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.

FIG. 1.

Replication kinetics in Huh7.5 cells of the parental JFH1 and Jc1 full-length RNA transcripts and comparison with that of Jc1-BB containing introduced BstBI and BglII restriction sites in NS3 for protease gene insertion. The replication-defective mutant JFH1-GND was used as a negative control. The y axis records the percentage of HCV NS5A-positive cells scored by fluorescence microscopy.

FIG. 2.

Jx recombinants and their viability in Huh7.5 cells. (A) Genome map of cDNA clones (pJ6CF, gray; pJFH1, white). The NS3 protease was replaced with the corresponding intra- or intergenotypic gene (replaced region, black). UTR, untranslated region. (B) RNA transcripts from J1a, J1b, J2a, J3a, J4a, J5a, and J6a were electroporated into Huh7.5 cells, and replication was assessed by immunostaining against NS5A. Jc1 and pJFH1-GND served as positive and negative controls, respectively. Transcripts from the J1a, J1b, J3a, J4a, and J6a clones showed no detectable replication (all values lying on the x axis line).

FIG. 3.

Comparison of genotype 1a, 1b, 2a, 3a, 4a, 5a, and 6a NS3 protease and NS4A cofactor sequences. (A) Alignment of NS3 protease residues (in black) from recombinants J1a1a, J1b1b, J2a2a, J3a3a, J4a4a, J5a5a, and J6a6a to JFH1 (NCBI accession no. AB047639). (B) Alignment of NS4A protease cofactor residues (in black) from recombinants J1a1a, J1b1b, J2a2a, J3a3a, J4a4a, J5a5a, and J6a6a to JFH1. Dots indicate amino acid sequence identity.

FIG. 4.

Jxx recombinants and their viability in Huh7.5 cell results from two independent experiments (Jxx_A and Jxx_B). (A) Genome map of cDNA clones (pJ6CF, gray; pJFH1, white). The NS3 protease and NS4A were replaced with intra- and intergenotypic genes (replaced region, black). (B to G) RNA transcripts from J1a1a, J1b1b, J2a2a, J3a3a, J4a4a-19, J5a5a, and J6a6a were electroporated into Huh7.5 cells. Jc1 and pJFH1-GND served as positive and negative controls, respectively.

FIG. 5.

J1b1b, J3a3a, J4a4a, and J6a6a study subject-derived protease recombinants and their viability in Huh7.5 cells. The protease region of J1b1b, J3a3a, J4a4a, and J6a6a was replaced with that of two or three study subject-derived protease genes, and their replication capacities were assessed in the Huh7.5 cells.

FIG. 6.

Viability of J2a2a, J5a5a, J6a6a, and J3a3a recombinants with study subject-derived NS3 protease genes, including adaptive/attenuating mutations observed in clonal analysis.

FIG. 7.

Antiviral inhibition of Jxxs. Reduction in supernatant infectivity. After 8 h of inoculation with Jxx (MOI, 0.015), Huh7.5 cells were washed and incubated in media containing 0.1% DMSO, as a carrier control, with or without the indicated doses of BILN 2061. Inhibition was calculated at 72 h postinfection as a reduction in supernatant infectivity (the number of FFU/ml; mean ± SEM; n = 3) after antiviral (AV) addition compared to infectivity of the control without antiviral.

FIG. 8.

Antiviral inhibition of Jxxs. Reduction in viral replication. RNA (1 to 10 μg) was electroporated into Huh7.5 cells and incubated for 24 h. Cells were then washed and incubated in media containing 0.1% DMSO, as a carrier control, with or without the indicated doses of BILN 2061 for a further 72 h. The percent inhibition of replication was determined at 96 h postelectroporation (mean ± SEM; n = 3) and calculated as the ratio of NS5A-positive cells in BILN 2061-treated cells to those of the control without antiviral.

FIG. 9.

Antiviral inhibition of J3a3a recombinants with study subject-derived proteases. RNA (10 μg) was electroporated into Huh7.5 cells and incubated for 24 h. Cells were then washed and incubated in media containing 0.1% DMSO, as a carrier control, with or without the indicated doses of BILN 2061 for a further 72 h. The percent inhibition of replication was determined at 96 h postelectroporation (mean ± SEM; n = 3) and calculated as the ratio of NS5A-positive cells in BILN 2061-treated cells to those of the control without antiviral.