Fig 5.

M-calpain activity is necessary for efficient SARS-CoV replication. (A) Vero E6 cells were pretreated with DMSO (mock), 2.5 μM MG132, 5 μM CI VI, or 10 μM MDL28170 for 1 h and then inoculated with SARS-CoV (MOI, 0.001) in FCS-free media containing the respective compounds. After removal of inocula, cells were incubated in growth medium supplemented with FCS and the respective compounds for 16 h, and then viral supernatants were collected. Viral titers were determined by plaque assay and are shown as PFU/ml. Values represent the means ± SEM of two independent experiments performed in duplicate. (B) Vero E6 cells in 25-cm² flasks were transfected with nonsilencing (ns) siRNA or 350 pmol siRNA against m-calpain. Forty-eight h after transfection, cells were lysed and equal protein amounts were subjected to immunoblot analysis using an m-calpain (Calpain 2) antibody (upper panel). Actin was used as a loading control (lower panel). (C) Vero E6 cells were transfected as described for panel B and infected with SARS-CoV (MOI, 0.001) 48 h posttransfection. Inocula were removed after 1 h, and cells were incubated with normal growth medium for 16 h. Viral titers were determined as described for panel A.