Treatment with BV dose-dependently decreased NS5A when assayed by WB (Fig. effect of BVR knockdown on BV antiviral activity was assessed. After Malic enzyme inhibitor ME1 >80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of -interferon in replicons. == Conclusion == BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to Malic enzyme inhibitor ME1 the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful future drug therapies targeting the NS3/4A protease. Keywords:Hepatitis C virus, biliverdin, bilirubin, NS3/4A protease == Introduction == Chronic hepatitis C virus (HCV) infection is an important cause of liver disease worldwide. A significant number of infected patients develop persistent viremia that leads to cirrhosis, end-stage liver disease, and hepatocellular carcinoma (1). Current standard treatment for chronic HCV infection, pegylated -interferon and ribavirin, achieves viral eradication in only about half of patients treated (2). Structurally, the virus has a plus-stranded RNA genome with a single long open-reading frame containing 5 and 3 flanking non-translated nucleotide regions that are important for translation, replication, and immune recognition (3). The genome contains a serine-activated protease and an RNA dependent RNA polymerase (RdRp) that are important targets for development of new antiviral drugs. While anti-protease and anti-polymerase drugs promise to improve treatment outcomes, their efficacy may be limited by Malic enzyme inhibitor ME1 the rapid development of viral resistance (4). Hepatocellular damage from HCV has been linked to oxidative stress (5). Consequently, we and others have been interested in the potential role of antioxidant enzymes as cytoprotective agents during HCV infection (69). Heme oxygenase-1 (HO-1) is an important cytoprotective enzyme, which is readily induced in response to a variety of stressors and cytotoxins. HO-1 oxidizes heme to equimolar concentrations of biliverdin (BV), carbon monoxide, and iron (10) (Fig. 1). Following heme oxidation, free BV is rapidly reduced to bilirubin (BR) by the enzyme biliverdin reductase (BVR) which is abundant in the hepatocyte. == 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. We and others have previously shown that HO-1 induction or overexpression in replicons inhibits HCV replication (9,11). Although the mechanism of this effect has not been clearly defined, it is reasonable to infer that one or more of the products of the reaction catalyzed by HO-1 may be responsible. Consistent with this prediction, iron has Malic enzyme inhibitor ME1 been demonstrated to inhibit the NS5B RdRp through inhibition of divalent cation binding (12). There are also accumulating data indicating that BV has antiviral activity (13), which has been linked to the induction of interferon response genes (13). However, these results do not exclude the possibility that BV has additional antiviral effects. In this paper, we report that BV potently inhibits viral replication at concentrations remarkably similar to those achieved following induction of HO-1 by heme, suggesting that BV is a primary antiviral agent released during heme oxidation. Furthermore, BV, and to a much lesser extent the BV metabolite, bilirubin (BR) inhibits HCV NS3/4A protease in cell-free assays. These findings provide a plausible mechanism for the antiviral activity of HO-1 in addition Malic enzyme inhibitor ME1 to those reported previously and strongly suggest a potential role for BV or structural derivatives in future drug design targeting the HCV NS3/4A protease. == Materials and Methods == == Materials == TaqDNA polymerase (Perkin-Elmer Cetus, Norwalk, CT), and Moloney murine leukemia virus reverse transcriptase (Gibco/BRL Life Technologies, Gaithersburg, MD) were used in these studies. Bile pigments were purchased from Frontier Scientific, Inc (Logan, UT) and included bilirubin-IX- (#B584-9), biliverdin-IX- hydrochloride (#B655-9) and mesobilirubin (B588-9). Bilirubin mixed isomers, (>99%) was purchased from Sigma Chemical Co (Saint Louis, MO). All preparations of tetrapyrroles were the purest form available (99% purity). The BR mixed isomer preparation contained 93% bilirubin IX-, 3% bilirubin III-, 3% bilirubin XIII- and traces of SP-II and isomers (MSDS information). BV was prepared by oxidation of highly purified -bilirubin followed by final crystallization in ether (personal communication, Dr. Jerry Bommer, Echo Laboratories, Frontier Scientific, Salt Lake City, UT). All tetrapyrroles were dissolved in 0.2 N NaOH and added in small volumes to achieve the final concentration..