Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Figure 1
Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.
(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of in vitro transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P [27]. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.