Efficienttrans-Encapsidation of Hepatitis C Virus RNAs into Infectious Virus-Like Particles

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Abstract
Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based ontrans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteinstrans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, thesetrans-complemented HCV particles (HCVTCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCVTCPproduction was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCVTCPstocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 106tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excludingcis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCVTCPmay prove valuable for gene delivery or vaccination approaches.