Visualizing Transcriptional Suppression in Virus-Infected Cells

Begin with coverslips carrying mammalian cells pre-treated with a uridine analog, with one group infected with the virus and the other uninfected.

In virus-infected cells, viral proteins bind to the host transcription machinery and inhibit host RNA production, whereas in uninfected cells, RNA synthesis remains uninterrupted.

Wash with a buffer. Add paraformaldehyde to fix the cells, then wash. Add a detergent to permeabilize the cell membranes, then wash.

Transfer the coverslips to a humid chamber.

Add the click staining solution containing fluorescent azide, a catalyst, and a reducing agent, then incubate in the dark.

The reducing agent activates the catalyst, which enables the fluorescent azide to bind to the uridine-labeled nascent RNA.

Rinse the coverslips in buffer, dip them in water, and remove excess moisture.

Using an antifade mounting medium, mount the coverslips on a slide and air-dry.

Under an inverted fluorescence microscope, reduced signal intensity in infected cells compared to uninfected cells indicates virus-induced transcriptional suppression.

Place 12 millimeter round coverslips into each well of a 12 well tissue culture plate. Then seed 293 cells in one milliliter of comp