Figure 1.

Transient replication of JFH1 RNA in transfected Huh7 cells. (a) Organization of the full-length HCV construct pJFH1. Open reading frames (thick boxes) are flanked by the 5′- and 3′-UTRs (thin boxes). T7, T7 RNA polymerase promoter; GDD, active-site motif of NS5B polymerase; XbaI, restriction site. (b) Northern blot analysis of total RNA prepared from cells transfected with full-length JFH1 and JFH1/GND RNA. Control RNA, given numbers of synthetic HCV RNA; Huh7, RNA isolated from naive cells. Arrowheads indicate full-length HCV RNA and 28S ribosomal RNA (28S). Upper panel, northern blot; lower panel, ethidium bromide staining. (c) Western blot analysis of transfected cells for HCV proteins NS5A, NS3 and core. Lysates of SGR/JFH1-RNA⁸ or pEF/Core²⁹ DNA– transfected Huh7 cells and naive Huh7 cells served as positive and negative controls, respectively. (d) Immunofluorescence assay of cells fixed 72 h after transfection with JFH1 or JFH1/ΔE1-E2 RNA. Magnification, ×200. (e) HCV core protein secretion into culture medium. HCV RNAs were transfected into Huh7 cells, culture media were harvested at given time points, and HCV core protein concentrations were determined.