The NTD and CTD of SARS-CoV-2 NSP3, yet not the CTD of NSP4 are essential for the formation of DMVs. (A) Tomographic slice of HeLa cells transfected with GFP-NSP3 and NSP4-mCherry, demonstrating the formation of DMVs induced by the overexpression of NSP3 and NSP4. The DMVs are connected with ribosome-decorated ER (blue arrowheads indicate the connections). Scale bar, 200 nm. (B) 3D rendering of the tomogram shown in A, illustrating the overall structure of the NSP3/4-induced DMVs. (C) Gallery of NSP3/4-induced DMVs, highlighting the connection of the outer membrane of DMVs with the ER (dark blue arrowheads). Neck-like connections (yellow arrowhead), the presence of ribosomes inside DMVs (purple arrowhead), and two fused DMVs (the last right panel) are also observed. Scale bar, 100 nm. (D) Enlarged view of the white-boxed area in A, revealing the presence of pore complexes in NSP3/4-induced DMVs (green arrowhead). Scale bar, 100 nm. (E) Averaged slices of a tomogram showing a zoom in area of one DMV pore as observed in D. Scale bar, 20 nm. (F and G) Different views of the sixfold-symmetrized subtomogram average of the pore complexes in NSP3/4-induced DMVs. (H and I) Tomographic slices of HeLa cells transfected with different truncations of NSP3/4, showing that the NTD or CTD truncations of NSP3, but not the CTD truncation of NSP4 impairs the DMV formation. Scale bar, 100 nm. (J) Zoom in area in I at higher magnification. Green arrowhead, DMV. Scale bar, 100 nm.