HCV Replicon Systems: Workhorses of Drug Discovery and Resistance

Abstract

The development of direct-acting antivirals (DAAs) has revolutionized the state-of-the art treatment of HCV infections, with sustained virologic response rates above 90%. However, viral variants harboring substitutions referred to as resistance-associated substitutions (RASs) may be present in baseline levels and confer resistance to DAAs, thereby posing a major challenge for HCV treatment. HCV replicons have been the primary tools for discovering and evaluating the inhibitory activity of DAAs against viral replication. Interest in replicon systems has further grown as they have become indispensable for discovering genotype-specific and cross-genotype RASs. Here, we review functional replicon systems for HCV, how these replicon systems have contributed to the development of DAAs, and the characteristics and distribution of RASs for DAAs.

Keywords: direct-acting antiviral; drug discovery; drug resistance; genotype; hepatitis C virus; replicon; resistance-associated substitution.

Figure 1

HCV genome organization and direct-acting antivirals for the treatment of HCV Infection. The HCV genome is consisted of single open reading frame (ORF) that is flanked by 5' and 3' non-translated regions (NTRs). The IRES present at the 5' NTR mediates the translation of the ORF leading to the formation of a polyprotein, which is further processed into three structural (core, E1 and E2) and seven nonstructural proteins (P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The NS3-to-NS5B coding region is the minimal sequence required for RNA replication (Lohmann et al., 1999). Recommended direct-acting antivirals are listed below and include the NS3/4A (or protease; names end in -previr) inhibitors, the NS5A inhibitors (names end in -asvir), and the NS5B (or polymerase) inhibitors of nucleoside and non-nucleoside (names end in -buvir).

Figure 2

Schematic comparison of different HCV replicons. (A) Classical bicistronic subgenomic replicon comprised 5' NTR, first 16 codons of core, neo gene, a selectable marker followed by EMCV IRES, HCV NS3-to-NS5B coding region and HCV 3' NTR; Other selectable markers less widely used are hygro and pac. Modified bicistronic replicons may carry either: (B) a Renilla luciferase (RLuc) or firefly luciferase (FLuc) (reporter gene) instead of a selectable marker, (C) a GFP at certain positions within NS5A, (D) a reporter gene that is fused with a selectable marker, (E) ubiquitin, a host cell cleavable sequence fused in-frame between a reporter and selectable marker, (F) a non-structural sequence derived from patient in Con1 or JFH-1 replicons genetic background or (G) an entire HCV ORF, core-to-NS5B and 3' NTR. Monocistronic replicons are composed of (H) HCV 5' NTR and first 12 codons of core followed by NS3-to-NS5B coding sequences and 3' NTR and (I) ubiquitin in-frame with either a reporter gene or selection marker. NTR, non-translated region; Ubi, ubiquitin, rg, reporter gene; sm, selection marker; neo, gene encoding neomycin phosphotransferase; hygro, hygromycin phosphotransferase; PAC, puromycin N-acetyltransferase; c, core; EMCV IRES, internal ribosomal entry site from encephalomyocarditis virus; GDD, amino acid motif in the catalytic site of RNA polymerse; GFP, green fluorescent protein. Viral and non-viral coding sequences are marked by rectangles; non-coding regions are indicated by horizontal lines.

Figure 3

Phenotypic assay of HCV resistance to antivirals. Bicistronic replicons described in Figure 2D are the most widely used systems for determination of phenotypic direct-acting antiviral resistance. Replicon RNAs from wild type or mutant are synthesized by in vitro transcription of linearized plasmids harboring HCV replicon, which are then transfected into hepatoma cells, subsequently subjected to G418 selection pressure for 3–4 weeks. Cells harboring active replicon RNAs become resistance to G418 due to expression of neo gene, whereas untransfected cells or cells that do not support RNA replication will be eliminated during the process. After the establishment of stable cell line supporting HCV autonomous replication, cells are subjected to increasing antiviral concentrations based on of EC₅₀ or EC₉₀ values. The level of susceptibility is evaluated by measuring the luciferase activity at 24, 48, and 72 h after treatment, in comparison to its wild type counterpart, which is commonly expressed as fold resistance. Luc-Neo, fused luciferase and neo gene encoding neomycin phosphotransferase; c, core; EMCV IRES, internal ribosomal entry site from encephalomyocarditis virus. Viral and non-viral coding sequences are marked by rectangles; non-coding regions are indicated by horizontal lines.