Fig. 2.

Three-dimensional views of RUBV factories as visualized by freeze-fracture and electron tomography. (A) Metal replica of BHK-21 cells stably transfected with the RUBrep/GFP/neo replicon and processed by freeze-fracture followed by etching. Mitochondria and RER cisternae surround the CPVs. Although these organelles are very close to the CPVs no clear contacts are detected (arrows in mainfield and inset). (B to G) A computational tomographic slice (~ 1.2 nm) corresponding to the tomogram of a semi-thick (~ 250 nm) section obtained from suspensions of trypsinized cells processed by HPF, FS and embedding in Lowicryl HM23 and visualized without post-staining. (B) CPV with a characteristic rigid membrane (star) surrounded by RER. (C to G) Expanded areas from the CPV shown in (B). (C and D) RER–CPV contacts seen as closely apposed membranes (dashed rectangles). (E and F) Protein bridges between RER and CPV are also observed (arrows). (G) Close to these complexes several ribosomes facing the CPV are found (dashed circles). RER, rough endoplasmic reticulum; mi, mitochondria. Bars, 200 nm in A and B; 50 nm in C to G.