The overall goal of this method is to purify Herpes Simplex virus type one DNA from infected cells for the proteomic analysis of viral genome associated proteins. This method provides key insights into the involvement of cellular factors and Herpes virus infection. The main advantage of this technique is that it can be adapted to directly purify different populations of viral DNA from infected cells, allowing for the analysis of multiple aspects of infection.
Begin this procedure with culturing of MRC five 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 absorption, aspirate the innoculum, 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. To harvest nuclei, replace the growth medium with 20 milliliters of nuclei extraction buffer and incubate for 20 minutes at four degree celsius with occasional rocking. After incubation, scrape nuclei from the plate using a cell scraper and transfer them to a 50 milliliter conical tube on ice.
Centrifuge for 10 minutes at 2, 500 x g and four degree celsius to pellet the nuclei. Following centrifugation, discard the supernatant. Gently dislodge the nuclear pellet in 10 milliliters of phosphate buffer saline or PBS and transfer it to a 50 milliliter conical tube.
Then, centrifuge the sample as before. After completely removing the PBS, re-suspend the nuclear pellet in 10 milliliters of click reaction mix, by gently pipetting up and down five times with a 10 milliliter pipette. Rotate the sample for one hour at four degree celsius, before pelleting the nuclei as before.
Completely remove the quick reaction mix, then, wash the pellet by gently re-suspending it in 10 milliliters of PBS and centrifuging again. Now, carefully re-suspend the nuclear pellet in one milliliter PBS and transfer the solution to a 1.5 milliliter microcentrifuge tube. Following centrifugation, completely remove the PBS and flash freeze the pellet in liquid nitrogen.
At this point, the pellet can be stored at 80 degree celsius or the procedure can be continued. To lyse the nuclei and fragment the DNA, re-suspend the thawed nuclei in 500 microliters of Buffer B1 by pipetting up and down. Incubate the suspension on ice for 45 minutes.
Then, sonicate the samples six times for 30 seconds each at 40%amplitude, using a three millimeter microtip probe. Place the samples on ice for at least 30 seconds between pulses. After sonication, samples should appear clear, not cloudy.
Pellet the cell debris by centrifuging at 14, 000 x G for 10 minutes at four degrees celsius. The pellet size should decrease substantially, filter the supernatant through a 100 micron cell strainer and retain the flow through. Then, add 500 microliters of Buffer B2 to the filtered supernatant.
To bind the biotinylated DNA to streptavidin coated beads, prepare the streptavidin magnetic beads by transferring 300 microliters of bead slurry to a 1.5 milliliter microcentrifuge tube. Prepare one tube of beads per sample. Wash the beads three times with milliliter of Buffer B2 by vortexing to re-suspend, applying to a magnet to separate the beads and aspirating the wash buffer.
Then, add 900 microliters of the sample to the washed beads. The remainder of the sample will be used for input DNA and protein isolation. Rotate the suspension overnight at four degree celsius.
The next day, place the samples into a magnetic microfuge tube rack and remove the supernatant. Gently re-suspend the beads in one milliliter of Buffer B2, before rotating at four degree celsius for five minutes. Remove the supernatant again using the magnetic microfuge tube rack.
This time, gently re-suspend the beads in one milliliter Buffer B3 and rotate at four degree celsius for five minutes. After removing the supernatant from the bead mixture using the magnet, re-suspend the beads in 50 microliters of 2x Laemmli sample buffer to allude DNA protein complexes. Next, boil the samples at 95 degrees celsius for 15 minutes.
Vortex a mix before quickly spinning the samples in a microfuge. Apply the samples to the magnet and transfer the allot to a new tube. Finally, flash freeze the samples and store them at 80 degrees celsius.
Perform protein analysis as described in the text protocol. EdC labeling was carried out for zero, 20, 40 or 60 minutes followed by the purification of viral DNA and associated proteins. The eluted proteins were subject to coomassie blue staining and western blotting with the antibody specific for viral DNA binding proteins, ICP4 and UL42.
The arrow indicates streptavidin that was stripped from the beads during elution which is present under all conditions. Proteins isolated by this method are then subject to mass spectrometry. The pie chart illustrates the number of proteins in each functional category that were found to be associated with Herpes Simplex virus type on genomes at six hours post infection.
A closer look at protein-protein interactions amongst identified proteins was carried out using the string functional protein association network database. The results reveal sub-complexes of proteins that function together in the same biological process. After watching this video, you should have a good understanding of how to purify viral DNA from infected cells for the proteomic investigation of viral genome associated proteins.
This technique can be completed in three to four days. While attempting this procedure, it is important to remember to take precautions to prevent DNAse or protease contamination of samples. In addition to this procedure, other methods like immunofluorescence can be performed to verify the nature of EdC labeled viral DNA.
This technique has paved the way for researchers in the field of Herpes virology, to explore the functions of host proteins and viral infection.
