This article describes a viral inactivation assay for hepatitis C virus (HCV) using a luciferase reporter system. The method evaluates the effectiveness of antiviral compounds in inhibiting HCV infection in hepatoma cells.
Take tubes with a recombinant hepatitis C virus, or HCV suspension containing RNA encoding a secreted luciferase reporter.
Add an antiviral test compound and solvent to the test and negative control tubes.
The test compounds bind to HCV glycoproteins, inactivating the virus.
Post-incubation, dilute the mixtures to prevent compound-cell interactions in subsequent steps.
Add these diluted mixtures onto HCV-replication-supporting hepatoma monolayers. Incubate.
The compound-inactivated HCV cannot attach to specific cell surface receptors, while active HCV glycoproteins bind to these receptors, facilitating cellular attachment.
Remove unadsorbed HCVs. Wash with buffer and add media. Incubate for a prolonged duration.
The bound HCV is internalized, releasing viral RNA and leading to viral protein and luciferase synthesis, secreted from the cell.
Collect the luciferase-containing culture supernatants. Centrifuge and add a luciferase substrate to the supernatants.
Luciferase oxidizes the substrate, emitting light.
A higher luminescence in the control well than in the test indicates viral inactivation by the compound.
To begin the viral inactivation assay, first, plate 1 times 10 to the fourth cells per well in a 96-well plate. Incubate the cells at 37 degrees Celsius with 5% carbon dioxide overnight for attachment. For infection, prepare Gaussia luciferase reporter-tagged hepatitis C virus or HCV particles, as referenced in the text protocol.
In a sterile tube, mix 100 microliters of 100 micromolar CHLA or PUG with 100 microliters of 10 to the fourth focus-forming units or FFU of HCV. For a positive control, mix HCV with heparin at a final concentration of 1,000 micrograms per milliliter. Incubate at 37 degrees Celsius for three hours.
After three hours, dilute the virus compound mixture 50-fold with 9.8 milliliters of basal medium at room temperature. Then, prepare a new virus compound mixture, and immediately dilute this mixture in basal medium for the zero-hour incubation sample. After removing the incubation medium from the cells, add 100 microliters of the diluted virus drug solution per well in triplicate.
The solution now contains 10 to the second FFU per well. Incubate at 37 degrees Celsius and 5% carbon dioxide for three hours to allow viral infection of the cells. After incubation, remove the viral suspension from the wells and gently wash the cells with 200 microliters of PBS twice.
Incubate the cells in 100 microliters of basal medium for 72 hours for viral replication and release of luciferase reporter into the supernatant. Then, collect the supernatants from the cultures in microtubes, and centrifuge at 17,000 times g for 5 minutes at 4 degrees Celsius to remove cellular debris. Mix 20 microliters of each supernatant with 50 microliters of Gaussia luciferase assay reagent, and measure the luminescence from the luciferase reporter activity in a luminometer.
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