Figure 6.

Cysteine mutations in thiol reductase active site compromise GILT activity to inhibit viral entry of EBOV and LASV. (A) Schematic representation of the GILT protein structural domains, motif of enzyme active centre, and N-linked glycosylation sites. (B) FLIP-IN T Rex 293 cells expressing GILT or indicated mutant GILT proteins were cultured in the presence or absence of 1 μg/ml of tet for 24 h. The expression of indicated GILT mutants was detected by Western bot assay using anti-GILT polyclonal antibody. β-actin served as a loading control. (C) The above-mentioned FLIP-IN T Rex 293 cells were infected with SARSpp, LASVpp, EBOVpp or IAVpp. Luciferase activities were examined at 48 hpi. Relative infection efficiency is the ratio of luciferase activity in cells cultured with tetracycline over that in cells cultured without tetracycline. Results are the means ± SD of three independent experiments. **p < 0.001 compared to wild-type GILT. (D) FLIP-IN T Rex 293 cells expressing wild type or mutant GILT proteins were left untreated or treated with tet for 24 h to induce the GILT expression. The expression of cathepsins L, B and S in FLIP-IN T Rex 293 cells were determined by western blot assay. (E) The cathepsin proteolytic activity in cells used in (D) were measured and presented as relative fluorescence unit. Results are the means ± SD of three independent experiments. **p < 0.001.