An HCV Renilla luciferase (HCV RLuc) reporter construct was used to measure the effect of each compound on cellular HCV RNA levels. The replicon was a generous gift from Seng-Lai Tan. In HCV RLuc, the HCV internal ribosome entry site (IRES) drives the translation of the neomycin and Renilla luciferase genes while the HCV nonstructural proteins (NS3 to NS5B) are translated from the Encephalomyocarditis virus IRES.29 (link) The plasmid DNA was cleaved with Sca I, purified by phenol/chloroform extraction followed by ethanol precipitation, and used as template for RNA transcription using MEGAscript™ T7 RNA transcription kit (Ambion, Austin, TX). The RNA transcripts were treated with 2 U DNase I (Ambion) at 37 °C for 30 min, purified by acid phenol/chloroform extraction, followed by isopropanol precipitation, and suspended in diethylpyrocarbonate-treated water. RNA concentration was determined by spectrophotometry by measuring the OD260. RNA integrity and size was checked on 1% agarose gel. Transcribed RNA was stored in aliquots at −80 °C until needed.
Huh-7.5 cells RNA were transfected with HCV RNA by electroporation. Briefly, subconfluent Huh7.5 cells were trypsinized, suspended in complete growth medium, and centrifuged at 1,000 × g for 5 min at 4 °C. The cell pellets were then washed twice with ice-cold phosphate-buffered saline (PBS) and suspended at 1.75 × 107 cells/mL in ice-cold PBS. Replicon RNA (5 μg) was mixed with 0.4 mL of cell suspension and transferred to 2 mm gap width electroporation cuvette (Eppendorf AG, Germany) and pulsed with 5 times for 99 μsec at 820 V over 1.1 sec intervals using the ECM 830 electroporator instrument (BTX Havard Apparatus, Holliston, MA). After 5 min recovery period at room temperature, cells transferred to 10 ml complete growth medium, and seeded into 10 cm diameter cell culture dishes. Twenty-four hours after transfection, the medium was replaced with fresh complete DMEM supplemented with 1 mg/ml geneticin (Invitrogen) and the medium was replaced every three to four days with fresh medium containing 1 mg/mL geneticin. Geneticin-resistant colonies were selected for a period of two weeks and expanded in the presence of 250 μg/mL geneticin.
HCV RLuc replicon cells were seeded at a density of 10 × 103 cells per well in 96-well plates and incubated for 4–5 h to allow the cells to attach to the plate. The compounds dissolved in dimethyl sulfoxide (DMSO) were added at a final concentration of 10 μM (DMSO solvent final concentration was 0.5%) and the cells were incubated for 72 h at 37 °C under 5% CO2 atmosphere. The effects of compounds on HCV replication were then assessed by measuring the Renilla luciferase activity in compound-treated versus DMSO-treated cells. At the end of the incubation period, the medium was aspirated and the cells were washed with 1 × PBS. The Renilla luciferase reporter gene assay was performed using the Renilla luciferase assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, the cells were lysed by addition of 50 μL of 1× Renilla luciferase lysis buffer followed by two cycles of freeze/thaw. The luciferase activity content of the lysate was measured with a FLUOstar Omega microplate reader instrument (BMG Labtech, Germany) after injecting 50 μL of luciferase substrate and reading for 5 s.
Huh-7.5 cells RNA were transfected with HCV RNA by electroporation. Briefly, subconfluent Huh7.5 cells were trypsinized, suspended in complete growth medium, and centrifuged at 1,000 × g for 5 min at 4 °C. The cell pellets were then washed twice with ice-cold phosphate-buffered saline (PBS) and suspended at 1.75 × 107 cells/mL in ice-cold PBS. Replicon RNA (5 μg) was mixed with 0.4 mL of cell suspension and transferred to 2 mm gap width electroporation cuvette (Eppendorf AG, Germany) and pulsed with 5 times for 99 μsec at 820 V over 1.1 sec intervals using the ECM 830 electroporator instrument (BTX Havard Apparatus, Holliston, MA). After 5 min recovery period at room temperature, cells transferred to 10 ml complete growth medium, and seeded into 10 cm diameter cell culture dishes. Twenty-four hours after transfection, the medium was replaced with fresh complete DMEM supplemented with 1 mg/ml geneticin (Invitrogen) and the medium was replaced every three to four days with fresh medium containing 1 mg/mL geneticin. Geneticin-resistant colonies were selected for a period of two weeks and expanded in the presence of 250 μg/mL geneticin.
HCV RLuc replicon cells were seeded at a density of 10 × 103 cells per well in 96-well plates and incubated for 4–5 h to allow the cells to attach to the plate. The compounds dissolved in dimethyl sulfoxide (DMSO) were added at a final concentration of 10 μM (DMSO solvent final concentration was 0.5%) and the cells were incubated for 72 h at 37 °C under 5% CO2 atmosphere. The effects of compounds on HCV replication were then assessed by measuring the Renilla luciferase activity in compound-treated versus DMSO-treated cells. At the end of the incubation period, the medium was aspirated and the cells were washed with 1 × PBS. The Renilla luciferase reporter gene assay was performed using the Renilla luciferase assay kit (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, the cells were lysed by addition of 50 μL of 1× Renilla luciferase lysis buffer followed by two cycles of freeze/thaw. The luciferase activity content of the lysate was measured with a FLUOstar Omega microplate reader instrument (BMG Labtech, Germany) after injecting 50 μL of luciferase substrate and reading for 5 s.
