FIG. 4.

Early steps of viral replication within the first replication cycle are affected. (A) For time-of-addition kinetics analysis, A549 cells were either left untreated or were pretreated for 10 h or 1 h with 50 nM PS-341 before infection and additionally posttreated after infection. Cells were infected with FPV at an MOI of 0.005. After virus inoculation cells were posttreated with 50 nM PS-341. Then the proteasome inhibitor was added after virus inoculation (−10 h, −1 h, and +30 min) or it was added at the different times p.i. as indicated (+1 h, +2 h, +4 h, +6 h, and +8 h; cells were not pretreated before infection). At 9 h p.i. supernatants were obtained and progeny virus titers were determined by standard plaque assay. Shown is one representative experiment out of three independent experiments. (B) A549 cells were pretreated with 50 nM PS-341 or left untreated for 1 h. Afterwards cells were infected with avian FPV or human PR8 at an MOI of 1. Subsequent to virus inoculation cells were posttreated with 50 nM PS-341 or left untreated. After the indicated times p.i. cells were lysed and analyzed by Western blotting for accumulation of viral proteins polymerase PB1 and matrix protein M1. Cellular protein ERK2 served as a control to demonstrate equal amounts of protein loading. Shown is one representative blot out of three independent experiments.