Nanometer-resolution in situ structure of the SARS-CoV-2 postfusion spike protein

Abstract

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in β-propiolactone-inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.

Keywords: SARS-CoV-2; cryo-electron tomography; postfusion state; spike protein; subtomogram analysis.

Fig. 1.

Cryo-ET of inactivated SARS-CoV-2 virions. (A) Slice view of tomographic reconstructions of BPL-inactivated SARS-CoV-2 virions. White arrows indicate Ss in the prefusion and postfusion states. (Scale bar, 20 nm.) (B) Selected slices of separate postfusion Ss. (C and D) Selected slices of oligomerized postfusion Ss with side-by-side (C) and branching (D) patterns. Dotted outlines indicate the adjacent postfusion Ss. All tomograms were deconvolved using Warp (17) and are displayed using IMOD (38). (E) Statistics of virion numbers per tomogram and numbers of postfusion Ss per virion. (F) Histograms of nearest pair distances for postfusion Ss in the experimental data and in the simulated data with random distributions.

Fig. 2.

Subtomogram averaging of the SARS-CoV-2 postfusion S. (A) Subtomogram averaging procedure, including the initial average of all particles at a binning level of eight (Left), the refined map at a binning level of two (Middle), and the postprocessed map with a local mask at the extracellular region at a binning level of two (Right). (B) The gold standard FSC curves for the final averaged map of postfusion S. (C) The directional FSC of the final averaged map of postfusion S. (D) Statistics of the coordinate and Euler angle changes before and after alignment during image processing. The coordinate offsets on the x, y, z axes are defined as dx, dy, and dz, while the angular offsets at three Euler angles are defined as drot, dtilt, and dpsi. (E) Plot back of the averaged map onto the original tomogram, showing relative orientations of postfusion Ss to the virus membrane. (F) Distribution of the tilting angles of postfusion Ss relative to viral envelope.

Fig. 3.

In situ structure of the SARS-CoV-2 postfusion S. (A) Geometry of postfusion S on the membrane. This map was generated by merging the TM region of the full averaged map onto the higher-resolution map from focused alignment on the extracellular domains. (B) Domain arrangement of full-length S protein and the modeled parts of the published structure (PDB entry: 6XRA) and our model. The potential cleavage sites and glycosylation sites are indicated. (C) Superimposition of our model with the merged map mentioned in A, showing the glycosylation sites and distinguished 6-HB domains. The observed glycosylation sites are shown in burlywood. Three S protomers are shown in medium slate blue, light coral, and medium turquoise.

Fig. 4.

Oligomerization arrangement of the in situ SARS-CoV-2 postfusion S. (A and B) Side-by-side (A) and branching (B) arrangements of postfusion Ss. The orientation of each spike was determined according to the final refined parameters. Coloring scheme is the same as that in Fig. 3C. (C) Possible model of HR2 domain exchange in the side-by-side oligomerization state. The HR2 domains of the two Ss are colored magenta and blue. The other parts of the two Ss are colored pink and cyan. (D) Models of in situ postfusion S in the regular state, side-by-side state with HR2 domain exchange, and branching state with FP interaction. (E) A scheme of the SARS-CoV-2 S transition from the prefusion to the postfusion state during viral infection and fusion pore formation with the side-by-side oligomerization of Ss involved.