Development of an Intergenotypic Hepatitis C Virus (HCV) Cell Culture Method To Assess Antiviral Susceptibilities and Resistance Development of HCV NS3 Protease Genes from HCV Genotypes 1 to 6

Abstract

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...

Development of a high-throughput pyrosequencing assay for monitoring temporal evolution and resistance associated variant emergence in the Hepatitis C virus protease coding-region

“…Synthesis of cDNA via gene specific primer reverse transcription (GSP-RT) was performed using Superscript III Reverse transcriptase (Life Technologies) according to the manufacturer's instructions, with minor modifications. 8µl of extracted vRNA was used for GSP-RT in 20µl volumes using 2pmol primer (5'-TCYGCTTGGTCMAGGAC-3') (Imhof and Simmonds, 2010). Reverse transcription reactions were incubated at 50C for 1 hour followed by 42C for 1 hour.…”

“…Nested PCR was performed using previously described 1a-specific primer pairs (Imhof and Simmonds, 2010).…”

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

“…Huh7.5 cells (3.5 ϫ 10 5 /well) were plated in a 6-well plate, incubated overnight, and infected with stocks of HCV genotype 1 to 6 recombinants for 24 h. PIs purchased from Acme Bioscience were dissolved in dimethyl sulfoxide (Sigma) (21,28 50 of faldaprevir (BI 201335), paritaprevir (ABT-450), or deldeprevir (ACH-2684) was initiated when ϳ1% to 20% cells were infected. Cultures were retreated with the PI upon cell splitting every 2 to 3 days; cells plated on a chamber slide after treatment and incubated overnight were immunostained as described above in order to evaluate viral spread.…”

PCR-BasedIn VitroSynthesis of Hepatitis C Virus NS3 Protease for Rapid Phenotypic Resistance Testing of Protease Inhibitors

“…Several enzymatic and replicon-based phenotypic assays have been developed for assessing the PI susceptibilities of HCV through replicons with NS3 genes derived from clinical isolates (17)(18)(19) or recombinant NS3 proteases coded by the NS3 genes of HCV in patient sera (13). These assays can confirm the effects of new mutations or complex mutation patterns on HCV susceptibility to protease inhibitors, but they are laborious and time-consuming, with turnaround times ranging from a few days to weeks.…”