Dr. Georg Wolff and colleagues have published their remarkable electron cryo-microscopy work to capture a molecular pore complex in the double membrane vesicles (DMV) from murine hepatitis coronavirus (MHC)- infected cells and SARS-CoV-2 infected cells. While the majority of the experiments were done using the murine hepatitis coronavirus for biosafety reasons, the authors note these features are likely conserved among the betacoronaviruses and interrupting this replication cycle may be an avenue of drug-development for coronavirus-specific therapies for SARS-CoV-2 and future novel coronaviruses.
The authors describe the significance of their findings:
“We surmise that this pore represents a generic coronaviral molecular complex playing a pivotal role in the viral replication cycle. Most likely, it allows the export of newly synthesized viral RNA from the DMVs to the cytosol. Functionally analogous viral complexes used for RNA export include those in the capsids of the Reoviridae (10) and, interestingly, the molecular pore in the neck of the invaginated replication spherules induced by flock house virus (11), although none of these is integrated in a double-membrane organelle.”
Lastly the authors propose a model and mechanism (Figure 4 below) based on their photos describing RNA viral export, encapsidation, travel to assembly sites, and viral budding.
The link to the entire paper is available here. Enjoy the beautiful photos below.
Figure 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 in infection. (B) 3D model of the tomogram with the segmented content annotated, see also movie S1. ERGIC, ER-to-Golgi intermediate compartment; RNP, ribonucleoprotein complex.
Figure 2 Architecture of the molecular pores embedded in DMV membranes.
Tomographic slices (7 nm thick) revealed that pore complexes were present in both (A, inset) MHV-induced and (B) prefixed SARS-CoV-2-induced DMVs (white arrowheads). (C to L) 6-fold 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 M) 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 6-fold symmetrized volume (c6, green) appeared as an off-centered asymmetric density in the unsymmetrized average (c1).
Figure 3 The coronavirus transmembrane-protein nsp3 is a component of the pore complex.
(A) Membrane topology (top) of MHV transmembrane nsps with protease cleavage sites indicated by orange (PL1pro), red (PL2pro) and gray (Mpro) arrowheads. (bottom) Detailed depiction of nsp3, showing some of its sub-domains and the position of the additional GFP moiety that is present in MHV-Δ2-GFP3. (B) Tomographic slice of DMVs induced by MHV-Δ2-GFP3 with embedded pore complexes (white arrowheads). Comparison of the central slices of the 6-fold symmetrized subtomogram averages of the pore complexes in DMVs induced by (C) wt MHV and (D) MHV-Δ2-GFP3. (E) Density differences of 3 standard deviations between the mutant and the wt 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). PLpro, papain-like protease; Mpro, main protease.
Figure 4 Model of the coronavirus genomic RNA transit from the DMV lumen to virus budding sites.
(Top) Tomographic slices from MHV-infected cells highlighting 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 RNPs then can travel to virus assembly sites for membrane association and (D) subsequent budding of virions.