SARS-CoV-2 NSP3/4 control formation of replication organelle and recruitment of RNA polymerase NSP12

Figures

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  Figure S1.. Workflow of cryo-CLIEM of NSP3/NSP4-induced DMVs, confocal analysis of the co-localization of different truncations of NSP3/NSP4 in HeLa cells, and TEM images of DMV in HEK-293T cells, related to Fig. 1 . (A) Co-localization signals of fluorescent GFP-nsp3 and nsp4-mCherry were observed in the vitrified HeLa cells, serving as a marker for the identification of regions containing DMVs. Scale bar, 20 μm. (A1) FIB image of the corresponding area in panel A. The blue arrow indicates the target cell, corresponding to the white box in A. (B) Cryo-FM image of the pre-lamella (∼400 nm) via FIB milling direction. Scale bar, 10 μm. (C) Cryo-FM image of the final lamella (∼150 nm). Scale bars, 10 μm. (D) The final lamella viewed from FIB milling direction. (E) The SEM image of the final lamella. (F and G) The cryo-EM image of the lamella, containing the DMVs of interest (white-dotted frame). LD, lipid droplet; ER, endoplasmic reticulum; NPC, nuclear pore complex; Mito, mitochondrion. Scale bar, 2 μm (F), 1 μm (G). (H) Confocal microscopy analysis of the co-localization of different truncations of NSP3/NSP4 in HeLa cells. Scale bar, 10 μm. (I) Conventional TEM images of DMV in HEK-293T cells…

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  Figure 1.. 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…

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  Figure S2.. Other NSPs are not recruited to the NSP3/4-induced DMVs, related to Fig. 2 . (A) Confocal microscopy analysis of the localization of NSP1-mCherry, NSP2-mCherry, GFP-NSP3, NSP4-GFP, NSP5-mCherry, mCherry-NSP6, NSP7-mCherry, NSP8-mCherry, NSP9-mCherry, NSP10-mCherry, NSP11-mCherry, NSP12-mCherry, NSP13-mCherry, NSP14-mCherry, NSP15-mCherry, and NSP16-mCherry in HeLa cells. Scale bar, 10 μm. (B) Confocal microscopy analysis of the co-localization of Flag-NSP3/NSP4-GFP puncta with NSP1-mCherry, NSP2-mCherry, NSP5-mCherry, NSP7-mCherry, NSP8-mCherry, NSP9-mCherry, NSP10-mCherry, NSP11-mCherry, NSP13-mCherry, NSP14-mCherry, NSP15-mCherry, or NSP16-mCherry in HeLa cells. Scale bar, 10 μm.

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  Figure 2.. NSP12 is recruited to the NSP3/4-induced DMVs through NSP3–NSP12 interaction. (A) Confocal microscopy analysis showing the colocalization of NSP12-mCherry with Flag-NSP3/NSP4-GFP–induced punctate structures in HeLa cells. Trace outline is used for line-scan analysis of the relative fluorescence intensity of NSP12-mCherry, Flag-NSP3, and NSP4-GFP. Scale bar, 10 μm. (B and C) Confocal microscopy analysis showing the colocalization of NSP12-mCherry with GFP-NSP3 (B) or NSP4-GFP (C) in HeLa cells. Trace outline is used for line-scan analysis of the relative fluorescence intensity of NSP12-mCherry with GFP-NSP3 or NSP4-GFP. Scale bar, 10 μm. (D) Co-IP analysis of the interaction of Flag-NSP3 or NSP4-Flag with NSP12-mCherry. Proteins were transiently expressed in HEK-293T cells and immunoprecipitates pulled down by Flag antibody were analyzed by IB with the indicated antibodies. Flag-vector was used as a negative control. Input represents 5% of the total cell extract used for IP. Molecular weights are in kDa. n = 3 independent experiments. (E) Co-IP analysis of the interaction of NSP12-mCherry with Flag-NSP3 or NSP4-Flag. Proteins were transiently expressed in HEK-293T cells and…

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  Figure S3.. Other NSPs are recruited by the NSP3/4-induced DMVs through NSP12, related to Fig. 2 . Confocal microscopy analysis of the co-localization of GFP-NSP3/NSP4-iRFP, NSP12-tagBFP puncta with NSP7-mCherry, NSP8-mCherry, NSP9-mCherry, NSP10-mCherry, NSP13-mCherry, NSP14-mCherry, NSP15-mCherry, or NSP16-mCherry in HeLa cells. Scale bar, 10 μm.

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  Figure 3.. The NTD of NSP3 directly interacts with NSP12. (A) Schematic representation of the structures of the NSP3 truncations. (B) Confocal microscopy analysis showing the colocalization of NSP12-mCherry with truncated Flag-NSP3/NSP4-tagBFP–induced punctate structures in HeLa cells. Scale bar, 10 μm. (C) In vitro pull-down analysis of the interaction between NTD of NSP3 and NSP12. Purified NTD of NSP3 and NSP12 proteins were subjected to pull-down assay and pull-down samples by Flag antibody were analyzed by IB with the indicated antibodies. Flag-GST protein was used as a negative control. Input represents 5% of the total proteins used for pull-down. Molecular weights are in kDa. n = 3 independent experiments. (D) Schematic representation of the structures of the NSP12 truncations. (E) Confocal microscopy analysis showing the colocalization of truncated NSP12-mCherry with GFP-NSP3/NSP4-tagBFP–induced punctate structures in HeLa cells. Scale bar, 10 μm. Source data are available for this figure: SourceData F3.

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  Figure 4.. RTCs localize on the cytosolic face of DMVs. (A and B) In FPP assays, time-lapse images showing the disappearance of GFP-NSP3 and NSP12-mCherry puncta, while most of the NSP4-tagBFP puncta persist upon pK addition in digitonin-permeabilized HeLa cells co-expressing NSP3, NSP4, and NSP12. Scale bar, 10 μm (A). Quantitative data are shown as mean ± SEM, n = 4 independent experiments (B). (C) Confocal microscopy analysis showing the colocalization of MHV-A59 NSP12 with dsRNA after MHV-A59 infection. Scale bar, 10 μm. (D) APEX2-based TEM showing RdRp localized on the cytosolic face of DMVs. (1, 2) 17Cl-1 cells expressing APEX2-tagged MHV-A59 NSP12 were infected with MHV-A59, fixed, and stained with DAB/H2O2, the DAB polymer generated by APEX2 pinpointing the positions of APEX2-tagged RdRp. (1) Low-magnification transmission electron micrograph. (2) Boxed area in A at higher magnification. Asterisks: DMVs. Arrowheads: DAB polymer (positions of RdRp). mt: mitochondrion. (3 and 4) WT 17Cl-1 cells devoid of APEX2-tagged MHV-A59 NSP12 were treated in the same way as in A and B. (3) Low-magnification transmission electron micrograph. (4) Boxed area in C at higher magnification. No DAB…

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  Figure 5.. DsRNA rather than other NSPs is critical for determining the DMV size. (A) IB analysis of the NSP12, NSP5, and NSP15 expression in HEK-293T cells stably expressing SARS-CoV-2 NSP3-16, where NSP5-11 and NSP13-16 were polyproteins. (B) Quantification of the size of DMVs is shown as mean ± SEM. n (number of analyzed DMVs) = 111, 339, and 401 for NSP3/4, NSP3-16, NSP3-16+dsRNA, respectively. Student’s two-tailed t test, ***: P < 0.001. ****: P < 0.0001. ns: not significant. (C) TEM analysis of the DMVs induced by SARS-CoV-2 at 6 or 24 hpi with or without RDV treatment. Each boxed area in a low-magnification TEM micrograph (left) is depicted in detail in a high-magnification one (right). Scale bars: 2 μm for low-magnification TEM micrographs, 200 nm for high-magnification TEM micrographs. (D) Quantification of the size of DMVs is shown as mean ± SEM (left). n (number of analyzed DMVs) = 129, 128, 255, and 47 for control 6 hpi, RDV 6 hpi, control 24 hpi, and RDV 24 hpi, respectively. Quantification of DMV density is shown as mean ± SEM (right). n (number of analyzed DMVs) = 10, 9, 10, and 10 for control 6 hpi, RDV 6 hpi, control 24 hpi, and RDV 24 hpi, respectively. Student’s…

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  Figure S4.. TEM analysis of the DMVs in HEK-293T cells, confocal microscopy analysis of dsRNA signal in the SARS-CoV-2–infected Vero-E6 cells and MHV-A59–infected 17Cl-1 cells, and the expression level of NSP3/4 only affects the density but not the size of DMVs, related to Fig. 5 . (A) The schematic structure of the SARS-CoV-2 genome template. (B) TEM analysis of the DMVs induced by SARS-CoV-2 NSP3/4 (left), NSP3-16 (middle), and NSP3-16+dsRNA (right) in HEK-293T cells. Each boxed area in a low-magnification TEM micrograph (upper) is depicted in detail in a high-magnification one (bottom). Scale bar, 2 μm for low-magnification TEM micrographs, 200 nm for high-magnification TEM micrographs. (C and D) Confocal microscopy analysis of dsRNA signal in the SARS-CoV-2–infected Vero-E6 cells at 6 or 24 hpi (C) and MHV-A59–infected 17Cl-1 cells at 4 or 8 hpi (D) with or without RDV treatment. Scale bar, 20 μm. (E) IB analysis of the NSP3 and NSP4 expression in Vero-E6 cells in SARS-CoV-2 infected at 6 or 24 hpi with or without RDV treatment. n = 3 independent experiments. (F) IB analysis of the NSP3 and NSP4 expression in low-dose (2 μg) and high-dose (4 μg) NSP3/4 transfection. n = 3 independent…