FIG. 6.

ET confirms the general integrity of the SARS-CoV-induced RVN upon BFA treatment. ET was applied for the three-dimensional analysis of the RVN in BFA-treated cells cryofixed at 7 h p.i. The top three panels illustrate how a three-dimensional surface-rendered model was derived by applying ET to a semithick section of a SARS-CoV-infected Vero-E6 cell, which had been treated with BFA from 1 to 7 h p.i. (A) A 0°-tilt transmission EM image of a 200-nm-thick resin-embedded section showing part of a SARS-CoV RVN after BFA treatment. Scale bar, 250 nm. (B) Using the IMOD software package, tomograms were computed from dual-axis tilt series of the 200-nm-thick section shown in panel A. The tomographic slice shown was taken from the central plane of the section and represents a thickness of 1.2 nm. The dashed squares mark the insets shown below in panel D. (C) The improved image from panel B after anisotropic diffusion filtering. The optimized signal-to-noise ratio facilitates masking, thresholding, and surface rendering. (D) Final three-dimensional surface-rendered model showing the RVN against a background of mitochondria (M, red), dilated ER and tubulo-vesicular clusters (both resulting from BFA treatment; depicted in light blue and pink, respectively). Furthermore, all substructures normally encountered in the SARS-CoV-induced RVN were observed after BFA treatment, including DMVs and VPs (outer membranes, dark blue; inner membranes, dark green) and CM (depicted in lavender). The insets (I, II, and III) show tilted tomographic slices taken from panel B and highlight RVN membrane connections between DMVs, between ER and DMVs, and between CM and DMVs (black arrowheads). Ribosomes attached to the cytosolic face of RVN membranes or ER are indicated with red arrowheads. Scale bar, 100 nm. For abbreviation definitions, see Fig. 4. TVC, tubulovesicular cluster (15).