Preparing Cells to Analyze Intracellular Uptake and Activity of an Antiviral Agent

Begin with human hepatoma cells cultured on coverslips in a multi-well plate.

These cells contain the hepatitis C virus genome in the cytoplasm, which produces viral proteins essential for viral genome replication.

In the control well, maintain the cells in culture medium alone, while in the test well, add lactoferrin, an antiviral agent.

Incubate the plate to allow lactoferrin to enter the cells through endocytosis and accumulate in the cytoplasm.

Inside the cytoplasm, lactoferrin disrupts the viral proteins, halting further viral genome replication.

In the control well, the viral genome replicates into negative-sense RNA intermediates, which are then used to produce positive-sense RNA copies that serve as templates for viral protein synthesis.

After treatment, wash the cells with a buffer to remove unbound lactoferrin.

Add a fixative solution with sucrose. Incubate to fix and stabilize the cells.

The cells are now ready for the analysis of intracellular uptake and antiviral activity of the treatment agent.

To begin, place a round cover glass in the bottom of each well of a 24-well plate and use PBS to wash the wells. Seed HUH seven cells carrying the HCV Replicon at a density of five time