Generation and Characterization of HCV Particles

In vitro transcription and RNA electroporation were performed as previously described [48 (link)]. Approximately 3 × 10⁶ Huh7.5.1 cells were electroporated with in vitro-transcribed RNAs derived from JC1 or JFH1-ad34-5A-Rluc containing the cell culture adapted mutations in the E2 and p7 proteins [45 (link)]. The culture supernatants were collected at 3–5 days after electroporation, and naïve Huh7.5.1 cells were infected with the generated viruses. The cell culture media of infected cells were collected at 3–5 days after infection. The HCV-containing supernatants were then filtered through with a 0.45 μm filter, and the filtrate was concentrated with a Vivaspin (100-kD cut-off; Millipore). The concentrated HCV culture medium was loaded onto a 20% sucrose cushion, and HCV particles were collected by ultracentrifugation at 4°C for 4 h at 36,000 rpm (SW41 rotor; Beckman). The viral pellets were resuspended with complete medium. Regarding the transient replication assay, in vitro transcripts of HCV subgenomic replicons were generated, purified, and electroporated into Huh7-Lunet/T7 cells. Firefly luciferase activities in cell lysates were measured as previously described [49 (link)].