Article Figures & Data
Figures
- FIG. 1.
Determination of the nucleotide sequences at the 5′-and 3′ ends of HCV RNA produced by the Pol I system. (A and B) 5′RACE and sequence analysis. A synthesized RNA adapter was ligated to RNA extracted from cells transfected with pHHJFH1. The positive-strand HCV RNA was reverse transcribed, and the resulting cDNA was amplified by nested PCR. The amplified 5′-end cDNA was separated by agarose gel electrophoresis (A), cloned, and sequenced (B). (C and D) 3′RACE and sequence analysis. RNA extracted from pHHJFH1-transfected cells, the culture supernatant of transfected cells, and the culture supernatant of H751JFH1/Zeo cells were polyadenylated, reverse transcribed, and amplified by PCR. The amplified 3′-end cDNA was separated by agarose gel electrophoresis (C), cloned, and sequenced (D).
- FIG. 2.
HCV RNA replication and protein expression in cells transfected with Pol I-driven plasmids. (A and B) Assessment of HCV RNA replication by RPA. Pol I-driven HCV-expression plasmids were transfected into Huh-7 cells. Total RNA was extracted from the cells on day 5 p.t. and positive (A)- and negative (B)-strand HCV RNA levels were determined by RPA as described in Materials and Methods. In the RNase (−) lanes, yeast RNA mixed with RNA probes for HCV and human β-actin were loaded without RNase A/T1 treatment. In the yeast RNA lanes, yeast RNA mixed with RNA probes for HCV and human β-actin were loaded in the presence of RNase A/T1. (C) Northern blotting of total RNAs prepared from the transfected cells. Huh-7 cells transfected with pHHJFH1 or pHHJFH1/GND were harvested for RNA extraction through days 1 to 4 p.t. Control RNA, given numbers of synthetic HCV RNA; Huh-7, RNA extracted from naive cells. Arrows indicate full-length HCV RNA and β-actin RNA. (D) HCV protein expression in the transfected cells. Pol I-driven HCV-expression plasmids were transfected into Huh-7 cells, harvested, and lysed on day 6 p.t. The expression of NS5B, core, and GAPDH was analyzed by Western blotting as described in Materials and Methods. The asterisk indicates nonspecific bands.
- FIG. 3.
HCV released from cells transfected with Pol I-driven plasmids. (A) HCV particle secretion from the transfected cells. The culture supernatant of Huh-7 cells transfected with Pol I-driven plasmids containing wild-type or mutated HCV genome were harvested on days 2, 4, and 6 and assayed for HCV core protein levels. The data for each experiment are averages of triplicate values with error bars showing standard deviations. (B) Sucrose density gradient analysis of the culture supernatant of pHHJFH1-transfected cells. Culture supernatant collected on day 5 p.t. was cleared by low-speed centrifugation, passed through a 0.45-μm-pore-size filter, and concentrated ∼30-fold by ultrafiltration. After fractionating by sucrose density gradient centrifugation, the core protein level and viral infectious titer of each fraction were measured. (C) Kinetics of core protein secretion from cells transfected with pHHJFH1 or with JFH-1 genomic RNA. A total of 106 Huh-7 cells were transfected with 3 μg of pHHJFH1 or the same amount of in vitro-transcribed JFH-1 RNA by electroporation. The cells were passaged every 2 to 3 days before reaching confluence. Culture supernatant collected on the indicated days was used for core protein measurement. The level of secreted core protein (pmol/liter) is expressed on a logarithmic scale. The data for each experiment are averages of triplicate values with error bars showing standard deviations.
- FIG. 4.
Indirect immunofluorescence analysis of H751JFH1/Zeo cells. (A and B) H751JFH1/Zeo cells (A) and parental Huh7.5.1 cells (B) were immunostained with an anti-NS5A antibody. (C to F) The subcellular colocalization of de novo-synthesized HCV RNA and NS5A in H751JFH1/Zeo cells was analyzed. The cells were stained with DAPI (C), an anti-bromodeoxyuridine antibody (D), and an anti-NS5A antibody (E). The merge panel is shown in panel F.
- FIG. 5.
Loss of CD81 expression in H751JFH1/Zeo cells. The cell surface expression of CD81 on Huh7.5.1 cells (A), H751JFH1/Zeo clone H751-1 (B), and clone H751-50 (C) was analyzed by flow cytometry after being stained with anti-CD81 antibody.
- FIG. 6.
Genome mutations of HCV secreted from H751JFH1/Zeo cells. (A) RT-PCR of HCV genome extracted from the culture supernatant of H751JFH1/Zeo cells. Viral RNA sequences were reverse transcribed [RT (+)] or not [RT (−)], followed by amplification with primer pairs encompassing the specified HCV genome regions. (B) Comparison of the genome mutations of HCV secreted from H751JFH1/Zeo cells cultured for 120 days (upper panel) and JFH-1 RNA-transfected cells cultured for 36 days (lower panel). The positions of original (wild-type) and mutated (day 120, day 36) nucleotides are indicated under the schematic diagram of the HCV genome. Amino acid residues and their positions are marked in parentheses. Del, deletion.
- FIG. 7.
Effects of glycosylation inhibitors on HCV production from H751JFH1/Zeo cells. (A and B) Effects of glycosylation inhibitors on the secretion of HCV core protein. H751JFH1/Zeo cells were seeded at a density of 1 × 104 cells/well in a 96-well culture plate (A) or 3 × 104 cells/well in a 12-well cell culture plate (B). One day later, each compound was added to the cell culture supernatant at the indicated concentrations. The culture supernatant was collected after a further 3-day culture and processed by core protein-specific ELISA. The control represents an untreated cell culture. The level of secreted core protein was normalized by setting the control value at 100%. The data for each experiment are averages of triplicate values with error bars showing standard deviations. (C) Effects of NN-DNJ and KIF on infectious HCV production. The culture supernatant obtained in panel B was used to infect naive Huh7.5.1 cells. At 72 h after infection, the inoculated cells were fixed and immunostained as described in Materials and Methods for titration of virus infectivity. The infectious titer was normalized by setting the control value at 100%. Cells were treated with INF-α at 100 IU/ml as a positive control. The data for each experiment are averages of triplicate values with error bars showing standard deviations. The control represents an untreated cell culture. (D) After 1 year of culturing H751JFH1/Zeo cells, antiviral effects of NN-DNJ and BILN 2061 were evaluated. H751JFH1/Zeo cells were seeded at a density of 3 × 104 cells/well in a 12-well cell culture plate. One day later, each compound was added to the cell culture supernatant at the indicated concentrations. The culture supernatant was collected after a further 3-day culture and processed by core protein-specific ELISA. The control represents an untreated cell culture. The level of secreted core protein was normalized by setting the control value at 100%. The data for each experiment are averages of triplicate values with error bars showing standard deviations.
- FIG. 8.
Establishment of a trans-packaging system involving two-plasmid transfection. (A) Schematic representation of the plasmids used for the production of HCV-LP. HCV polyproteins are indicated by the open boxes. Bold lines indicate the HCV UTR. EMCV IRES is denoted by gray bars. The firefly luciferase gene (F Luc) is depicted as a gray box. CAG, CAG promoter; Pol I P, Pol I promoter; dC, 5′ region of Core gene; Pol I T, Pol I terminator. (B) Luciferase activity in Huh7.5.1 cells inoculated with culture supernatant from cells transfected with the indicated plasmids. Luciferase activity is expressed in terms of relative luciferase units (RLU). The data for each experiment are averages of triplicate values with error bars showing standard deviations. (C) Culture supernatant from cells cotransfected with pHH/SGR-Luc and the indicated plasmids were collected 4 days p.t. The luciferase activity in Huh7.5.1 cells inoculated with culture supernatant was determined 3 days postinoculation and expressed as relative luciferase units (RLU). The RLU was normalized according to the luciferase activity observed in the pCAG/C-NS2-transfected sample (C-NS2), which was set at 100%. The data for each experiment are averages of triplicate values with error bars showing standard deviations. (D) Huh7.5.1 cells were inoculated with HCVLP in the absence (−) or presence of 5 μg of anti-CD81 or anti-FLAG antibody/ml. The luciferase activity was determined 72 h postinoculation and is expressed as relative luciferase units (RLU). The RLU was normalized to the level of luciferase activity observed in the antibody-untreated sample (−), which was set at 100%. The data for each experiment are averages of triplicate values with error bars showing standard deviations.
Tables
- TABLE 1.
Oligonucleotides used for RT-PCR and RACE of the JFH-1 genome
Method or segment Oligonucleotide Sequences (5′-3′) 5′RACE RT GTACCCCATGAGGTCGGCAAAG 45-nt RNA adapter GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA 5′RACEouter-S GCTGATGGCGATGAATGAACACTG 5′RACEouter-R GACCGCTCCGAAGTTTTCCTTG 5′RACEinner-S GAACACTGCGTTTGCTGGCTTTGATG 5′RACEinner-R CGCCCTATCAGGCAGTACCACAAG 3′RACE CAC-T35 CACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 3X-10S ATCTTAGCCCTAGTCACGGC nt 129-2367 44S (1st PCR) CTGTGAGGAACTACTGTCTT 2445R TCCACGATGTTCTGGTGAAG 17S (2nd PCR) CGGGAGAGCCATAGTGG 2367R CATTCCGTGGTAGAGTGCA nt 2285-4665 2099S (1st PCR) ACGGACTGTTTTAGGAAGCA 4706R TTGCAGTCGATCACGGAGTC 2285S (2nd PCR) AACTTCACTCGTGGGGATCG 4665R TCGGTGGCGACGACCAC nt 4574-7002 4547S (1st PCR) AAGTGTGACGAGCTCGCGG 7027R CATGAACAGGTTGGCATCCACCAT 4594S (2nd PCR) CGGGGTATGGGCTTGAACGC 7003R GTGGTGCAGGTGGCTCGCA nt 6949-9634 6881S (1st PCR) ATTGATGTCCATGCTAACAG 3X-75R TACGGCACTCTCTGCAGTCA 6950S (2nd PCR) GAGCTCCTCAGTGAGCCAG 3X-54R GCGGCTCACGGACCTTTCAC
