MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region ...(5' UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5' UTR of different HCV genotypes have not been available for testing 5' UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5' UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5' UTR SLI was essential for genotypes 1-6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 5' UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.
Background & Aims The hepatitis C virus (HCV) genotype influences efficacy of interferon (IFN)-based therapy. HCV protease inhibitors are being licensed for treatment of genotype 1 infection. Because ...there are limited or no data on efficacy against HCV genotypes 2–7, we aimed at developing recombinant infectious cell culture systems expressing genotype-specific nonstructural (NS) protein 3 protease (NS3P). Methods Viability of J6/JFH1-based recombinants with genotypes 1–7 NS3P/NS4A was evaluated in Huh7.5 human hepatoma cells. Adaptive mutations were identified in reverse genetic studies. Efficacy of lead compound linear protease inhibitors VX-950 (telaprevir) and SCH503034 (boceprevir) and macrocyclic inhibitors TMC435350, ITMN-191 (danoprevir), and MK-7009 (vaniprevir) was determined in high-throughput infection assays. Results For genotype(isolate) 2a(J6), 3a(S52), 5a(SA13), and 6a(HK6a), we developed culture systems producing supernatant infectivity titers of 3.5–4.0 log10 focus forming units/mL. Against 2a(J6), 5a(SA13), and 6a(HK6a), all inhibitors showed similar efficacy; macrocyclic inhibitors had ∼10-fold greater potency than linear inhibitors. However, compared with 2a recombinant J6/JFH1, efficacy against 3a(S52) was 16- to 70-fold lower for macrocyclic inhibitors and 2- to 7-fold lower for linear inhibitors. Testing of additional genotype 2a and 3a isolates showed that these differences were genotype specific. The resistance of 3a isolates was similar to J6/JFH1 with engineered resistance mutations originally observed for genotype 1 patients. In contrast, we found similar efficacy of NS5A inhibitor BMS-790052 and interferon-alfa2. Conclusions Novel HCV culture systems with genotype specific NS3P/NS4A revealed similar efficacy of protease inhibitors against genotypes 2a, 5a, and 6a and comparatively low but varying efficacy against genotype 3a isolates. These systems will facilitate genotype-specific studies of HCV protease inhibitors and of viral resistance.
Background. Recently, a hepatitis C virus (HCV) cell-culture system was developed that employed strain JFH1 (genotype 2a), and JFH1-based intra- and intergenotypic recombinants now permit functional ...studies of the structural genes (Core, E1, and E2), p7, and NS2 of genotypes 1–4. The goal was to adapt the system to employ genotype 5. Methods. Huh7.5 cells infected with SA13/JFH1, containing Core-NS2 of strain SA13 (genotype 5a), were monitored for Core expression and for supernatant infectivity and HCV-RNA titers. Adaptive mutations of SA13/JFH1 were identified by sequence analysis of recovered genomes and reverse-genetic studies. Receptor blockage was performed with anti-CD81 and anti-SR-BI. For neutralization experiments, SA13/JFH1 or JFH1-based viruses of other genotypes were incubated with patient sera. Results. SA13/JFH1 with NS2 and NS3 mutations yielded infectivity titers >105 TCID50/mL. Infection with SA13/JFH1 was inhibited by CD81 blocking and SR-BI blocking, respectively, and by preincubation with genotype 5a chronic-phase patient sera. Such sera had varying cross-genotype neutralization potential. However, preincubation and treatment with homologous neutralizing antibodies could not control SA13/JFH1 infection in culture. Conclusion. The SA13/JFH1 culture permits genotype 5a-specific studies of Core-NS2 function and interfering agents. The ability of HCV to spread in vivo during treatment with neutralizing antibodies was confirmed in vitro.
Six major hepatitis C virus (HCV) genotypes and numerous subtypes have been described, and recently a seventh major genotype was discovered. Genotypes show significant molecular and clinical ...differences, such as differential response to combination therapy with interferon‐α and ribavirin. Recently, HCV research has been accelerated by cell culture systems based on the unique growth capacity of strain JFH1 (genotype 2a). By development of JFH1‐based intergenotypic recombinants containing Core, envelope protein 1 and 2 (E1, E2), p7, and nonstructural protein 2 (NS2) of genotype 6a and 7a strains, as well as subtype 1b and 2b strains, we have completed a panel of culture systems for all major HCV genotypes. Efficient growth in Huh7.5 cells depended on adaptive mutations for HK6a/JFH1 (6a/2a, in E1 and E2) and J4/JFH1 (1b/2a, in NS2 and NS3); viability of J8/JFH1 (2b/2a) and QC69/JFH1 (7a/2a) did not require adaptation. To facilitate comparative studies, we generated virus stocks of genotype 1–7 recombinants with infectivity titers of 103.7 to 105.2 50% tissue culture infectious dose/mL and HCV RNA titers of 107.0 to 107.9 IU/mL. Huh7.5 cultures infected with genotype 1–6 viruses had similar spread kinetics, intracellular Core, NS5A, and lipid amounts, and colocalization of Core and NS5A with lipids. Treatment with interferon‐α2b but not ribavirin or amantadine showed a significant antiviral effect. Infection with all genotypes could be blocked by specific antibodies against the putative coreceptors CD81 and scavenger receptor class B type I in a dose‐dependent manner. Finally, neutralizing antibodies in selected chronic phase HCV sera had differential effects against genotype 1–7 viruses. Conclusion: We completed and characterized a panel of JFH1‐based cell culture systems of all seven major HCV genotypes and important subtypes and used these viruses in comparative studies of antivirals, HCV receptor interaction, and neutralizing antibodies. (HEPATOLOGY 2009.)
Efficient in vitro systems to study the life cycle of hepatitis C virus (HCV) were recently developed for JFH1 (genotype 2a), which has unique replication capacity in Huh7 cells. We developed 4a/JFH1 ...intergenotypic recombinants containing the structural genes (Core, E1, and E2), p7, and all or part of NS2 of the 4a prototype strain ED43 that, after transfection of Huh7.5 cells with RNA transcripts, produced infectious viruses. Compared with the J6/JFH control virus, production of viruses was delayed. However, efficient spread of infection and high HCV RNA and infectivity titers were obtained in serial passages. Sequence analysis of recovered viruses and subsequent reverse genetic studies revealed a vital dependence on one or two NS2 mutations, depending on the 4a/2a junction. Infectivity of ED43/JFH1 viruses was CD81 dependent. The genotype 4 cell culture systems permit functional analyses as well as drug and vaccine research on an increasingly important genotype in the Middle East, Africa, and Europe. We also developed genotype 1a intergenotypic recombinants from H77C with vital mutations in NS3. Using H77C/JFH1 and ED43/JFH1 viruses, we demonstrated high homologous neutralizing antibody titers in 1a and 4a patient sera, respectively. Furthermore, availability of JFH1 viruses with envelope proteins of the six major HCV genotypes permitted cross-neutralization studies; 1a and 4a serum cross-neutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses. Thus, the JFH1 intergenotypic recombinants will be of importance for future studies of HCV neutralization and accelerate the development of passive and active immunoprophylaxis.
Background & Aims Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of ...HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α. Methods Huh7.5 hepatoma cells were transfected with RNA of genotype 1–7 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays. Results Cells infected with viable HCV that expressed NS5A of genotypes 1–7 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC50 ) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC50 values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region. Conclusions We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 1–7 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.
There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For ...HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.
The six major epidemiologically important hepatitis C virus (HCV) genotypes differ in global distribution and antiviral responses. Full-length infectious cell-culture adapted clones, the gold ...standard for HCV studies in vitro, are missing for genotypes 4 and 5. To address this challenge for genotype 5, we constructed a consensus full-length clone of strain SA13 (SA13fl), which was found non-viable in Huh7.5 cells. Step-wise adaptation of SA13fl-based recombinants, beginning with a virus encoding the NS5B-thumb domain and 3´UTR of JFH1 (SA13/JF372-X), resulted in a high-titer SA13 virus with only 41 JFH1-encoded NS5B-thumb residues (SA13/JF470-510cc); this required sixteen cell-culture adaptive substitutions within the SA13fl polyprotein and two 3´UTR-changes. SA13/JF372-X and SA13/JF470-510cc were equally sensitive to nucleoside polymerase inhibitors, including sofosbuvir, but showed differential sensitivity to inhibitors targeting the NS5B palm or thumb. SA13/JF470-510cc represents a model to elucidate the influence of HCV RNA elements on viral replication and map determinants of sensitivity to polymerase inhibitors.
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