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-infocted L cells grown in 16-ram dishes were starved at 5.5 h after infection in MEM without methionine (GIBCO BRL, Life Technologies Ltd., Paisley, Scotland) and pulse labeled for 2 min at 6 h after infection with 50 ~Ci/well of [35S]mcthionine of (Expre3sS3SS NEN, DuFont GmbH, Dreiech, Germany). Cells were put on ice, rinsed once with PBS and incubated for l0 rain on ice in RPMI without glucose (How Laboratories, McLean, VA) containing 2% dialyzed fetal calf serum, I00 ~g/ml cycloheximide (Signm Chemical Co., St. Louis, MO), 2 mM methionine and, when indicated 10 mM sodium azide (Merck, Darmstadt, Germany) and I0 mM 2-deoxy-D-ghicose (Sigma Chemical Co.). Cells were then chased at 370C. Cells were lysed and the M protein immunoprecipitated as de-scribed earlier (Krijnse-Locker et al., 1992b) using the MHV antiserum (Rottier et al., 1981b) and analyzed in a 15% SDS-PAGE. When the effect of low temperature was tested, pulse-labeled calls were chased in DME/I0% FCS, containing cyclobeximide, methionine and 10 mM Hepes-KOH pH 7.4, in a water bath at the indicated temperature.