Elsevier

Antiviral Research

Volume 100, Issue 2, November 2013, Pages 439-445
Antiviral Research

In vitro efficacy of approved and experimental antivirals against novel genotype 3 hepatitis C virus subgenomic replicons

https://doi.org/10.1016/j.antiviral.2013.08.018Get rights and content

Highlights

  • We establish a novel genotype 3a replicon that encodes a luciferase reporter.

  • Unique adaptive mutations and Huh7-derived cell clones are identified.

  • The replicon replicates robustly in both stably-selected and transiently-transfected cells.

  • Antiviral EC50 values are reproducibly determined in 384-well assays.

  • This new replicon is aiding the development of pan-genotypic HCV therapies.

Abstract

Infection with genotype 3 hepatitis C virus (HCV) is common throughout the world, however no direct-acting antiviral (DAA) has been approved to treat this genotype. We therefore attempted to develop novel genotype 3 replicons to facilitate the discovery and development of new HCV therapies. A novel Huh-7-derived cell line 1C but not Lunet cells enabled the selection of a few stable colonies of a genotype 3a subgenomic replicon (strain S52). Genotypic analysis revealed a mutation of P89L in the viral NS3 protease domain, which was confirmed to enhance genotype 3a RNA replication and enable the establishment of highly replicating luciferase-encoding replicons. Secondary adaptive mutations that further enhanced RNA replication were identified in the viral NS3 and NS4A proteins. In addition, cell lines that were cured of genotype 3a replicons demonstrated higher permissiveness specifically to genotype 3a HCV replication. These novel replicons and cell lines were then used to study the activity of approved and experimental HCV inhibitors. NS3 protease and non-nucleoside NS5B polymerase inhibitors often demonstrated substantially less antiviral activity against genotype 3a compared to genotype 1b. In contrast, nucleoside analog NS5B inhibitors and host-targeting HCV inhibitors showed comparable antiviral activity between genotypes 3a and 1b. Overall, the establishment of this novel genotype 3a replicon system, in conjunction with those derived from other genotypes, will aid the development of treatment regimens for all genotypes of HCV.

Introduction

Chronic hepatitis C virus (HCV) infection represents a major unmet medical need with an estimated 170 million people infected worldwide (Lavanchy, 2011). Genotype 1 HCV is generally predominant throughout the world (Cornberg et al., 2011), but genotype 3 is also common in many regions. Genotype 3 accounts for more than 50% of HCV infections in India and Thailand, and approximately 30% in Australia and many European countries, including Great Britain, Germany, and Russia (Cornberg et al., 2011). Furthermore, genotype 3 HCV infection is associated with higher incidence of liver steatosis and hepatocellular carcinoma (Nkontchou et al., 2012).

Currently, the standard treatment for HCV genotype 3 patients is pegylated interferon and ribavirin for 24–48 weeks (Ghany et al., 2009), which has better efficacy in genotype 3 infection than genotype 1 but remains only partially efficacious with poor tolerability. Neither boceprevir nor telaprevir has been approved to treat genotype 3 HCV infection (Foster et al., 2011). Many HCV inhibitors are in advanced clinical development, however, the majority is being developed to treat genotype 1 infections. Thus, there is a clear need to develop novel therapeutic agents to treat chronic genotype 3 HCV infection, which also aligns with the increasing interest in developing next-generation “pan-genotypic” treatments for HCV (Gane et al., 2012).

The slow advances in treatment for genotype 3 HCV are, in part, due to lack of efficient in vitro tools. HCV replicons are self-replicating RNA sequences derived from the HCV genome (Bartenschlager, 2005). These replicons have been crucial in multiple aspects of drug discovery and development including the identification of novel classes of inhibitors and the characterization of clinical resistance. Despite successes in generating replicons derived from genotypes 1a, 1b, or 2a (Bartenschlager, 2005), generation of efficiently replicating RNAs from other genotypes has proven difficult. Here we report the isolation of novel genotype 3a replicons that efficiently replicate in cell culture. We demonstrate that robust replication requires the adaptive mutation P89L in the NS3 protease domain, which can be further augmented by mutations in NS3 and NS4A. Combining selected adaptive mutations with a novel host cell line cured of genotype 3 replicons yielded highly efficient replication of genotype 3a HCV RNA in both transient-transfected cells and stably replicating luciferase-encoding replicon cell lines. These systems support efficient potency profiling of compounds in high-throughput antiviral assays. Notably, differential antiviral activity was observed with multiple classes of DAAs against genotype 3a compared to genotype 1b. The novel replicons and permissive cell lines described here provide valuable tools for drug development and molecular virology studies of genotype 3 HCV.

Section snippets

Cell culture

Huh7 Lunet cells and 51C cells were described previously (Robinson et al., 2010). 1C cells were derived by curing an NS5A inhibitor GS-5885-resistant genotype 1a replicon clone derived from 51C cells, and showed much higher permissiveness to genotype 1a replicon replication (Peng et al., 2013). Replicon cells were cured to establish 1C cells and other cell lines as described in the Supplementary material. All cell lines were propagated in DMEM medium as described previously (Cheng et al., 2011).

Construction and selection of a subgenomic genotype 3a replicon

A subgenomic genotype 3a replicon was constructed as previously described by Lohmann et al. (Lohmann et al., 1999) and based on the consensus sequence (GenBank accession #GU814263) of the genotype 3a S52 strain (Fig. 1A) (Gottwein et al., 2010). To enhance the basal level of replication, the NS5A mutation S232I (equivalent to the major adaptive mutation S2204I in genotype 1 polyprotein) was incorporated (Blight et al., 2003).

In vitro-transcribed replicon RNA was electroporated into Huh7 Lunet,

Discussion

Infection with genotype 3 HCV is common throughout the world. Here we report the establishment of robust genotype 3a replicons to aid the discovery and development of new therapies for genotype 3 HCV infection. Using a novel permissive cell line (1C), stable genotype 3a colonies were identified, which revealed the key adaptive mutation, P89L in the NS3 protease. As a result, we were able to establish stable genotype 3a luciferase-encoding replicon cell lines, with persistent and robust HCV RNA

Acknowledgments

We are grateful to Huiling Yang and Matthew Paulson for suggestions on designing the replicon constructs.

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