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.

Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research.

Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance.

Various protease inhibitors (PIs) currently are becoming available for treatment of hepatitis C virus (HCV). For genotype 1, substitutions at NS3 protease positions 155, 156, and 168 are the main determinants of PI resistance. For other genotypes, similar substitutions were selected during PI treatment but were not characterized systematically. To elucidate the impact of key PI resistance substitutions on genotypes 2 to 6, we engineered the substitutions R155A/E/G/H/K/Q/T, A156G/S/T/V, and D/Q168A/E/G/H/N/V into HCV recombinants expressing genotype 2 to 6 proteases. We evaluated viral fitness and sensitivity to nine PIs (telaprevir, boceprevir, simeprevir, asunaprevir, vaniprevir, faldaprevir, paritaprevir, deldeprevir, and grazoprevir) in Huh7.5 cells. We found that most variants showed decreased fitness compared to that of the original viruses. Overall, R155K, A156G/S, and D/Q168A/E/H/N/V variants showed the highest fitness; however, genotype 4 position 168 variants showed strong fitness impairment. Most variants tested were resistant to several PIs. Resistance levels varied significantly depending on the specific substitution, genotype, and PI. For telaprevir and boceprevir, specific 155 and 156, but not 168, variants proved resistant. For the remaining PIs, most genotype 2, 4, 5, and 6, but not genotype 3, variants showed various resistance levels. Overall, grazoprevir (MK-5172) had the highest efficacy against original viruses and variants. This is the first comprehensive study revealing the impact of described key PI resistance substitutions on fitness and PI resistance of HCV genotypes 2 to 6. In conclusion, the studied substitutions induced resistance to a panel of clinically relevant PIs, including the newer PIs paritaprevir, deldeprevir, and grazoprevir. We discovered complex patterns of resistance, with the impact of substitutions varying from increased sensitivity to high resistance.

Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors.

BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A) from genotypes 1-6 and 2a(JFH1) NS5B-3' untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants. METHODS: The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration-response profiles were determined for each DAA agent in replicate infected Huh7.5 cells. RESULTS: Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity. CONCLUSIONS: The mutations F1464L, A1672S, and D2979G permitted the development of efficient HCV recombinants comprising genotype-specific 5' untranslated region-NS5A (5-5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5-5A recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1-6.

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors.

UNLABELLED: Hepatitis C virus (HCV) is a genetically diverse virus with multiple genotypes exhibiting remarkable differences, particularly in drug susceptibility. Drug and vaccine development will benefit from high-titer HCV cultures mimicking the complete viral life cycle, but such systems only exist for genotypes 1a and 2a. We developed efficient culture systems for the epidemiologically important genotype 2b. Full-length molecular clones of patient strains DH8 and DH10 were adapted to efficient growth in Huh7.5 cells by using F1468L/A1676S/D3001G (LSG) mutations. The previously developed J8cc prototype 2b recombinant was further adapted. DH8 and J8 achieved infectivity titers >4.5 log10 Focus-Forming Units/mL. A defined set of DH8 mutations had cross-isolate adapting potential. A chimeric genome with the DH10 polyprotein coding sequence inserted into a vector with J8 untranslated regions was viable. Importantly, we succeeded in generating DH8, J8, and DH10 viruses with authentic sequences in the regions targeted by lead direct-acting antivirals. Nonstructural protein (NS)5B inhibitors sofosbuvir, mericitabine, and BI207127 had activity against 1a (strain TN), 2a (strains JFH1 and J6), and the 2b strains, whereas VX-222 and filibuvir only inhibited 1a. Genotype 2b strains were least sensitive to seven lead protease inhibitors, including MK-5172 with high overall potency. NS5A inhibitor daclatasvir was exceptionally potent, but efficacy was affected by the HCV strain. CONCLUSION: Highly efficient HCV full-length 2b culture systems can be established by using consensus clones with defined mutations. Lead protease and NS5A inhibitors, as well as polymerase inhibitors sofosbuvir, mericitabine, and BI207127, show cross-activity against full-length 1a, 2a, and 2b viruses, but important sensitivity differences exist at the isolate level. Infectious cultures for different HCV strains will advance studies on viral biology and pathogenesis and promote individualized patient treatment.

Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor.

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.

Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.

With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3' untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ~4 log(10) focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low concentrations.

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system.

Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1-7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5' untranslated region (5'UTR)-NS5A and JFH1 NS5B-3'UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log(10) focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment.