Huh7 and Huh7.5 cells were a gift from Jake Liang. Huh7, Huh7D, and Huh7.5 cells were grown in DMEM (Gibco) supplemented with 10% bovine calf serum (Atlanta Biologicals), L-glutamine (Gibco), penicillin and streptomycin (Gibco).
The JFH1 virus was a gift from Takaji Wakita. J6/JFH1 virus was obtained by transfection of Huh7D cells (see below) with in vitro transcribed HCV J6/JFH1 RNA. HCV J6/JFH1 RNA was obtained from plasmid pFL- J6/JFH1 (a gift from Charles Rice) that was cut with Xba1 and transcribed with T7 RNA polymerase (T7 Megascript AMBION).
Generation of HCV replicon containing the neomycin resistance gene ("HCV-neo-Rep")
HCV-neo-Replicon and replication-defective HCV replicon were obtained as previously described [18
]. Briefly, plasmid pFK i389neoNS3-3'/NK5.1 coding for a highly permissive HCV-neo replicon harboring several replication enhancing mutations [19
] and plasmid pFK i389neoNS3-3'/delta5B [18
] (kindly provided by Ralph Bartenschlager) were cut with restriction enzyme Sca1, and in vitro
transcribed using T7 Megascript kit (AMBION).
Generation of HCV replicon containing the hygromycin resistance gene ("HCV-hyg-Rep")
To obtain HCV replicons carrying the hygromycin resistance gene ("HCV-hyg-Rep") we replaced the neomycin resistance gene with the hygromycin resistance gene in plasmid pFK i389neoNS3-3'/NK5.1 using restriction enzymes Asc1 and Pme1. Hygromycin resistance gene was obtained by PCR using plasmid pIREShyg (Clontech) as template and sense oligo AACTAAAGGCGCGCCATGGATAGATCCGGAAAGCCTGAACTCAC (carrying the Asc1 restriction site) and anti-sense oligo AGTTATGGTTTAAACCTATTCCTTTGCCCTCGGACGAGTGCTGGG or anti-sense oligo AGTTATGGTTTAAACCTATTCCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGAACTTCTAC (both carrying the Pme1 restriction site). The later anti-sense oligo is designed to mutate the Sca1 restriction site present at the 3' end of the hygromycin resistant gene, without changing the coded amino-acid (restriction enzyme Sca1 is used to linearize the vector in order to make in vitro transcripts, see below). The PCR products were cut with restriction enzymes Asc1 and Pme1 and ligated to plasmid pFK i389neoNS3-3'/NK5.1 that was cut with same restriction enzymes to obtain pFK i389hygNS3-3'/NK5.1 and pFK i389hygscalessNS3-3'/NK5.1. The resultant recombinant plasmids were transformed into TOP10 competent bacteria (Invitrogen). Bacteria clones carrying the HCV-hyg-Replicons were confirmed by restriction analysis and sequencing. Plasmids pFK i389hygNS3-3'/NK5.1 (clones 2 and 3) were cut with restriction enzyme Spe 1 (generating a replicon with additional 4 nucleotides at the 3' end) and plasmids pFK i389hygscalessNS3-3'/NK5.1 (clones 1 and 8) were cut with Sca1 (to obtain a replicon with the authentic 3' end sequence), and in vitro transcribed using T7 Megascript kit (AMBION).
Generation of Huh7D cells
Huh7 cells grown in 12 well plates were transfected with approximately 100 or 200 ng of HCV-neo-replicon using as a facilitator 3 μl of lipofectamine (Invitrogen) in 200 μl of Optimem (Gibco). At 5 hours post transfection, medium was replaced with DMEM containing 10% fetal calf serum and antibiotics. Replicon harboring cells were selected with G-418 (Roche) at a concentration of 250 μg/ml for 20 days. Single cell clones obtained by end-point dilution were grown and tested by PCR and Southern blot for the (lack of) incorporation of the Neomycin resistance gene into the genome and by Northern blot for the presence of the RNA transcript corresponding to the replicon (not shown). Expression of HCV protein was assessed by immunfluorscence using an anti-NS5a antibody (not shown). Clone D, which had high levels of HCV protein, harbored the HCV replicon, and did not have the Neo gene integrated into the cellular genome, was selected for further analysis. Clone D was "cured" from the replicon using a strategy similar as the one previously described [6
]. Briefly, Clone D cells were passed four times at 7 or 8 day interval in absence of G-418 and treated with human Interferon (Sigma I2396) at a concentration of 100 IU/ml. After two weeks, cells were tested for the absence of the HCV replicon by RT-PCR and their susceptibility to G-418. The expected PCR band was not detected, and cells regained susceptibility to G-418 at a concentration of 250 μg/ml, which indicated that the Clone D cells were cured from the HCV replicon, and were named Huh7D cells.
Transfection of Huh7 cells with HCV replicons
Huh7, Huh7D, and Huh7.5 cells grown in 12 well plates were transfected with different amounts of HCV-neo- or HCV-hyg replicons using as a facilitator 3 μl of lipofectamine (Invitrogen) in 200 μl of Optimem (Gibco). At 5 hours post transfection, medium was replaced with DMEM containing 10% fetal calf serum and antibiotics. At 24 hours post transfection, medium was replaced with same medium containing G-418 (Roche) at a concentration of 250 μg/ml (for HCV-neo-replicon transfected cells) or hygromycin B (Roche) at a concentration of 65 μg/ml (for HCV-hyg-replicon transfected cells). To measure susceptibility to HCV replication, the HCV-neo-replicon transfected cells were fixed 13 or 15 days post transfection and stained with a solution of 50% methanol and 10% acetic acid containing 0.6 g/L of Comassie brilliant blue. The HCV-hyg-replicon transfected cells were split in medium containing 65 μg/ml hygromycin B, and colonies were stained with anti-HCV specific antibody as described below.
Detection of HCV antigen by immunfluorscence (IF)
Cells transfected with HCV-hyg-replicon or infected with HCV J6/JFH1 were fixed with methanol, blocked with a solution containing 1% BSA and 0.2% non-fat milk in 1 × PBS, treated with a 1:200 dilution in 0.05% tween 20 in 1 × PBS of a serum from a persistently infected chimpanzee that carried high levels of anti-HCV antibodies [20
] for 2 hours, washed with 1 × PBS, stained with FITC-conjugated goat anti-human antibody (KPL), washed, and observed in the microscope.
Infection of Huh7 cells with HCV J6/JFH1 and titration of progeny virus
Huh7, Huh7D, and Huh7.5 cells grown in 6-well plates were mock infected or infected with HCV-J6/JFH1 at an moi of 0.01. At 6 hours post infection, cells were washed three times with DMEM containing 10% FCS and split into 12-well plates (for titration of total progeny virus) and 96-well plates (for IF analysis, described above). At 0, 1, 3, 5, 7, and 10 dpi, the 12-well plates were frozen at -70°C. For later time points, cells in one 12-well plate were split and treated as described above. Total virus from each time point was recovered by freezing and thawing the cells 3 times. Viral titers were obtained in Huh7.5 cells infected with 10-fold serial dilutions of the cell extracts followed by detection of viral antigens by IF analysis at three days post-infection as described above.
Amplification and sequencing of RIG-I mRNA
Total RNA was extracted from Huh7, Huh7D, and Huh7.5 cells grown in T25 flasks using Trizol reagent as recommended by the manufacturer (Invitrogen). cDNA was synthesized using 4 μg of each RNA, SuperScript III reverse transcriptase (Invitrogen), and random primers. PCR amplification of RIG-I transcripts was performed using RIG-I specific primers RIG-I 91+ (5'-CTACCCGGCTTTAAAGCTAG-3 and RIG-I 3020- (5'-CGATCCATGATTATACCCAC-3'). Nested-PCR was performed using RIG-I-specific primers RIG-I 121+ (5'-CCTGCGGGGAACGTAGCTAG-3') and RIG-I 530- (5'-AATGATATCGGTTGGGATAA-3'), RIG-I 421+ (5'-CCATTGAAAGTTGGGATTTC-3') and RIG-I 1410- (5'-TGGCATCCCCAACACCAACC-3'), RIG-I 421+ and RIG-I 2990-(5'-TCTTCTCCACTCAAAGTTAC-3'). The Expand High Fidelity system (Roche) was used for PCR amplifications as described by the manufacturer. PCR products were run in agarose gels and purified using gene-elute agarose gel columns (Sigma) and sequenced (ABI-prism) using the above mentioned oligos and oligos RIG-I 474- (5'-GTAATCTATACTCCTCCAAC-3'), RIG-I 1331- (5'-AGATCAGAAACTTGGAGGAT-3'), RIG-I 2281+ (5'-AGTGCAATCTTGTCATCCTT-3'), and RIG-I 2360- (5'-TCTTGCTCTTCCTCTGCCTC-3').