Fig 5.

Effects of a furin inhibitor on replication of wtrAPMV-7 and the Fcs-5B mutant. Vero cells in 6-well plates were infected with wt rAPMV-7 (A and C) or the Fcs-5B mutant (B and D) at an MOI of 0.01. The cells were incubated with serum-free media containing furin inhibitor at 0, 1, 5, and 10 μM, as indicated. (A and B) Virus replication. Samples of the culture medium supernatants were collected at 24-h intervals, and virus titers were determined by limiting dilution assay. (C and D) Western blot analysis. The cells in additional plates that had been infected and treated in parallel were harvested and lysed at 2, 4, and 6 dpi. Western blot analysis was performed by using rabbit antiserum that had been raised against a synthetic peptide representing the cytoplasmic tail of the APMV-7 F protein (upper panel in C and D) or using anti-N protein rabbit serum (lower panel in C and D). The precursor (F0) and cleaved subunit (F1) of the F protein are indicated. (E) Efficiency of cleavage of the F proteins of wt rAPMV-7 and the Fcs-5B mutant in the absence of the furin inhibitor. The relative levels of the F0 and F1 proteins in the absence of inhibitor in the Western blot images in panels C and D were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0.