DNAJC14 does not inhibit NS2/3 cleavage of YFV and HCV.

YFV replication is inhibited in cells inducibly expressing the NT5 mutant form of DNAJC14. (A) T-REx-293-NT5 cells were left uninduced or induced by treatment with doxycycline (Dox) for the indicated h and lysates were analyzed by Western blot using anti-myc and anti-actin (loading control) antibodies. Migration of size standards (in kDa) is indicated to the left. (B) T-REx-293-NT5 (NT5) or T-REx-293-LacZ (LacZ) cells were induced to express DNAJC14-NT5 or β-galactosidase, respectively, by 24 h treatment with doxycycline. The cells were then infected with YFV (moi = 5) and the medium was harvested and replaced at each timepoint. Virion production since the prior time point was enumerated by plaque assay. Pfu, plaque forming units. (C) YFV and HCV NS2/3 cleavage in T-REx-293-NT5 cells. T-REx-293-NT5 cells were left untreated or were induced to express DNAJC14-NT5 by 24 h treatment with doxycycline (Dox) as indicated. The cells were then cotransfected with pEGFP (loading control) and either pFlag-HCV-NS2/3(181) or pFlag-YFV-NS2/3(181). As controls, NS2-3 proteins containing active site mutations in the HCV NS2 (H143A) or YFV NS3 (S138A) proteases and incapable of cleavage activity were also expressed as indicated. Cells were harvested 1 day later and analyzed by Western blot using anti-Flag antibody (top panel) or anti-myc and anti-GFP antibodies (bottom panel). Arrows indicate the migration of the relevant proteins and migration of size standards (in kDa) is indicated on the left.