FIG. 6.
Proteolytic activation at the second IBV furin site is mediated by furin and other serine protease(s). (a) Giant syncytium formation by rIBV was mediated by proteolytic activation at the second furin site. Syncytium formation was observed as described in the legend to Fig. 2a. For observation of CPE, cells were incubated with DMEM instead of overlay medium. (b) S1/S2 cleavage occurred efficiently in M2.1- and M2.5-infected cells, but not in M1+2.1-infected cells. HuH-7 cells were infected with rIBV for 18 h. Cell extracts (20 μg protein) were analyzed by immunoblotting them with the S2 Ab. (c) M2.1 and M1+2.1, which lack induction of syncytia, demonstrated prolonged replication with high productivity of virions. The growth kinetics was investigated as described in the legend to Fig. 2b. The error bars indicate standard deviations of the means. (d) Infection of M2.1, M1+2.1, and M2.5 was not affected by pretreatment with furin inhibitor. The effect of pretreatment with furin inhibitor was investigated as described in the legend to Fig. 4d. (e) Infection of the WT, M2.1, and M2.5 was inhibited by pretreatment with AEBSF, but not leupeptin. The effects of protease inhibitors were investigated as described in the legend to Fig. 4d, except that the cells were pretreated with leupeptin or AEBSF.