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