profile of the map and is aligned to the slice image. (D) One asymmetric unit of the virus containing four unique copies of E1 (magenta), E2 (cyan), E3 (orange) and CP (blue). The cryo-EM densities for the viral membrane (yellow) and genomic RNA (green) are also displayed at slightly lower isosurface threshold. Scale bar: 2 nm.

Figure 2

E1 and E2 ectodomains. (A) Our models for VEEV E1 (magenta) and E2 (cyan). The homology model parts and de novo model parts are shown as ribbon and stick, respectively. The asymmetric unit averaged map is shown in transparent grey. The de novo modelled part of E1 stem region (residues 390–402) is coloured in yellow. Subdomains of E1 (I, II and III) and E2 (A, B, C and D) are labelled in black circle, following the previous definition (Lescar et al, 2001; Voss et al, 2010). Scale bar: 2 nm. (B) The separation of β-strands at E2 subdomain C, displayed at slight higher isosurface threshold. It also shows the protrusion density for glycan at E2-318 and the annotated atomic structure of N-acetylglucosamine (NAG). (C) A 180° rotation of (A) shows the E1 stem region wraps around E1 subdomain III. The blue dashed arrow points to a small, unidentified density. (D) The E2 subdomain D. (E) The protrusion density for the glycan at E1-N134 and the annotated NAG. (F) The E1 fusion loop (orange) which sits between E2 A and B subdomains.

Figure 3

Mapping of specific residues on VEEV E1 and E2. (A) Model of an E1-E2 heterodimer. Two N-linked glycosylation sites (E1-N134 and E2-N318) are labelled in green and red, respectively. The major determinant of virulence attenuation (residue E2-T120) is labelled in dark blue. Two sets of residues (E2-193/213 and E2-218) whose mutations strongly affects the equine and mosquito host range are labelled in grey and black, respectively. The previously identified VEEV epitopes for murine monoclonal antibodies mMAbs (residues 182–207) and human hMAbs (residues 115–119) are coloured in orange and yellow, respectively. (B) Model of an E2 homotrimer of VEEV in one asymmetric unit. The residue labelling is the same as (A).

Figure 4

The presence of E3 protein in mature VEEV. (A) The densities for E3 (orange) in one asymmetric unit of the original 3D reconstruction. Note the E3 densities are displayed at slightly lower isosurface threshold than E1 (magenta) and E2 (cyan) densities. Scale bar: 2 nm. (B, C) Side and top views of the density for E3 in the asymmetric unit averaged map. The crystal structures of CHIKV pE2 (orange) and E1 (blue) (PDB code: 3N40) are fitted separately as a rigid body into the averaged density. Our models of VEEV E1, E2 and E3 are shown in magenta, cyan and green, respectively. The blue arrows point to the two rod-like features in the density. (D) SDS–PAGE result of VEEV TC-83 samples we used for imaging. The leftmost and rightmost lanes are molecular size markers. Lanes 1–4 are the four batches of VEEV TC-83 samples used for cryo-EM imaging.

Figure 5

E1 and E2 endodomains and their interactions with the CPs. (A) Our models for E1 (magenta) and E2 (cyan) endodomains and CP (dark blue). The homology model parts and de novo model parts are shown as ribbon and stick, respectively. The asymmetric unit averaged map is shown in transparent grey. Various features are highlighted: E2 Y-R-L motif (red), E1 G415/G416 at the kink region (green), E2 C396/C416/C417 near the inner membrane (yellow) and the helix (residues 115–124) of CP (orange). The disordered densities for the lipid bilayer and genomic RNA are simplified with transparent orange and green lines, respectively. Scale bar: 1 nm. (B, C) The prominent side-chain densities for E1-W407, E1-W409 and E1-Y434 in the averaged density map. (D) Same as (A) with less density transparency showing the E2 C-terminal tail and its interaction with the CP pocket. The blue dashed arrow points to the small C-terminal helix of E2 (residues 409–416). (E) Different viewing angle (rotation of 70° along the z axis) of (D) showing the density for the previously unidentified helix of CP (pointed by blue dashed arrow). (F) Secondary structure prediction for E1 and E2 C-termini from PSIPRED.

Figure 6

CP/CP interactions in the nucleocapsid core. (A) Our model of the entire nucleocapsid core fitted into the cryo-EM density. The four copies of CPs in one asymmetric unit are coloured in red, blue, green and yellow. Scale bar: 5 nm. (B) Zoom in view of the intra-capsomere interactions. (C) Zoom in view of the densities around a quasi three-fold axis.

Figure 7

Proposed spatial arrangement of the first ∼120 residues of CP in the nucleocapsid core. (A) In our proposed model, the helix I coiled-coil structure (residues 34–51) between two neighbouring CPs is located at the low-density shell (radius 95–130 Å) between the CP/RNA mixture shell and the central core, while the first 33 hydrophobic residues are located at the central core (radius <95 Å). Scale bar: 10 nm. (B) Secondary structure prediction for CP N-terminus from the PSIPRED results.