Figure 6

Tomatidine directly inhibited the activity of PEDV 3CL protease. A Vero cells cultured in 24-well plates were co-transfected with 250 ng of GFP_nsp5/6 encoding plasmid, 250 ng of 3CLpro or inactive mutants expressing plasmid or empty vector. At 24 hpi, the cells were harvested and the cleaved fragment of GFP_nsp5/6 was visualized using western blot. B Vero cells cultured in 24-well plates were co-transfected with 250 ng of GFP_nsp5/6 encoding plasmid, 250 ng of 3CLpro expressing plasmid or empty vector. At 12 hpi, the culture supernatants were replaced with fresh DMEM containing the indicated concentrations (10, 20, and 30 μM) of tomatidine or DMSO. At 24 hpi, the cells were harvested and the cleaved fragment of GFP_nsp5/6 was visualized using western blot. C PEDV 3CLpro was used at final concentrations of 0.5, 1, and 1.5 μM; and 10 μM (3CLpro substrate) was added to the protein in a black 96-well plate. PRRSV GP5 protein was used as a negative control. The mixtures were then further incubated at 37 °C for 20 min, and fluorescence was monitored at 340 nm excitation and 485 nm emissions every minute. The RFU were calculated by subtracting the mock from the fluorescence readings to eliminate the effect of background signals. D Tomatidine (25, 50 μM) or obacunone (50 μM, negative control) or DMSO was pre-incubated with 1 μM protease for 20 min at 37 °C, and 10 μM (3CLpro substrate) was added to the mixture in a black 96-well plate. The mixtures were then further incubated at 37 °C for 20 min, and fluorescence intensity was monitored at 340 nm excitation and 485 nm emissions every minute. RFU were calculated as above. E Vero cells were inoculated with PEDV (0.01 and 0.02 MOI) and 6 μM tomatidine. Cells were transfected with 250 ng pCAGGS-3CLpro as 3CLpro positive control. After incubation for 16 h, the cellular proteins were collected and the virus polyprotein was detected with western blot using anti-3CLpro mouse antibody, as previously described (20). Meanwhile, PEDV N-protein, GAPDH, and 3CLpro-Flag were detected with western blot using molecular antibodies against PEDV N-protein, GAPDH, and Flag. The ratio of nsp5-6 complex/N protein levels was depicted by integrated density analysis. The arrow indicates the location of nsp5-6 complex. All results are mean ± SD from three independent experiments performed in triplicate.