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

Abstract

bTo 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 ϳ3.5 to 4.5 log 10 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. C hronic hepatitis C virus (HCV) infection is a major public health burden. HCV is an enveloped virus with a positivestrand RNA genome with 5=-and 3=-untranslated regions (UTRs) and an open reading frame (ORF) encoding a single polyprotein, which is cleaved into structural proteins (core, E1, and E2), p7, and the nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1). The NS3 protein consists of a serine protease (NS3P), responsible for downstream cleavages, and an RNA helicase (NS3H) domain. The function of NS3P is dependent on the NS4A protein. Due to great genetic heterogeneity, HCV was classified into seven major genotypes, which differ in about 30% of the sequence (2).For the last decade, combination therapy with pegylated alpha interferon 2 (IFN-alpha2) and ribavirin was the standard of care, but it was characterized by various contraindications and severe side effects. Efficacy of combination therapy depended on host factors as well as HCV genotype, with sustained viral response rates of 40 to 50% for genotype 1 and ϳ80% for genotypes 2 and 3 and intermediate response rates for patients infected with genotypes 4 to 6 (1, 3). Various genome regions were suspected to confer re...

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

“…Nevertheless, it should be noted that results obtained in these systems might be influenced by the presence of cell culture-adaptive substitutions. In general, except for certain JFH1-based chimeric genotype 2a or 2b recombinants (5,17,30,35,(40)(41)(42), HCV infectious culture systems depend on cell culture-adaptive substitutions (39). Such adaptive substitutions might facilitate the interaction of HCV proteins with host factors or of HCV proteins of different genotypes in the case of chimeric recombinants.…”

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

“…For example, of the genotype 2a recombinants studied, only the highly efficient genotype 2a (isolate JFH1) recombinant acquired A156V, and the fitness costs of T54A and A156S depended on the specific recombinant. We did not study NS4A, since it does not seem to influence sensitivity to PIs (67).…”

Hepatitis C Virus Genotype 7, a New Genotype Originating from Central Africa

“…It was found to successfully replicate in Huh7.5 cells, and its viability did not depend on adaptive mutations. More recently, this group of researchers developed J6/JFH1 recombinants expressing either the NS4A or NS5A proteins in Huh7.5 cells (14,15). When exposed to several protease inhibitors, interferon alfa2b, and a putative NS4A inhibitor, the genotype 7a-NS4A expressing recombinant responded in a manner similar to that of the genotype 1 to 6 NS4A-expressing recombinants (14).…”

“…More recently, this group of researchers developed J6/JFH1 recombinants expressing either the NS4A or NS5A proteins in Huh7.5 cells (14,15). When exposed to several protease inhibitors, interferon alfa2b, and a putative NS4A inhibitor, the genotype 7a-NS4A expressing recombinant responded in a manner similar to that of the genotype 1 to 6 NS4A-expressing recombinants (14). For the NS5A-expressing recombinants, mutations in the low-complexity sequence II region led to a high reduction in viral production for genotype 4a and 7a recombinants (15).…”