FIG. 1.

Concentration-dependent inhibition of S protein cleavage by the furin inhibitor dec-RVKR-cmk. Parallel cultures of LR-7 cells (15) were inoculated with MHV-A59 at an MOI of 10 in 35-mm culture dishes. After 1 h, the inoculum was replaced by culture medium, which in some cases additionally contained the furin inhibitor peptidyl chloromethylketone (dec-RVKR-cmk; Calbiochem) at the indicated concentrations. In the latter cultures, the inhibitor was maintained at the same concentrations in the culture medium during all subsequent steps. At 4.5 h postinfection, the cells were starved for 30 min in cysteine- and methionine-free minimal essential medium containing 10 mM HEPES (pH 7.2). The medium was replaced with the same medium containing 100 μCi of ³⁵S in vitro labeling mix (Amersham). After a 15-min pulse, the cells were chased for 2 h in fresh medium supplemented with 2 mM both cold methione and cysteine. The culture supernatants were precleared at 1,500 rpm at 4°C for 5 min, while the cells were washed once with ice-cold phosphate-buffered saline and solubilized in lysis buffer as described before (15). The culture supernatants and the cell lysates were subsequently prepared for and subjected to immunoprecipitation by using the MHV S-specific monoclonal antibody WA3.10, after which the immune complexes were analyzed by electrophoresis in an SDS-PAGE (10% polyacrylamide) gel followed by fluorography as described before (15). The position of the immature S protein precursor Gp150, its mature form, Gp180, and the S1 and S2 cleavage products thereof (Gp90) are indicated on the left side of the gel.