Figure 2.
Ectopic expression of SARS-CoV-2 nsp3 and nsp4 is sufficient to induce DMVs. (A) In HeLa cells expressing GFP-nsp3 or nsp4-GFP alone, each protein shows a diffuse ER pattern and colocalizes with RFP-Sec61β. When cells are cotransfected with GFP-nsp3 and nsp4-mCherry, a large number of foci are observed, which are positive for both nsp3 and nsp4. Bars: 5 μm; inserts, 2 μm. (B) 3D-SMLM imaging of nsp4-SNAP co-transfected with GFP-nsp3 in a cell. The left panel shows the image of two nsp4 puncta in a large area with the z position color-coded. The middle panels show the x-y cross-sections of the boxed regions 1 and 2. The corresponding vertical cross-sections along the dotted lines are shown in the right panels. Bars: left panel, 5 μm; middle and right panels, 100 nm. (C–E) TEM images demonstrate that numerous DMVs are detected in HeLa cells coexpressing nsp3/4 (C). The sizes of DMVs formed during SARS-CoV-2 infection are larger than those induced by nsp3/4 expression (D). N, nucleus. Quantification of the size of DMVs is shown as mean ± SD (nsp3/4, n = 125; SARS-CoV-2, n = 71; E). ****, P < 0.0001. Bars: 500 nm. (F) CLEM images of a control cell expressing GFP-nsp3 and nsp4-mCherry. The left panel shows two nsp4 puncta in a large area. The middle and right panels show the boxed regions 1 and 2. Bars: left panel, 2 μm; middle and right panels, 500 nm. (G and H) In FPP assays, time-lapse images show that GFP-nsp3 puncta immediately disappear, while most of nsp4-mCherry puncta persist upon pK (proteinase K) addition in digitonin-permeabilized HeLa cells coexpressing nsp3/4 (H). Quantitative data (n = 11) are shown as mean ± SEM (G). Bars: 5 μm. (I) In most cells cotransfected with plasmids expressing GFP-nsp3-SBP and Strep-nsp4-mCherry, the two proteins show a diffuse ER pattern. After the addition of biotin for 24 h, GFP-nsp3-SBP and Strep-nsp4-mCherry form punctate structures. Bars, 5 μm. (J) The top panel shows a schematic of the topology of nsp3, and the structures of the nsp3 truncations. The bottom panel demonstrates that Flag–nsp4 is immunoprecipitated by WT GFP-nsp3, GFP-nsp3(1–1546), or GFP-nsp3(1–1582), but not GFP-nsp3(1-1452) in a GFP-Trap assay. (K) The left panel shows a schematic of the topology of nsp4 and the structures of the nsp4 truncations. The right panel demonstrates that Flag-nsp3 is immunoprecipitated by WT nsp4-GFP or nsp4(Δ338-364)-GFP, but not nsp4(Δ35-280)-GFP in a GFP-Trap assay. (L) In a GFP-Trap assay, mCherry-nsp3 is immunoprecipitated by GFP-nsp3. (M) In a GFP-Trap assay, nsp4–mCherry is immunoprecipitated by nsp4-GFP. (N) In a GFP-Trap assay, more Flag-nsp3 is immunoprecipitated by WT GFP-nsp3, compared with GFP-nsp3(Δ1436-1499). (O) In a GFP-Trap assay, more Flag-nsp4 is immunoprecipitated by WT nsp4-GFP, compared with nsp4(Δ35–280)-GFP. Source data are available for this figure: SourceData F2.

Ectopic expression of SARS-CoV-2 nsp3 and nsp4 is sufficient to induce DMVs. (A) In HeLa cells expressing GFP-nsp3 or nsp4-GFP alone, each protein shows a diffuse ER pattern and colocalizes with RFP-Sec61β. When cells are cotransfected with GFP-nsp3 and nsp4-mCherry, a large number of foci are observed, which are positive for both nsp3 and nsp4. Bars: 5 μm; inserts, 2 μm. (B) 3D-SMLM imaging of nsp4-SNAP co-transfected with GFP-nsp3 in a cell. The left panel shows the image of two nsp4 puncta in a large area with the z position color-coded. The middle panels show the x-y cross-sections of the boxed regions 1 and 2. The corresponding vertical cross-sections along the dotted lines are shown in the right panels. Bars: left panel, 5 μm; middle and right panels, 100 nm. (C–E) TEM images demonstrate that numerous DMVs are detected in HeLa cells coexpressing nsp3/4 (C). The sizes of DMVs formed during SARS-CoV-2 infection are larger than those induced by nsp3/4 expression (D). N, nucleus. Quantification of the size of DMVs is shown as mean ± SD (nsp3/4, n = 125; SARS-CoV-2, n = 71; E). ****, P < 0.0001. Bars: 500 nm. (F) CLEM images of a control cell expressing GFP-nsp3 and nsp4-mCherry. The left panel shows two nsp4 puncta in a large area. The middle and right panels show the boxed regions 1 and 2. Bars: left panel, 2 μm; middle and right panels, 500 nm. (G and H) In FPP assays, time-lapse images show that GFP-nsp3 puncta immediately disappear, while most of nsp4-mCherry puncta persist upon pK (proteinase K) addition in digitonin-permeabilized HeLa cells coexpressing nsp3/4 (H). Quantitative data (n = 11) are shown as mean ± SEM (G). Bars: 5 μm. (I) In most cells cotransfected with plasmids expressing GFP-nsp3-SBP and Strep-nsp4-mCherry, the two proteins show a diffuse ER pattern. After the addition of biotin for 24 h, GFP-nsp3-SBP and Strep-nsp4-mCherry form punctate structures. Bars, 5 μm. (J) The top panel shows a schematic of the topology of nsp3, and the structures of the nsp3 truncations. The bottom panel demonstrates that Flag–nsp4 is immunoprecipitated by WT GFP-nsp3, GFP-nsp3(1–1546), or GFP-nsp3(1–1582), but not GFP-nsp3(1-1452) in a GFP-Trap assay. (K) The left panel shows a schematic of the topology of nsp4 and the structures of the nsp4 truncations. The right panel demonstrates that Flag-nsp3 is immunoprecipitated by WT nsp4-GFP or nsp4(Δ338-364)-GFP, but not nsp4(Δ35-280)-GFP in a GFP-Trap assay. (L) In a GFP-Trap assay, mCherry-nsp3 is immunoprecipitated by GFP-nsp3. (M) In a GFP-Trap assay, nsp4–mCherry is immunoprecipitated by nsp4-GFP. (N) In a GFP-Trap assay, more Flag-nsp3 is immunoprecipitated by WT GFP-nsp3, compared with GFP-nsp3(Δ1436-1499). (O) In a GFP-Trap assay, more Flag-nsp4 is immunoprecipitated by WT nsp4-GFP, compared with nsp4(Δ35–280)-GFP. Source data are available for this figure: SourceData F2.

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