Assessing Viral Infectivity Using a β-Galactosidase-Based Assay

Take reporter epithelial cells that contain the β-galactosidase gene under a viral protein-responsive promoter.

Add different concentrations of the virus to the cells and incubate to allow viral entry into the host cells.

Discard the media and wash to remove excess viral particles.

Add fresh media and incubate.

The viral RNA is converted into DNA, which is then integrated into the host genome and expressed as viral proteins.

The viral protein binds to the viral protein-responsive promoter, enabling the expression of the enzyme β-galactosidase.

Remove the media and wash with a buffer.

Add a fixative to preserve cellular integrity.

Remove the fixative and wash with a buffer.

Add a membrane-permeable chromogenic substrate.

The β-galactosidase converts the substrate into a colored product.

Add a stop solution to stop the reaction.

Wash to remove excess substrate.

Count the blue-stained cells in each concentration to quantify infective virion particles.

To begin seed 0.1 times 10 to the power of 6 TZM-bl cells in a 24-well plate using 500 microliters of complete DMEM for each well and place it in a cell culture incubator for 10 to 12 hours. From the vial containing the HIV-1 virus transfer an appropr