Protease inhibitors (PIs) have proven to be effective adjuncts to interferon/ribavirin treatment of hepatitis C virus (HCV) infections. Little clinical or in vitro data exists, however, on their effectiveness for nontype 1 genotypes that predominate in Europe, the Middle East, Africa, and most of Asia. NS3 protease and NS4A genes from genotypes 1-6 were inserted into the JFH clone to generate replication-competent intergenotype chimeras. Susceptibility to PIs was determined by replication and infectivity assays. To study resistance development, chimeras were cultured in subinhibitory concentrations of PIs and mutations phenotypically characterized. Marked differences in susceptibility of different genotypes to danoprevir and telaprevir (VX-950) were observed. Genotypes 1, 4, and 6 showed median inhibitory concentration (IC 50 ) values of 2-3 nM, >100-fold lower than genotypes 2/3/5 (250-750 nM). Telaprevir susceptibilities varied over a 4-fold range, with genotypes 1 and 2 being most susceptible and genotypes 4 and 5 most resistant. Culture of genotypes 1-6 in PIs induced numerous mutations in the NS3 protease domain, highly variable between genotypes. Introduction of danoprevir and BILN 2061-induced mutations into the original clones by site-directed mutagenesis (n 5 29) all conferred resistant phenotypes, with particularly large increases (1-2 log greater IC 50 values) in the initially susceptible genotypes 1/4/6. Most introduced mutations and showed little or no effect on replicative fitness. Conclusion: Major differences were found between genotypes in their susceptibility and resistance development to PIs. However, equal sensitivities of genotypes 1, 4, and 6 to danoprevir and a broader efficacy range of telaprevir between genotypes than initially conceptualized provide strong evidence that PIs might be effectively used beyond their genotype 1 target group. (HEPATOLOGY 2011;53:1090-1099 I nfections with hepatitis C virus (HCV) are a major cause of chronic liver diseases, such as cirrhosis and hepatocellular carcinoma. 1 Clinical management of HCV and the effectiveness of currently used interferon-a (IFN)/ribavirin (RBV) therapy of infected individuals is greatly influenced by genetic heterogeneity of the virus. There are, for example, major differences between the six genotypes of HCV in response rate to therapy and evidence for some genotype-associated variability in the rate of disease progression and associated liver pathology. 2,3 HCV replication is additionally associated with high mutation rates; this confers on HCV, in common with human immunodeficiency virus 1 (HIV-1), considerable adaptive capacity to escape from immunological or drug-treatment pressure. The effectiveness of newly developed protease and polymerase inhibitors for HCV, at least as monotherapy, is indeed likely to be substantially impaired through the acquisition or selection for preexisting amino acid mutations that confer antiviral resistance.Genetic heterogeneity between HCV genotypes translates into significant molecular and clin...

Protease inhibitors (PIs) of hepatitis C virus (HCV) provide an additional or alternative therapy for chronic infection. However, assessment of their efficacy and ability to inhibit replication of different genotypes is hampered by the lack of a convenient animal model or a method for in vitro culture of HCV other than the type 1/2-based replicons and the infectious genotype 2a clone JFH1. To address this problem, we constructed a panel of replication-competent chimeric Jc1 (pFK JFH1/J6/C-846) clones containing protease and NS4A coding sequences from all six major genotypes, enabling the determination of replication and the susceptibility to PIs. Chimeras showed substantial variability in replication kinetics, attributable in part to naturally occurring polymorphisms and differing requirements for adaptive mutations in NS3 and NS4A. Through calculation of 50% inhibitory concentrations (IC 50 s) of BILN 2061, measuring reduction in the number of focus-forming units/ml (FFU/ml) and replication inhibition, consistent genotype-associated differences in antiviral susceptibilities were observed. IC 50 s for genotype 1b, 4a, and 6a-derived chimeras (1 to 3 nM) were approximately 100-fold lower than those for genotypes 2a, 3a, and 5a (range, 80 to 720 nM), implying major differences in response to therapy. In vitro passage in increasing concentrations of BILN 2061 rapidly induced resistance-associated mutations at position 168 in chimeras of all 6 genotypes and at position 156 in genotypes 1b and 4a, each with substantial variability in the identity of substituted amino acids. The system will allow future comprehensive phenotypic characterization of naturally occurring and treatment-induced mutations for PIs in trial or entering clinical use.Worldwide, about 170 million individuals are estimated to be infected with hepatitis C virus (HCV) (1, 48). Chronic HCV infection is a leading cause of chronic liver diseases, such as cirrhosis and hepatocellular carcinoma (6). HCV has a positive-sense, single-stranded RNA genome of approximately 9,600 nucleotides, belonging to the family Flaviviridae (7). A single polyprotein of around 3,000 amino acids (53) is translated and processed by cellular and viral proteases to generate 10 different structural and nonstructural proteins (16,18,19).The error-prone RNA-dependent RNA polymerase (RdRp) NS5B, and the resulting high mutation frequencies during replication, contributes to the substantial genetic and antigenic heterogeneity of HCV, with seven major genotypes showing Ͼ30% nucleotide sequence divergence from each other and numerous subtypes identified to date (5, 50-52). The distribution of genotypes varies by geographical location and risk groups for infection; the predominant genotypes within the United States, Europe, Australia, and East Asia (Japan, Taiwan, Thailand, and China) are 1, 2, and 3. Genotype 4 is largely confined to the Middle East, Egypt, and Central Africa, whereas genotypes 5 and 6 are found predominantly in South Africa and Southeast Asia, respectively (49).The c...