HCV NS3 helicase is a member of a superfamily of helicases, termed superfamily II. Residues of NS3 helicase which are conserved among superfamily II helicases line an interdomain cleft between the first two domains. The oligonucleotide binds in an orthogonal binding site and contacts relatively few conserved residues. There are no strong sequence-specific interactions with the oligonucleotide bases.

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An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.

Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, El (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of El is associated with E2 and E2-NS2 via disulfide linkages.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3⅐4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3⅐4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3⅐4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1 (1). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care is pegylated interferon ␣ in combination with ribavirin, which has a sustained viral response rate of 40 -50% in genotype 1 HCV-infected patients, which accounts for the majority of the hepatitis C population in the United States and Japan, and of 80 -90% in patients infected with genotype 2 or 3 HCV (4, 5) (for a review, see Ref. 6). Thus, more effective therapeutic drugs with fewer side effects and shorter treatment durations are needed for patients infected with HCV.HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products, in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 7). NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (8). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 9). However, successful discovery of a new HCV-specific drug candidate has been hampered by the lack of a robust, reproducible infectious virus cell culture system. The development of a HCV replicon system by Lohmann et al. (10) and subsequent optimization by several laboratories (11, 12) has enabled quantitative evaluation of the antiviral potency of HCV inhibitors.The HCV NS3⅐4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (13-17). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essentia...