This study investigates the mechanisms of Hepatitis C virus (HCV) entry and fusion in liver cancer cells. By utilizing a luciferase reporter system, the research assesses the impact of test compounds on viral envelope-endosome fusion.
Take liver cancer cells, and incubate at a low temperature to impair endocytosis.
Introduce a recombinant Hepatitis C virus or HCV, comprising an engineered RNA genome encoding a secreted luciferase reporter.
The viral lipoproteins interact with the proteoglycans on the host cells, while the viral envelope glycoproteins bind to specific host receptors.
In selected wells, add the test compound that binds to viral glycoproteins.
Upon incubation at physiological temperature, the bound viruses are endocytosed.
Discard the non-internalized viruses, add a growth media, and incubate.
In the untreated wells, the glycoproteins aid viral envelope-endosome fusion, releasing the nucleocapsid, while compound masking impairs fusion in the treated wells.
Upon release, the RNA expresses viral proteins, including luciferase which is secreted extracellularly.
Harvest the luciferase-containing supernatant and add a bioluminescent substrate, which is oxidized by luciferase to emit light.
A decreased luminescence in the treated wells compared to the untreated indicates inhibition of viral envelope-endosome fusion.
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.
繁體中文
