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

We first examined the ultrastructure of the budding compartment in L cells in conventional Epon sections. Our data fully support and extend the observations made previously by Tooze et al. (1988) . As shown in Fig. 2 , images were found showing direct membrane continuities between the viral budding compartment and the rough ER. The budding region was also continuous with a cisternal element on one side of the Golgi stack. The architecture of the ER-Golgi boundary was much easier to appreciate in cytosol-depleted L cells that had been permeabilized with the pore forming toxin streptolysin O. Under these conditions the contrast of the membranes was visibly increased. The images in Figs. 3 and 4 indicate that the structure where MHV buds is directly continuous with the rough ER, the nuclear envelope and with cisternal elements on one side of the Golgi stack. In agreement with Tooze et al. (1984) , we could also find small amounts of virus budding into the rough ER, even at relatively early times, 6 h of infection ( Fig. 3 E) . The continuities between these structures became even more obvious when we carried out an analysis of serial sections (Fig. 5 ). In all these preparations the membranes of the rough ER (Figs. 3, A and D and 5) as well as the structures containing the budding virions were in direct continuity with approximately 30-nm wide membrane tubules. These tubules also appeared to contact the nuclear envelope; in oblique sections some images suggested that these tubules were attached to the nuclear pores ( Fig. 4 B) . Similar tubules were seen in our recent study on vaccinia virus infected cells (Sodeik et al., 1993) . The membranes of the budding compartment were also continuous with distinct electron-dense, tubular-cisternal structures (Fig. 3 A) . Such structures are occasionally seen in uninfected cells (not shown) but are far more prominent in MHV infected cells. They are very similar to the structures described by Hobman et al. (1992) in cells expressing the E1 protein of rubella virus, a protein that also accumulates in post-ER, pre-Golgi structures. As shown below, these structures are significantly enriched in rab2.