Labeling of Replicating HSV-1 Genomes with a Nucleoside Analog

Take a stationary-phase mammalian cell culture with minimal DNA replication and collect the conditioned medium.

Add herpes simplex virus (HSV) and incubate with occasional shaking to enable viral attachment. Wash to remove the unbound virus and restore the conditioned medium.

Upon incubation, the virus fuses with the host membrane, releasing the nucleocapsid into the cytoplasm.

The viral dsDNA enters the nucleus, circularizes, and expresses viral proteins for viral DNA replication.

The viral and host proteins unwind the viral DNA and synthesize an RNA primer. Next, the viral DNA polymerase complex binds and extends the primer for DNA synthesis.

As the replication proceeds, add a nucleoside analog to the culture. It enters cells and is converted into a triphosphate form.

Because host DNA replication is minimal, the analog is preferentially incorporated into replicating viral DNA, labeling the viral genomes.

The labeled viral genomes are ready for downstream proteomic analysis of the associated proteins.

Begin this procedure with culturing of MRC-5 cells as described in the text protocol. Then, dilute HSV-1 in cold tris-buffered saline or TBS. For the following steps, it is important to turn off the blower and the tissue culture hood to prevent cells from drying out.

Use a pipette to transfer the growth medium from the MRC-5 cells to a sterile bottle to add back to the cells after infection. Infect the cells by adding seven milliliters of diluted virus to the cells. Rock the cells every 10 minutes for one hour at room temperature.

Following adsorption, aspirate the inoculum, rinse the cells with 50 milliliters of room temperature TBS and replace the original growth medium. Then, incubate the cells at 37 degrees Celsius in the presence of 5% carbon dioxide. After the onset of viral DNA replication, around four hours post-infection, label replicating viral DNA.

To do so, dilute EdC in one milliliter of growth medium and add it to the cell culture medium of infected cells for two to four hours.