Figure 1.
Generation of cell culture-adapted JFH 5a-GFP virus.
(A) Cell-free culture supernatants were collected 6 days (JFH 5a-GFP pt6d) and 20 days (JFH 5a-GFP pt20d) after transfection, and used to inoculate Huh7.5.1 cells. HCV-infected cells were fixed 5 days after inoculation and then treated with primary (anti-core monoclonal) and secondary (Alexa 555-conjugated donkey anti-mouse IgG) antibodies. The core-expressing cells are shown in red and the Hoechst 33258-stained nuclei are shown in blue. (B) Schematic diagram of JFH 5a-GFP and its derivatives with a substituted region (from Age I site to Avr II site) containing amplified DNA fragments from long distance RT-PCR of cell-adapted HCV RNA. (C) Huh7.5.1 cells transfected with JFH 5a-GFP, Ad 9, Ad 12 or Ad 16 RNAs were grown on coverslips for 3 days and fixed. Core-expressing cells are shown in red as in 1A (panels d to f). The NS5a-GFP signal was visualized by fluorescence microscopy (green). The nuclei, shown in blue, were stained with Hoechst 33258 (panels a to c). (D) Huh7.5.1 cells were infected with JFH 5a-GFP, Ad 9, Ad 12 or Ad 16 viruses. The NS5a-GFP signals were visualized by fluorescence microscopy (green). (E) Virus titers were determined using a TCID50 assay. The bars and lines represent the means and standard deviations, respectively, from three independent experiments.
Figure 2.
Genetic mutations identified in cell culture-adapted HCV RNAs.
A schematic diagram of JFH 5a-GFP is shown at top. The positions of predicted amino acid changes are depicted in bold characters, and those with silent mutations are depicted in plain characters.
Figure 3.
Mutations in E2 and p7 proteins increased viral infectivity.
(A) Schematic diagram of JFH 5a-GFP and its derivatives containing adaptive mutations. Mutations are indicated with an asterisk; dedicated names are on the left. (B) Western blot analysis of Huh7.5.1 cells transfected with JFH 5a-GFP or its derivative RNAs containing adaptive mutations. Protein levels were analyzed by western blotting with anti-NS5a, anti-core or anti-GAPDH antibodies, respectively. (C) Huh7.5.1 cells were infected with JFH 5a-GFP, Ad 9, JFH-G m1, JFH-G m2, JFH-G m3, JFH-G m4 or JFH-G m5 viruses, and the NS5a-GFP signal was visualized by fluorescence microscopy (green). (D) Viral titers were determined using a TCID50 assay. The bars and lines represent the means and standard deviations, respectively, from three independent experiments.
Figure 4.
A mutation in the p7 protein augmented a virion assembly step.
(A) Replication of JFH 5a-Rluc, JFH-R m2, JFH-R m3 and JFH-R m4 RNAs was investigated using a luciferase reporter assay. Cells were transfected with RNAs and harvested at the indicated times. Luciferase activity values are expressed relative to reporter activity measured at 4 hours (reference value = 1). The bars and lines represent the means and standard deviations, respectively, from three independent experiments. (B) Infectivity of culture fluids harvested 5 days after transfection with JFH 5a-Rluc, JFH-R m2, JFH-R m3 or JFH-R m4 RNAs was determined by measuring luciferase activity in cells 3 days after inoculation of Huh7.5.1 cells. Luciferase activity values are expressed relative to reporter activity measured in cells infected with the JFH 5a-Rluc virus (reference value = 1). The bars and lines represent the means and standard deviations, respectively, from three independent experiments. (C) The kinetics of virus release into the media of transfected cells in panel A were measured. Culture supernatants were harvested at the indicated times and used to inoculate Huh7.5.1 cells. Three days after infection, luciferase activity in cells was measured and normalized to protein concentration, determined by the Bradford assay. The bars and lines represent the means and standard deviations, respectively, from three independent experiments. (D) Two days after transfection, culture media were harvested to collect extracellular virus. Virus-producing cells were washed and lysed by four freeze-thaw cycles to release intracellular virus. Infectious virus titers in the media and inside the cells were investigated by infecting Huh7.5.1 cells with media and cell lysates, respectively. The extracellular (white bars) and intracellular (gray bars) viral titers were measured by luciferase assay. The relative ratios of intracellular and extracellular infectious viruses are shown in panel (E). The bars and lines represent the means and standard deviations, respectively, from three independent experiments.
Figure 5.
A mutation in the E2 protein increased virion viability at 37°C.
(A and B) Infectious viral titers remaining in the media were determined by infecting Huh-7.5.1cells after same amount of JFH, JFH-m2, JFH-m3 and JFH-m4 viruses were incubated at 4°C (A) or 37°C (B) for the indicated times. Viral titers were determined by counting numbers of plaques visualized by an immune-fluorescence method. (C) The same infectious dose of JFH, JFH-m2, JFH-m3, and JFH-m4 viruses were incubated at 37°C for the indicated times, and then CD81-interacting viruses were precipitated by a protein G agarose resin conjugated with CD81-Fc fusion protein. The amounts of resin-bound virions were measured by quantitative RT-PCR. The relative amount of resin-bound viruses is depicted. The dots and lines represent the means and standard deviations, respectively, from three independent experiments. (D) The relationship between CD81-binding capability and infectivity of HCV viruses. The relationship between CD81-binding capability and infectivity of the mutant viruses were analyzed by plotting the relative CD81-binding capability of various viral stocks in panel (C) on the x-axis and relative viral titers of the stocks in panel (B) on the y-axis. The correlation co-efficient was calculated using sigma plot.
Figure 6.
Processing of the E2-P7-NS2 polypeptides of JFH 5a-GFP, JFH-G m2, JFH-G m3 and JFH-G m4 viruses.
Huh7.5.1 cells were transfected with the indicated RNAs and then incubated for 48 hours. Newly synthesized proteins were labeled with [35S]methionine/cysteine for 24 hours and then chased with unlabeled media for 0, 3 or 6 hours. Mock-transfected cells were used as a negative control. The labeled cell lysates were immunoprecipitated using an E2 specific antibody (AP33). Immunocomplexes were resolved by SDS-PAGE and E2-related proteins were visualized by autoradiography. HCV proteins are indicated by arrowheads on the left, and the positions of the molecular weight markers are given on the right. The relative amounts of E2/E2p7, which were normalized to those in starting time point (t0), are depicted below the figure.