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 1-7 specific NS4A. We developed efficient culture systems expressing NS4A of genotype (isolate) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69) with peak infectivity titers of 3.5-4.5 log10 Focus Forming Units per ml. Except for 2a (J6), growth depended on adaptive mutations identified in long-term culture. 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 1-7 NS4A recombinants showed similar responses to protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to interferon-alpha2b, and to the putative NS4A inhibitor ACH-806. Efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (NS3 protease), suggested to mediate resistance to ACH-806 in replicons. 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 evaluation of efficacy of antivirals.