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

L cells grown on coverslips, coated with 0.1% gelatin in PBS, were infected with MHV-A59 at a multiplicity of infection (MOI) of 10 and fixed at 6 h after infection with 3 % paraformaldehyde. Cells were labeled as described by Den Boon et al. (1991) using either a monoclonal antibody directed against the NH2 terminus of the M protein (J 1.3; a kind gift of Dr. J. Fleming; Fleming et al., 1989) or a rabbit peptide serum raised against the COOH terminus of the M protein (Krijnse-Locker et al., 1992b) . The monoclonal antibodies were concentrated from the supernatant of hybridoma cultures by a centricon 30 microconcentrator (Amicon, W.R. Grace & Co.-Corm, Beverly, MA) . For the lectin labeling permeabilized cells were blocked for 10 rain in PBS with 0.2% fish skin gelatin (PBS/FSG) and then labeled for I0 min with Helix pomatia agglutinin (HPA, Boehringer GmbH, Mannheim, Germany) at a concentration of 25/~g/ml in PBS/FSG. After extensive washing, cells were incubated with a rabbit anti-lectin serum (1/100, Serotec, Oxford, UK) for 20 rain. The second antibodies (goat anti-mouse and goat anti-rabbit coupled to FITC or Rhodamine) were from Protos (Protos Immunoresearch, San Francisco, CA). Coverslips were mounted and viewed as before (Den Boon et al., 1991) .