Figure S1.
DMVs are induced by coexpression of SARS-CoV-2 nsp3 and nsp4, related to Figs. 1 and 2. (A) Genomic DNA sequences of VMP1 in control and VMP1 KO HeLa cells. Deletions are indicated by dashes. (B) Immunoblotting with VMP1 antibody verifies the KO efficiency in VMP1 KO HeLa cells. (C) Genomic DNA sequences of TMEM41B in control and TMEM41B KO HeLa cells. The insertion is highlighted in red. (D) Immunoblotting with TMEM41B antibody verifies the KO efficiency in TMEM41B KO HeLa cells. (E–J) Immunostaining shows that Flag-nsp3/nsp4-mCherry double positive foci do not colocalize with GFP-Rab5 (E), GFP-Rab7 (F), EHD1-GFP (G), GFP-Sec24c (H), ERGIC53-GFP (I), or EDEM1-GFP (J). Bars: 5 μm; inserts, 2 μm. (K) TEM analysis demonstrates that clusters of DMVs are formed in HeLa cells coexpressing nsp3/4. The arrow indicates a DMV associated with the ER. The red boxed area in the left panel is enlarged in the right panel. N, nucleus. Bars: left panel, 500 nm; right panel, 200 nm. (L) Immuno-electron microscopy micrographs of control cells coexpressing mCherry-nsp3 and nsp4-GFP. Anti-GFP antibody (10 nm gold, arrows) was used to detect nsp4-GFP. The red boxed area in the left panel is enlarged in the right panel. Bars: left panel, 100 nm; right panel, 50 nm. (M and N) In FPP assays, time-lapse images show that mCherry-nsp3 puncta immediately disappear while most of nsp4-GFP puncta persist upon pK addition in digitonin-permeabilized HeLa cells coexpressing nsp3/4 (M). Quantitative data are shown as mean ± SEM (n = 16; N). Bars: 5 μm. (O and P) In FPP assays, time-lapse images show that mCherry-nsp3 puncta immediately disappear while most of nsp4-GFP puncta persist upon pK addition in digitonin-permeabilized COS7 cells coexpressing nsp3/4 (O). Quantitative data are shown as mean ± SEM (n = 17; P). Bars: 5 μm. (Q and R) HeLa cells coexpressing GFP-nsp3-SBP and nsp4–mCherry at control (Q) and 24 h biotin treatment (R) conditions. Bars, 5 μm. Source data are available for this figure: SourceData FS1.

DMVs are induced by coexpression of SARS-CoV-2 nsp3 and nsp4, related to Figs. 1 and 2. (A) Genomic DNA sequences of VMP1 in control and VMP1 KO HeLa cells. Deletions are indicated by dashes. (B) Immunoblotting with VMP1 antibody verifies the KO efficiency in VMP1 KO HeLa cells. (C) Genomic DNA sequences of TMEM41B in control and TMEM41B KO HeLa cells. The insertion is highlighted in red. (D) Immunoblotting with TMEM41B antibody verifies the KO efficiency in TMEM41B KO HeLa cells. (E–J) Immunostaining shows that Flag-nsp3/nsp4-mCherry double positive foci do not colocalize with GFP-Rab5 (E), GFP-Rab7 (F), EHD1-GFP (G), GFP-Sec24c (H), ERGIC53-GFP (I), or EDEM1-GFP (J). Bars: 5 μm; inserts, 2 μm. (K) TEM analysis demonstrates that clusters of DMVs are formed in HeLa cells coexpressing nsp3/4. The arrow indicates a DMV associated with the ER. The red boxed area in the left panel is enlarged in the right panel. N, nucleus. Bars: left panel, 500 nm; right panel, 200 nm. (L) Immuno-electron microscopy micrographs of control cells coexpressing mCherry-nsp3 and nsp4-GFP. Anti-GFP antibody (10 nm gold, arrows) was used to detect nsp4-GFP. The red boxed area in the left panel is enlarged in the right panel. Bars: left panel, 100 nm; right panel, 50 nm. (M and N) In FPP assays, time-lapse images show that mCherry-nsp3 puncta immediately disappear while most of nsp4-GFP puncta persist upon pK addition in digitonin-permeabilized HeLa cells coexpressing nsp3/4 (M). Quantitative data are shown as mean ± SEM (n = 16; N). Bars: 5 μm. (O and P) In FPP assays, time-lapse images show that mCherry-nsp3 puncta immediately disappear while most of nsp4-GFP puncta persist upon pK addition in digitonin-permeabilized COS7 cells coexpressing nsp3/4 (O). Quantitative data are shown as mean ± SEM (n = 17; P). Bars: 5 μm. (Q and R) HeLa cells coexpressing GFP-nsp3-SBP and nsp4–mCherry at control (Q) and 24 h biotin treatment (R) conditions. Bars, 5 μm. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal