Characterization of the Early Steps of Hepatitis C Virus Infection by Using Luciferase Reporter Viruses

Abstract

The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Next, it is important to ensure that the luciferase activity values obtained are in the linear range of the assay and in particular do not reach saturation due to the luminometer properties or to a too high ratio of infected cells. On the opposite, luciferase reporter viruses might yield high signals despite a low number of infection events [69], and therefore, one should be careful that the signals recorded represent a sample of virus that is large enough to be representative. These issues can be tackled by titering beforehand the virus preparations when generating viruses from a new luciferase reporter construct, with the TCID 50 or FFU assays.…”

“…A further development to the HCVcc system is the incorporation of reporter genes that allow easy and rapid quantification of infectivity [69,70]. The main reporter viruses are listed in Figure 3.…”

“…However, such modified viruses are often not as stable as the original virus in cell culture which might lead to the loss of the reporter gene expression. They also often do not spread efficiently in cell culture [69]. These tools are therefore extremely useful for transient assays but not appropriate to follow the virus spread and evolution in culture along a long time period [63].…”

“…Reporter protein Abs [109,137], ITX 5061 [134], natural ligands [135,136] Abs, soluble CD81 [9,11] Abs [142] Cldn1 peptide [143] Heparin, GAG nzymatic digestion [69,132] Abs, natural ligands, soluble LDL receptor [30,133] GAGs LDLR CD81 SR-BI Cldn1 Ocln…”

“…Anti-E1/E2 Abs [9,11,62,148], patient sera [9], soluble E2 [46], lectins [150,151], anti-ApoE antibodies [29], apoE peptides [149] Abs, Erlotinib, Lapatinib, Gefitinib [141] EGF, TGF-α [141] HDL [138] ApoCI [139] BLTs [138,140] Abs, Dasatinib [141] Arbidol [144] Abs [109,137], ITX 5061 [134], natural ligands [135,136] Abs, soluble CD81 [9,11] Abs [142] Cldn1 peptide [143] Heparin, GAG nzymatic digestion [69,132] Abs, natural ligands, soluble LDL receptor [30,133] GAGs LDLR CD81 SR-BI Cldn1 Ocln…”