A molecular pore spans the double membrane of the coronavirus replication organelle

Abstract

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

Fig. 1. Coronavirus-induced DMVs revealed by cryo-ET.

(A) Tomographic slice (7 nm thick) of a cryo-lamella milled through an MHV-infected cell at a middle stage of infection. (B) Three-dimensional (3D) model of the tomogram, with the segmented content annotated. See also movie S1. ERGIC, ER-to-Golgi intermediate compartment.

Fig. 2. Architecture of the molecular pores embedded in DMV membranes.

Tomographic slices (7 nm thick) revealed that pore complexes were present in both (A) MHV-induced DMVs and (B) prefixed SARS-CoV-2–induced DMVs (white arrowheads). The inset in (A) is a close-up view of the area delineated by white brackets. (C to L) Sixfold-symmetrized subtomogram average of the pore complexes in MHV-induced DMVs. (C) Central slice through the average, suggesting the presence of flexible or variable masses near the prongs (black arrowhead) and on the DMV luminal side. (D to F) Different views of the 3D surface-rendered model of the pore complex (copper colored) embedded in the outer (yellow) and inner (blue) DMV membranes. (G to L) 2D cross-section slices along the pore complex at different heights (see also movie S2). (M and N) An additional density at the bottom of the sixfold-symmetrized volume (c6, green) appeared as an off-center asymmetric density in the unsymmetrized average (c1).

Fig. 3. The coronavirus transmembrane protein nsp3 is a component of the pore complex.

(A) (Top) Membrane topology of MHV transmembrane nsps, with protease cleavage sites indicated by orange (PL1^pro), red (PL2^pro), and gray (M^pro) arrowheads. (Bottom) Detailed depiction of nsp3, showing some of its subdomains and the position of the additional EGFP moiety present in MHV-Δ2-GFP3. PL^pro, papain-like protease; M^pro, main protease. (B) Tomographic slice of DMVs induced by MHV-Δ2-GFP3, with embedded pore complexes (white arrowheads). (C and D) Comparison of the central slices of the sixfold-symmetrized subtomogram averages of the pore complexes in DMVs induced by (C) wild-type (wt) MHV and (D) MHV-Δ2-GFP3. (E) Density differences of 3 standard deviations between the mutant and wild-type structures, shown as a green overlay over the latter, revealed the presence of additional (EGFP) masses in the mutant complex (black arrowheads; see also movie S3).

Fig. 4. Model of the coronavirus genomic RNA transit from the DMV lumen to virus budding sites.

Tomographic slices from MHV-infected cells (top) highlight the respective steps in the model (bottom). (A) The molecular pore exports viral RNA into the cytosol, (B) where it can be encapsidated by N protein. (C) Cytosolic RNP complexes can then travel to virus assembly sites for membrane association and (D) subsequent budding of virions. The insets in the top panels provide close-up views of the areas delineated by white brackets.