Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Figure 6
In vitro infectivity of Con1/wt particles released from transfected Huh7.5 cells.
(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect in vitro infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.