Fig. 2.

Generation of an intergenotypic HCV chimera infectious for Huh7–Lunet cells. (A) The luciferase reporter virus genome based on the JFH1 isolate is shown at the top. The locations of the junction sites (designated C1–C6) selected to generate Luc–Con1/JFH1 chimeras are indicated in the topology model drawn below. Arrows refer to signalase cleavage sites. (B) Replication of chimeras in transfected Huh7–Lunet cells as determined by luciferase assays performed at 24, 48, 72, and 96 h posttransfection (white, light gray, dark gray, and black bars, respectively). Values were normalized for transfection efficiency by using the luciferase activity determined 4 h after transfection, which was set at 100%. (C) Release of core protein from Huh7–Lunet cells (B) 96 h posttransfection as determined by ELISA. Values were normalized for transfection efficiency and RNA replication by using the relative light units (RLU) determined 96 h after transfection. (D) Determination of infectivity released from transfected cells (B). Naïve Huh7–Lunet cells were inoculated with supernatants from cells harvested 96 h after transfection with genomes specified at the bottom. After 72 h infected cells were harvested and luciferase activity was determined. Values were normalized to RLU in transfected cells to exclude variations caused by different transfection and replication efficiencies. B–D show mean values of two independent experiments and the SEM.