Fig. 1.

Two-dimensional ultrastructure of RUBV factories: high-pressure freezing, freeze-substitution and sectioning. (A) Conventional EM shows two CPVs (frontal view for the one on the top and side view for the CPV on the bottom) in the perinuclear area of a replicon-transfected cell. Both CPVs exhibit internal vesicles and straight elements (stars) and locally recruited RER and mitochondria. (B to D) Suspensions of BHK-21 cells stably transfected with a RUBV replicon were cryofixed by HPF without cryoprotectants. Cell in (B) was freeze-substituted in 1% OsO₄, 0.25% glutaraldehyde in acetone and embedded in EML-812 while cells in (C) and (D) were freeze-substituted in 0.5% UA in acetone and embedded in Lowicryl HM23. (B) Detail of a CPV surrounded by RER cisternae and one mitochondrion. The CPV exhibits a very heterogeneous content with numerous vesicles of variable size and density, vacuoles and a rigid membrane (star). (C) Low magnification view showing large areas of the cell and several CPVs (arrows) over the holes of the carbon film. (D) Image at 0º of a CPV surrounded by RER and mitochondria ready for ET. Ultra-thin (~ 50 nm) post-stained sections are shown in (A) and (B), while (C) and (D) show semi-thick (~ 200 nm) sections collected on Quantifoil® holey carbon grids and visualized without post-staining. RER, rough endoplasmic reticulum; mi, mitochondria; N, nucleus. Bars, 200 nm in A, B, and D; 1 μm in C.