Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: mechanism for the antiviral effects of heme oxygenase?

Abstract

Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 μM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 μM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons.

Conclusion: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.

Figure 1. Heme oxygenase and biliverdin reductase enzymatic reactions

Heme is first oxidized by heme oxygenase-1 producing biliverdin, Carbon monoxide and iron. Biliverdin is then reduced at the central methene bridge position 10 (arrows) by biliverdin reductase to produce bilirubin.

Figure 2. Biliverdin and bilirubin suppression of HCV replication

Log-phase full-length (FL) (A) or non-structural (NS) (B) replicon cells were treated 48 hours with indicated concentrations of BV or BR. HCV replication was determined by Real-Time RT-PCR using the Comparative Cycle Threshold (ΔC_T) method as described in Supplemental methods available on line. Each value is the mean +/− the SEM of 5–6 determinations per treatment group. Biliverdin = >99% Biliverdin IX-α. Bilirubin-mixed isomers (MI) = 93% Bilirubin IX-α, and 6% associated Bilirubin isomers as described in Materials. Bilirubin IX-α = >99% Bilirubin IX-α. [** or ## or ^^ p<0.01 from respective controls. ^ p < 0.05 from control.].

Figure 3. Effect of BV, BR, and FeCl₂ treatment on HCV proteins

(A, B) Log-phase NS or FL replicons were treated with biliverdin (20 µM), BR (200 µM) or FeCl2 (100 µM) for 24 h. Cells were then lysed and protein expression evaluated by WB. (C, D) Full-length replicons were treated overnight with various concentrations of BV and assayed by WB (C) or immunoprecipitation for NS5A (D). In D, upper band was identified as NS5A and lower band was immunoglobulin heavy chain (Ig HC).

Figure 3. Effect of BV, BR, and FeCl₂ treatment on HCV proteins

(A, B) Log-phase NS or FL replicons were treated with biliverdin (20 µM), BR (200 µM) or FeCl2 (100 µM) for 24 h. Cells were then lysed and protein expression evaluated by WB. (C, D) Full-length replicons were treated overnight with various concentrations of BV and assayed by WB (C) or immunoprecipitation for NS5A (D). In D, upper band was identified as NS5A and lower band was immunoglobulin heavy chain (Ig HC).

Figure 4. Biliverdin inhibition of J6/JFH HCV replication in Huh7.5 cells

(A–C) J6/JFH infected Huh7.5 cells were treated with different concentrations of BV (0, 20, 200 µM, A–C respectively) for 72 h. Cultures were then fixed and stained immunocytochemically with HCV genotype 2A polyvalent human serum. (D) HCV RNA was quantified using ΔC_T assay with 3–5 determinations per treatment group. Each bar represents the mean +/− SEM for each treatment group. **HCV RNA in BV-treated cells versus control (p < 0.01).

Figure 5. Inhibition of NS3/4A protease

(A–B) Protease activity was determined fluorometrically using recombinant NS3/4A enzyme as described in Methods. (C) Endogenous NS3/4A protease activity in microsomes of replicons was measured using the same assay with partially purified NS3/4A protease from replicon cells as described in Supplemental methods available online. Each point is the mean+/− SEM of 3–5 determinations per point Inhibitor refers to the commercial NS3/4A protease competitive inhibitor, AnaSpec #25346. Biliverdin = >99% Biliverdin IX-α. Bilirubin-mixed isomers (MI) = 93% Bilirubin IX-α, and 6% associated Bilirubin isomers as described in Materials. Bilirubin IX-α = >99% bilirubin IX-α.

Figure 6. Kinetics of BV inhibition of NS3/4A protease

(A) Reciprocal (Lineweaver-Burk) plot of substrate concentration versus enzyme activity. Recombinant protease activity was determined fluorometrically as described in Fig. 5 and Methods. Each point is the mean +/− SEM of 3–5 determinations per point. (B,C) Secondary plots of 1/Vap (y-intercepts) or Km/V (slopes) versus BV concentrations to estimate Ki’ and Ki of BV inhibition non-competitive and competition inhibition respectively. Plot of [BV] vs either 1/Vap or Km/V showed highly significant linearity, (r = 0.975 and r = 0.979 respectively, P< 0.005) suggesting mixed inhibition of NS3/4A protease by BV (Ki’ = 1.1 and Ki = 0.6 µM, respectively).

Fig. 7. Effect of biliverdin reductase (BVR) knockdown on antiviral activity

(A) The efficiency of BVR knockdown was determined by WB after transfection of BVR siRNA or scrambled controlled RNA into NS (left panel) or FL (right panel) replicons. Real-time RT-PCR measurements for HCV RNA were performed after control vehicle or BV (20 µM) overnight incubation in both replicon lines (B left panel). Note that the antiviral activity of BV was significantly enhanced (P < 0.01) when BVR was knocked down. In B (right panel) HCV RNA was quantified after control vehicle or BR (100 µM) overnight incubation in both replicon lines. BVR knockdown had no effect on the antiviral activity of BR. Each graph is the mean +/− SEM of 5–6 determinations for each treatment. (## not significant), (* p < 0.01, ** p < 0.005).

Figure 8. Additive effect of BV on α-interferon antiviral activity

FL or NS replicons were treated with indicated amounts of α-interferon alone or in the presence of 50 or 100 µM BV overnight. HCV replication was determined by real-time RT-PCR using the ΔC_T method. Each value is the mean +/− SEM of 3 determinations per point. (*) p < 0.01 vs control (#) p < 0.005 vs control