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. 2020 Nov 18;11(1):5885.
doi: 10.1038/s41467-020-19619-7.

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

Steffen Klein  1   2 , Mirko Cortese  3 , Sophie L Winter  1   2 , Moritz Wachsmuth-Melm  1   2 , Christopher J Neufeldt  3 , Berati Cerikan  3 , Megan L Stanifer  3 , Steeve Boulant  4 , Ralf Bartenschlager  5   6   7 , Petr Chlanda  8   9
Affiliations

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

Steffen Klein et al. Nat Commun. .

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Spatial distribution and length of RNA filaments.
a Slice of a SIRT-like filtered tomogram of an A549-ACE2 cell infected with SARS-CoV2 showing a DMV. b Magnified slice of a tomogram (1.3 nm thick) showing RNA filaments and a branching point (arrowhead) in detail. c Distribution of DMV inner membrane diameter in three different cell lines (A549-ACE2: n = 14; VeroE6: n = 20; Calu3: n = 3). Data is shown as Box and Whiskers plots indicating the median (center of box), 25% and 75% quartiles (bounds of box), minimum and maximum values (bars), and all data points. Unpaired two-sided t-test showed no significant differences between the diameter of DMVs found in the three different cell lines. d, e Tomogram slices shown in a and d after content-aware denoising using cryo-CARE. f Average normalized density line profile of filament cross-sections. The indicated error band shows the SD (n = 80 from two tomograms), the average gray value of the DMV interior is shown as dotted line (0.44 arbitrary units). g, h Manual segmentation of the denoised DMV (Supplementary Movie 1), inner and outer membrane are represented in light and dark green, respectively. Individual segmented filaments are colored according to their length and a branching point is indicated by an arrowhead in the magnified segmentation image (h). i Histogram of RNA length (n = 101) with a bin size of 20 nm. Branched filaments were measured individually. Bicubic interpolation was applied for image smoothing in b and e. Scale bars: a, d 100 nm; b, e 10 nm.
Fig. 2
Fig. 2. Juxtaposed DMVs form membrane junctions that lead to homotypic fusion.
Tomograms showing DMVs in VeroE6 (a–c) and Calu3 (d–f) cells at 16 hpi. For each tomogram, a magnified area is shown below and indicated as a white square. 10 slices of the tomogram were averaged. a DMV–DMV interaction via a constricted outer membrane connection. b, c Tightly apposed membrane stack composed of four membranes (arrows) of varying curvature between two juxtaposed DMVs. d VP containing two inner vesicles with a tight membrane–membrane contact. e DMV interaction with a VP containing two inner vesicles. f VPs containing four inner vesicles with tight contact and an opening aperture formed by two stacked membranes indicated by white arrows. Scale bars: a–f 100 nm; magnified areas, 50 nm.
Fig. 3
Fig. 3. SARS-CoV-2 virion budding and assembly at the ERGIC membrane.
Different budding events captured in two tomograms (Supplementary Fig. 6 and Supplementary Movies 2 and 3) of VeroE6 cells infected with SARS-CoV-2 at 16 hpi. A 3D volume rendering is shown for each area with cellular and viral membranes in green and magenta, respectively. S (yellow) and vRNPs (cyan) are represented as subtomogram averages. The S and vRNP locations correspond to the location in the tomogram, vRNP orientations were randomized. A non-local means filter was applied, and 20 slices were averaged. a Overview of budding events at the ERGIC membrane and intracellular released virions inside the ERGIC lumen are indicated. b, c Accumulated S at the lumenal side of the ERGIC membrane. d, e Early virion budding stage with S and vRNPs accumulated at the lumenal and cytosolic ERGIC membrane, respectively. f Assembled viron in the ERGIC lumen in close proximity to the membrane, showing a polarized distribution of S. g Assembled virion further away from the membrane with redistributed S. Scale bars: a 200 nm, b–g 100 nm.
Fig. 4
Fig. 4. Structural analysis of intracellular virions.
a Tomogram showing an intracellular virion of VeroE6 cells infected with SARS-CoV-2 at 16 hpi. 20 slices of the tomogram were averaged and a median filter (radius = 1 pixel) was applied. b 3D volume rendering of a with the viral envelope shown in magenta, with both leaflets of the membrane resolved. S (yellow) and vRNPs (cyan) are represented as subtomogram averages. The S and vRNP locations correspond to the location in the tomogram, vRNP orientations were randomized. c Distributions of intracellular virion diameters measured in A549-ACE2 (n = 52), VeroE6 (n = 20), and Calu3 cells (n = 3). Box and whiskers plots indicate the median (center of box), 25% and 75% quartiles (bounds of box), minimum and maximum values (bars), as well as all data points. Unpaired two-sided t-test showed no significant differences between the diameter of intracellular virions found in the three different cell lines. d, e Central longitudinal (d) and cross-sectional slice (e) showing the subtomogram average of the S trimer of intracellular virions. f Orthogonal views of the S trimer subtomogram average (yellow) and the virion envelope (magenta). Fitted structure of the S trimer ectodomain (6VXX, 10.2210/pdb6VXX/pdb) and the HR2 domain of SARS-CoV-1 (2FXP, 10.2210/pdb2FXP/pdb) are shown in black and orange, respectively (Supplementary Movie S4). g Plot showing the distribution of S nearest-neighbor distances (n = 100) on the surface of virions with an average of 23.6 nm (SD = 8.1 nm, n = 100). Scale bars: a, b 50 nm; d–f 5 nm.
Fig. 5
Fig. 5. Structural analysis of extracellular virions.
a–c Slices of tomograms showing extracellular virions released from A549-ACE2, VeroE6, and Calu3 cells, respectively. d–f magnified views of (a–c), for better visualization, 10 slices were averaged. Exemplarily, S glycoproteins in a pre-fusion (arrow) or post-fusion conformation (arrowhead) are marked, vRNP complexes are encircled with dashed lines. g Plot profile through the viral envelope (VE, magenta) of virions released from VeroE6 cells and adjacent plasma membrane (PM, green) to determine monolayer separation (n = 129 and 49, respectively, SD indicated). h Number of vRNP complexes per virion (n = 28) released from VeroE6 cells. Data are represented as a histogram with a bin size of 3 and Gaussian fit (R2 = 0.8645). i, j Subtomogram average of vRNP complexes from 1570 individual vRNPs found in 15 tomograms. Central XZ-slice (i) and XY-slice (j) are shown. k, l Isosurface representation of the subtomogram average (Movie S5) shown from the top (k) and side view (l). Scale bars: a–c 100 nm; d–f 50 nm; i–l 5 nm.
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