Expression of MCL1 is impaired in cells infected with flaviviruses.

(A) Expression of MCL1 is decreased in cells infected with flaviviruses. Huh7 cells were infected with either JEV (MOI = 5), DENV (MOI = 3) or ZIKV (MOI = 1), then incubated with the caspase inhibitor, QVD-OPH (20 μM). Expression of the indicated proteins was assessed by immunoblotting. (B) Huh7 cells were infected as described above were incubated with QVD-OPH (20 μM) for 2 days, and further incubated for indicated time in the presence of a proteasome inhibitor (ALLN; 20 μM). Expression of the indicated proteins was assessed by immunoblotting. (C) JEV infection attenuates cellular protein synthesis. Total protein synthesis in JEV-infected cells was determined via [35S]-methionine incorporation at indicated time points (left panel). The data represent the mean ± SD of three independent experiments. Significant differences were determined using Student’s t-test and are indicated by asterisks (*P35S]-methionine and [35S]-cysteine incorporation at 2 days post-infection. MCL1 proteins were immunoprecipitated with the appropriate antibody and subjected to SDS-PAGE (right panel). (D) Huh7 cells infected with JEV (MOI = 5) were incubated with QVD-OPH (20 μM) and subjected to immunoblotting at 2 days post-infection. (E) Huh7 cell lines stably expressing GFP and D2GFP were infected with JEV (MOI = 5 or 20) then incubated with QVD-OPH (20 μM) and subjected to immunoblotting at 2 days post-infection. Images (A, B, D, E) are representative of two independent experiments.