Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step

For the preparation of 35S-labeled MHV infected L cells were incubated from 5 to 8 h after infection with 250 ~Ci/ml of Expre35S3~S in methionine-free MEM, to which at 6.5 h after infection 2.5 t,g/ml unlabeled methionine was added. 50 t~l of culture supernatant diluted with 500 ul detergent solution (50 mM Tris-Cl, pH 8.0, 62.5 mM EDTA, 1% Nonidet P-40, 0.4% sodium deoxycholate) was incubated at 4"C for 4 h with 100 SLO-permeabilized L cells infected with MHV for 6 h. In A, the small arrows indicate virions that are in the process of budding into the smooth membrane structures close to the Golgi complex (G). The dashed lines indicate a direct membrane continuity between the rough ER and the smooth membrane budding compartment. Tshows a smooth membrane tubule that is continuous with the rough ER; one of these tubules is possibly continuous with the nuclear envelope (large arrow; N, nucleus). The asterisks indicate the electron dense "clusters" of tubular cisternal elements that are seen in uninfected cells but become pronounced after infection (see Fig. 9 C). These structures are continuous with the smooth membrane budding compartment (double arrowheads-small) as well as the rough ER (double arrowheadslarge). Note how one of the cisterna of the Golgi complex (G) folds back on itself adjacent to the nuclear envelope as well as the thin filaments (F) lining up next to the nuclear envelope. B shows a continuity (arrowhead) between the tubular projection where the virus buds with the rough ER (small arrows, ribosomes). In C, budding virions (arrows) are shown in cisternal elements on one side of the Golgi stack (G). Putative clathrincoated vesicles (C) are apparent on the opposite (trans) side of the stack. D shows a direct membrane continuity between a smooth tubule (T) close to the budding virion (arrow) and the Golgi stack (G). In E virions are shown in the rough ER. The arrowheads indicate budding profiles, while the arrows show virions with no apparent membrane continuities with the ER membrane. Bars, 100 nm.