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

MHV-infected L cells were permeabilized with streptolysin O (SLO) 6 h after infection. Cells were rinsed twice with ice-cold PBS and once with SLO-buffvr (25 mM Hepes-KOH, pH 7.4, 115 mM KOAc, 2.5 mM MgC12) containing 1 mM dithiothreitol (DTT; Boehringer GmbH). SLO (Wellcome diagnostics, Dartford, UK) at 1 U/ml was bound for 10 rain on ice, ceils were rinsed twice with SLO-buffer/DTT and incubated in this buffer for an additional 30 rain at 37°C before fixation for 30 rain at room temperature with 1% glutaraldehyde in 200 mM Hepes-KOH, pH 7.4. Cells were scraped from the dish in fixative with a rubber policeman consisting of a piece of 0.5-ram-thick Teflon. Cells were cenWifuged for 3 rain at 2,000 g and fixative replaced by PBS. Cells were processed for either Epon embedding or cryo-sectioning. For cells prepared in the absence of SLO, the cells were fixed on the dish as above for 30 rain and embedded in Epon (Griffiths ct al., 1983) . Some preparations (see Figs. 2 and 4) were treated with 1% tannic acid in 0.2 M cacodylate for 30 rain, after the osmium treatment. For the glucose 6 phosphatase reaction infected cells, both untreated and after SLO permeabilization, were fixed for 10 rain in 0.1% glutaraldehyde/ 4 % paraformaldehyde in 0.2 M cacodylate. After rinses in the same buffer the cells were incubated for glucose 6 phosphatase as described (Griffiths et al., 1983) . After the reaction for 3 h at room temperature, the cells were fixed again with 1% glutaraldehyde before embedding. For cryosections of non-SLO-permeabilized cells the monolayer was removed with proteinas¢ K and fixed for 30 rain with 0.1% glutaraldebyd¢/4% paraformaldehyde, then with 8 % paraformaldehyde overnight and immunolabeled as described (Griffiths et al., 1983; Griffiths, 1993) . For some of the labeling experiments with E-cop antibodies the 37°C permcabilization step was done in SLO-buffer containing 50/~M GTPTS (Boehringer GmbH) for 30 rain at 37°C. HPA labeling was done as described for the immunofluoresecnce.