Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K i , 80 to 90 nM) compared to genotype 1 enzymes (K i , 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these nongenotype-1 NS3-NS4A proteins, with K i values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.According to the latest World Health Organization estimates, more than 170 million individuals may be infected with hepatitis C virus (HCV). Chronic infection, observed in about 85% of cases, could lead to progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (7). HCV belongs to the Flaviviridae family. Its positive-strand RNA genome contains 9,600 nucleotides and encodes a ϳ3,100-amino-acid protein that is posttranslationally processed by host-and virally encoded proteases into structural (C, E1, E2, p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (23). The nonstructural (NS) proteins include enzymes necessary for protein maturation (NS2/3 and NS3 proteases) and viral replication (NS3 helicase/nucleoside triphosphatase and NS5B RNA polymerase).The high rate of viral production linked to the low fidelity of the RNA polymerases (5, 6) leads to genetic heterogeneity of HCV in infected patients (20). Natural variants of HCV are currently classified into 6 genotypes and more than 50 subtypes (25). The genotypes differ by as much as 34% in their nucleotide sequences, resulting in approximately 30% amino acid sequence divergence between the encoded polyproteins, while subtypes can differ by as much as 23% of their nucleotide sequence. The degree of sequence variability also varies for the different subgenomic regions. For example, the core and the 3Ј and 5Ј nontranslated regions are more conserved, whereas the envelope region displays more variability (24, 31). Sequences coding for the NS3 protease domain and the NS5B polymerase show degrees of variability comparable to that for the complete genome.The HCV infections most frequently encountered are caused by genotypes 1, 2, and 3 (18). In Europe, Japan, and the United States, more than 70% of the HCV-posi...

The NS3 protein of hepatitis C virus is unusual because it encodes two unrelated enzymatic activities in linked protease and helicase domains. It has also been intensively studied because inhibitors targeting its protease domain have potential to significantly improve treatment options for those infected with this virus. Many enzymological studies and inhibitor discovery programs have been carried out using the isolated protease domain in complex with a peptide derived from NS4A which stimulates activity. However, some recent publications have suggested that the NS3 helicase domain may influence inhibitor binding and thus suggest work should focus on the full-length NS3-NS4A protein. Here we present the characterization of a single-chain protease in which the NS4A peptide activator is linked to the N-terminus of the NS3 protease domain. This protein behaves well in solution, and its protease activity is very similar to that of full-length NS3-NS4A. We find that this fusion protein, as well as the noncovalent complex of the NS4A peptide with NS3, gives similar Ki values, spanning 3 orders of magnitude, for a set of 25 structurally diverse inhibitors. We also show that simultaneous mutation of three residues on the surface of the helicase domain which has been hypothesized to interact with the protease does not significantly affect enzymatic activity or inhibitor binding. Thus, the protease domain with the NS4A peptide, in a covalent or noncovalent complex, is a good model for the protease activity of native NS3-NS4A.

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As an obligate step for picornaviruses to replicate their genome, the small viral peptide VPg must first be specifically conjugated with uridine nucleotides at a conserved tyrosine hydroxyl group. The resulting VPg-pUpU serves as the primer for genome replication. The uridylylation reaction requires the coordinated activity of many components, including the viral polymerase, a conserved internal RNA stem loop structure, and additional viral proteins. Formation of this complex and the resulting conjugation reaction catalyzed by the polymerase, offers a number of biochemical targets for inhibition of an essential process in the viral life cycle. Therefore, an assay recapitulating uridylylation would provide multiple opportunities for discovering potential antiviral agents. Our goal was to identify inhibitors of human rhinovirus (HRV) VPg uridylylation, which might ultimately be useful to reduce or prevent HRV-induced lower airway immunologic inflammatory responses, a major cause of asthma and chronic obstructive pulmonary disease exacerbations. We have reconstituted the complex uridylylation reaction in an AlphaScreen suitable for high-throughput screening, in which a rabbit polyclonal antiserum specific for uridylylated VPg serves as a key reagent. Assay results were validated by quantitative mass spectrometric detection of uridylylation.