This study investigates the antiviral properties of target compounds against hepatitis C virus (HCV) using a luciferase reporter assay. The methodology involves assessing viral attachment and entry in human hepatoma cells under controlled conditions.
Begin with a multi-well plate containing a pre-chilled human hepatoma cell monolayer.
Treat test wells with a mix of genetically modified hepatitis C virus carrying the luciferase gene and a target compound; treat control wells with the virus alone.
In the test wells, target molecules interact with viral glycoproteins and cell surface receptors, preventing viral attachment.
However, in control wells, viral glycoproteins interact with cell surface receptors, enabling their attachment. A low temperature inhibits viral entry.
Remove the supernatant, introduce a medium, and re-incubate at the optimum temperature, facilitating viral entry.
Upon entry, the virus releases the genetic material, initiating the synthesis of viral proteins and luciferase enzymes, which are secreted into the medium.
Transfer the luciferase-containing supernatant, and introduce a substrate.
The enzyme-substrate interaction produces light in proportion to luciferase levels.
The lower light intensity in the test wells, compared to the control wells, indicates the antiviral property of the target compound.
For the viral attachment assay, plate 1 x 104 cells per well in a 96-well plate, and incubate overnight for cell attachment. The next morning, first, chill the 96-well plate containing the cell monolayer at 4 degrees Celsius for 1 hour.
Prepare the test compounds and virus mixture on ice. For example, HCV-CHLA, HCV - 1% DMSO, or positive control HCV-heparin solutions with 102 FFU virus for each treatment well. Aspirate the medium from the cell culture wells gently. Then, immediately add 100 microliters of virus test compound mixture in triplicate wells, taking care not to raise the temperature. Incubate the plate in a refrigerator maintained at 4 degrees Celsius for three hours.
Virus particles will attach on the cell surface at 4 degrees Celsius but will not be internalized. Next, aspirate the supernatants. Gently wash the cells twice with 200 microliters of ice-cold PBS. Add 100 microliters of basal medium to each well, and then incubate at 37 degrees Celsius for 72 hours. Finally, for quantitating the released luciferase reporter from the infected cells, collect the supernatant from each sample well and perform the Gaussia luciferase assay.
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