An Assay to Evaluate the Effect of Test Compounds on Viral Entry and Fusion in Host Cells

For assaying viral fusion and entry, pre-chill the cell culture plate at 4 degrees Celsius for one hour after the overnight incubation for cell attachment. Remove the existing medium from the wells, and infect the cells with 10 to the second FFU HCV on ice.

Incubate the plate at 4 degrees Celsius for three hours. Gently wash the cells twice with 200 microliters of ice-cold PBS to remove the non-adhered virus. Then, on ice, treat the cells with a test compound dissolved in basal medium or basal medium with 1% DMSO as a control, and incubate the plate at 37 degrees Celsius for three hours. This allows assessment of the test compounds on viral entry, which occurs at 37 degrees Celsius.

Aspirate the medium and wash the non-internalized viruses twice with 200 microliters of citrate buffer or PBS. Add 100 microliters of basal medium per well, and incubate at 37 degrees Celsius for another 72 hours. Collect the supernatant and perform the Gaussia luciferase assay as demonstrated before for detecting the released luciferase reporter.