Summary auto-generated
Researchers developed a transient-replication assay for hepatitis C virus (HCV) genotype 2a JFH1 strain using subgenomic replicons containing the firefly luciferase gene. In Huh-7 hepatoma cells, the JFH1 replicon replicated efficiently without requiring adaptive mutations, producing 200-400 fold higher luciferase activity than genotype 1b replicons by 48-72 hours post-electroporation. The increased luciferase activity correlated with accumulation of NS5A viral protein and replicon RNA. The replication was dose-dependently inhibited by interferon-alpha treatment, demonstrating the assay's ability to distinguish between translation and replication inhibition. Beyond Huh-7 cells, the U2OS human osteosarcoma cell line also supported robust JFH1 replication. This system offers significant advantages over existing genotype 1b replicon assays for quantitative measurement of viral RNA synthesis without selective pressure, providing a more robust tool for studying early-stage HCV replication and screening antiviral compounds.
Key findings
- JFH1 genotype 2a replicon replicates 200-400 fold more efficiently than genotype 1b replicons without requiring cell culture-adapted mutations
- Luciferase activity from JFH1 replicon correlated with NS5A protein and viral RNA accumulation, confirming genuine RNA replication
- HCV replication in JFH1 system was dose-dependently inhibited by interferon-alpha treatment
- U2OS non-hepatic osteosarcoma cells supported efficient JFH1 replication comparable to Huh-7 cells
- The transient assay enables rapid screening of antiviral compounds and cell lines supporting HCV replication without long-term selection pressure
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Abstract
Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.
Quantitative RT-PCR results are available as supplementary material in JGV Online.