Abstract
Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5′ and 3′ termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes.
Highlights
hepatitis C virus (HCV) infects 2–3% of the world population
We established a new cell culture model for producing infectious HCV in a cell line stably bearing a subgenomic replicon from West Nile virus that circumvents the requirement for HCV RNA replication
And release of HCV particles by baby hamster kidney (BHK)-West-Nile virus (WNV) cells In initial analyses of the possible effects of flavivirus replicons on HCV virus particle production from proteins provided in trans, we observed that release of HCV structural proteins was dramatically enhanced in BHK-21 cells carrying a lineage II WNV subgenomic replicon [32] compared to parental cells; a less pronounced increase was observed in the cell lysate (Fig. 1A)
Summary
HCV infects 2–3% of the world population. A majority of infected people fail to clear the virus and are at risk for developing serious liver complications (reviewed in [1]). The limited experimental availability of chimpanzees, the primary animal model for HCV [3,4], and difficulties encountered in reproducing true infection in small animals have significantly limited the use of in vivo models to study the biology of this virus. The natural target cells of HCV are primarily hepatocytes in the liver, in vitro most human hepatic cells poorly propagate HCV isolates from patients Subgenomic replicons (i.e. without structural genes) of subtypes 1b [7,8] and 1a [9] were established in selected subclones of the human hepatic Huh-7 cell line that are highly permissive for HCV replication, e.g. Huh-7.5 cells [10]. A full infectious cycle was reproduced in cell culture with JFH-1, a particular strain of genotype 2a [11,12], or with a J6/JFH-1 chimera [13]; the released particles are referred to as HCVcc
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