HCV JFH-1 stocks and HCV infection
Preparation and titration of HCV JFH-1 virus was reported previously [24]. For examining anti-HCV effects, Huh7.5-IRF3ER cells were incubated with 0.5 MOI JFH-1 HCV for 14 days to achieve fully infected Huh7.5-IRF3ER monolayer cells [28]. The Huh7.5-IRF3ER cells were then treated with 4-HT for 72, 48 and 24 hours prior to collecting total cellular RNA. Huh7.5-IRF3ER cells without 4-HT treatment for 72 hours were used as control. Total RNA was isolated for detecting HCV RNA by real-time PCR.
Detection of IFN-α and IFN-β in Huh7.5-IRF3ER cells
Huh7.5-IRF3ER cells were treated with 4-HT for 72, 48, and 24 hours prior to collecting cellular lysates. Control is Huh7.5-IRF3ER cells that did not receive 4-HT treatment for 72 hours. Total cellular RNA was isolated for detecting IFN-α or IFN-β RNA by real-time PCR.
Real-Time PCR assay
Total cellular RNA was isolated from infected Huh7.5-IRF3ER monolayers by Trizol (Invitrogen). First-strand cDNA were synthesized from 1 μg total cellular RNA by reverse transcription (20 μl of reaction volume). Superscript II (200 U reverse transcriptase per reaction) and a RT-PCR kit (Invitrogen) was used to prime with oligo (dT) 12-18 for first-strand synthesis according to the manufacturer’s instructions. Taqman primers were obtained from Applied BioSystems. Reactions were conducted in a 96-well MyiQ cycler (Bio-Rad, Hercules, CA). Fluorescence was monitored during every PCR cycle at the annealing step. The primers for HCV JFH-1 are: forward, 5′-CGGAATTGCCGGGAAGAC-3′; reverse, 5′-CAAATGGCCGGGCATAG AG-3′; FAM probe, 5′-CTTTCTTGGATAAACCC-3′. The primers for IFN-α are: forward, 5′- GGGATGAGGACCTCCTAGACAAATT-3′; reverse, 5′- ACACAGGCTTCCAAGTCA TTC-AG-3′; FAM probe, 5′- CTGCACCGAACTCTAC-3′. The primers for IFN-β are: forward, 5′-TGGCTGGAATGAGACTATTGTTGAG-3′; reverse, 5′-CAGGACTGTCTTCA GATGG-TTTATCT-3′; FAM probe, 5′-CCTCCTGGCTAATGTC-3′. GADPH primers were purchased from the Applied Biosystems. PCR was performed with the following conditions: 50°C, 2 min; 95°C, 10 min; (95°C, 15s; 60°C, 1 min) for 40 cycles. Relative RNA level indicates statistical quantification of altered RNA levels from these cellular lysates with different primers. Samples were run in triplicate and the results were analyzed using the Bio-Rad iQ5 software; means ± the standard error of the mean are shown.
Luciferase assays
Huh7.5-IRF3ER cells were cultured in 6-well plates and transfected with the plasmid pRL-HL and lipofectamine 2000 (Invitrogen). After 24-hours of transfection, Huh7.5-IRF3ER cells were treated with 4-HT for 96, 72, and 48 hours prior to preparing cell lysates. Control Huh7.5-IRF3ER cells were incubated for 96 hours in the absence of 4-HT. All samples were analyzed for luciferase activity using the Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI) in triplicate. The translation efficiency was calculated as a proportion of control (100%).
Statistical analysis
Different cellular lysates were collected for analysis of luciferase activity or relative RNA level from Huh7.5-IRF3ER cells with special treatment. Results of these studies are expressed as means ± standard deviation (SD).