FIG. 4.

Cleavage of HA by chicken furin. LoVo cells (35) were infected with VV:gfur and VV:HAwt (22), each at an MOI of 10. Virus inocula were replaced by DMEM without FCS 1 h after infection. At 4 h after infection, LoVo cells (diameter of the culture dishes, 35 mm) were starved for methionine for 1 h and then labeled with 100 μCi of [³⁵S]methionine (1,000 Ci/mmol; Amersham, Braunschweig, Germany) for 3 h in 0.5 ml of methionine-free MEM. The medium was replaced by MEM containing nonradioactive methionine, and incubation was continued for an additional hour. Cells were lysed in radioimmunoprecipitation buffer. After immunoprecipitation with anti-FPV or anti-hfur rabbit serum (final dilution, 1:500) and protein A-Sepharose CL-4B (Sigma, Deisenhofen, Germany), proteins were analyzed by SDS–10% PAGE under reducing conditions. Sizes are shown in kilodaltons.