Figure 4. Biliverdin inhibits DENV NS2B/NS3 protease activity.

(A) Inhibition of DENV protease activity by biliverdin. DENV protease activity was determined by incubating the indicated concentrations of biliverdin with recombinant DENV protease and Boc-GRR-AMC in a cleavage buffer for 30 min. Free AMC was measured using a spectrofluorometer and was expressed as percentage change relative to that in biliverdin-untreated controls (defined as 100%). (B,C) Determination of mechanisms underlying the effect of biliverdin on DENV NS2B/NS3 protease activity. The fluorogenic peptide at indicated concentrations was incubated with recombinant DENV protease in the presence of 5, 12.5 and 25 μM biliverdin for 30 min. The rate of release of AMC as a function of DENV protease activity was determined using a spectrofluorometer. Kinetic parameters and mode of inhibition were calculated from a standard curve generated using an AMC-positive control solution and were analyzed using Lineweaver–Burk plots of reciprocal velocity at the indicated concentrations of biliverdin. (D) Schematic diagram of the NS2B/NS3 protease vector with autocleavage activity (E) Inhibition of cis-acting activity of DENV protease by biliverdin in Huh-7 cells. The pNS2B(H)-NS3pro-transfected Huh-7 cells were treated with biliverdin at increasing concentrations for 3 days. The western blotting analysis were performed using anti-DENV NS2B specific antibody. (F) Schematic diagram of the protease reporter vector and the protease vector (G,H) Inhibition of trans-acting activity of DENV protease by biliverdin in Huh-7 and DENV-infected Huh-7 cells. The protease reporter vector and protease expression vector were co-transfected into Huh-7 cells or protease reporter vector alone was transfected into DENV-infected Huh-7 cells, followed by incubation of biliverdin at increasing concentrations for 3 days. Each transfection mixture contained 0.1 μg of firefly luciferase expression vector as a transfection control for normalization against the nano luciferase activity. Transfection of dysfunctional NS2B/NS3 protease expression vector or non-DENV-infected Huh-7 cells served as a negative control. The trans-acting activity of DENV protease is presented as percentage change relative to that in wild type protease-transfected or DENV-infected Huh-7 cells and biliverdin-untreated cells (defined as 100%). Results are expressed as the mean ± SD (error bar) of 3 independent experiments; *P < 0.05, **P < 0.01.