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 polymer was visible. (5, 6) 17Cl-1 cells expressing APEX2-tagged MHV-A59 NSP12, mock-infected, fixed, and stained in the same way as in 1 and 2. (5) Low-magnification transmission electron micrograph. (6) Boxed area in 5 at higher magnification. Neither DMV nor APEX2-tagged RdRp recruited to the periphery of DMV was visible. (7–8) 17Cl-1 cells expressing APEX2-tagged MHV-A59 NSP12, infected and fixed in the same way as in A and B, but without DAB/H2O2 staining. (7) Low-magnification transmission electron micrograph. (8) Boxed area in G at higher magnification. No DAB polymer is visible. Scale bars: 2 μm for 1, 3, 5, and 7; 200 nm for 2, 4, 6, and 8. (E) Immuno-EM showing that NSP12 is predominantly localized on the cytosolic membranes of DMVs. Upper panel: Negative control for immunogold labeling, where the NSP12 primary antibody was not applied. Lower panel: In immunogold-labeled samples, gold particles (black dots) indicating the position of NSP12 molecules.

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 polymer was visible. (5, 6) 17Cl-1 cells expressing APEX2-tagged MHV-A59 NSP12, mock-infected, fixed, and stained in the same way as in 1 and 2. (5) Low-magnification transmission electron micrograph. (6) Boxed area in 5 at higher magnification. Neither DMV nor APEX2-tagged RdRp recruited to the periphery of DMV was visible. (7–8) 17Cl-1 cells expressing APEX2-tagged MHV-A59 NSP12, infected and fixed in the same way as in A and B, but without DAB/H2O2 staining. (7) Low-magnification transmission electron micrograph. (8) Boxed area in G at higher magnification. No DAB polymer is visible. Scale bars: 2 μm for 1, 3, 5, and 7; 200 nm for 2, 4, 6, and 8. (E) Immuno-EM showing that NSP12 is predominantly localized on the cytosolic membranes of DMVs. Upper panel: Negative control for immunogold labeling, where the NSP12 primary antibody was not applied. Lower panel: In immunogold-labeled samples, gold particles (black dots) indicating the position of NSP12 molecules.

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