The overall goal of the following experiment is to analyze transcriptional activity in virus infected cells. First infect the 2 9 3 cells with rift valley fever virus, MP 12. Then label the cells with the uridine analog five hanal uridine, which gets incorporated into nascent RNA.
Next couple, the incorporated five hanal uridine to a fluorescent azide. In order to visualize newly synthesized RNA results obtained, assessed differences in the amount of newly synthesized RNA between infected and uninfected cells based on either fluorescent microscopy or flow cytometry. Unlike labeling with tritiated, uridine or five bromo uin, this click chemistry approach does not require the use of radioactive isotopes.
It is also faster and more sensitive than anti Roma UIN immuno staining. The idea for this methods stem from our need for quick and reliable way to measure transcription suppression in cells infected with different drift quality, fewer virus and P 12 s Place. 12 millimeter round cover slips into each well of a 12 well tissue culture plate.
Then seed 2 9 3 cells in one millimeter of complete DMEM medium incubate the cells in humidified incubator at 37 degrees Celsius and 5%carbon dioxide overnight. As per experimental design, dilute the virus stock so that infection of the cells with MP 12 is at a multiplicity of infection of three in a total volume of 200 microliters per well. A sign one well as the uninfected control and another as a control for transcriptional suppression.
Now, remove the growth medium. Add the viral inoculum and incubate for one hour in the humidified incubator. Aspirate the inoculum.
Add one milliliter of fresh growth medium per well, and incubate at 37 degrees Celsius at 15 hours post-infection. Replace the growth medium with medium containing one millimolar of the uridine analog. Five etal uridine to one of the mock infected wells.
Also add five micrograms per milliliter of the inhibitor of DNA dependent RNA synthesis act. Antimycin D.Return the cells to the incubator at 16 hours Post-infection. Remove the medium and wash the cover slips once with one milliliter of PBS per, well fix the cells with one milliliter of 4%para formaldehyde per well.
Rinse once with one milliliter of PBS per well. To assemble the humid chamber line the bottom of a cell culture or Petri dish with a 10 centimeter diameter with plastic paraffin film. Line the inside of the edge of the dish with damp paper towels.
Now to permease the cells, add one milliliter of 0.2%tritton X 100 in PBS incubate for 10 minutes at room temperature after three PBS washes, transfer the cover slips into the humid chamber for the click reaction to detect labeled RNA cover. Each cover slip with 50 to 100 microliters of freshly prepared Click staining solution incubate for one hour. Add room temperature in the dark.
Add one milliliter of PBS to the wells of a fresh 12. Well plate and transfer the cover slips into the wells wash two more times with one milliliter of PBS per well to mount cells. For fluorescence microscopy, place one drop of flora mount G onto a microscope slide.
Dip the cover slip into water. Remove the excess water. Place the cover slip cell side down into the drop of flora, Mount G and air dry for five minutes.
As per experimental design, infect the 2 9 3 cells with MP 12. Add an MOI of three. Remove the growth medium and add diluted virus stock and incubate for one hour in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.
Replace the inoculum with two milliliters of fresh growth, medium per well, and incubate at 37 degrees Celsius for optimal time for viral labeling and harvesting. Then replace the growth medium with medium containing 0.5 millimolar five hanal uridine to one of the mock infected wells. Also add five micrograms per milliliter acton mycin D.Return the cells to the incubator for one hour after one PBS wash, harvest the cells by trypsin.
Wash the harvested cells three times with PBS containing one millimolar EDTA. To fix the cells, resuspend the pellets in one milliliter of 4%PFA and incubate for 30 minutes at room temperature. Wash once with one milliliter of P-B-S-E-D-T-A per tube.
Then perme the cells in 0.5 milliliters of 0.2%Triton X 100 in PBS for 10 minutes at room temperature. Wash once with one milliliter of nuclease free PBS for the click reaction. Resus suspend the cell pellets in 200 microliters of click staining solution containing the fluorescent dye.
If the cells clump together, resus suspend by pipetting incubate for one hour at room temperature in the dark wash twice with fax buffer. In this experiment, 2 9 3 cells were infected with MP 12 and a deletion mutant virus that lacks the ability to suppress host cell transcription. In the mock infected cells, nuclei appear bright red due to ongoing transcription.
Since cells have only been labeled with EU for one hour, followed by immediate fixation, most RNAs remain in the nucleus. In contrast, when cells have been treated with ACT Antimycin D to pharmacologically inhibit transcription, the red fluorescence of nuclei is markedly reduced. This fluorescence is similarly reduced when cells are infected with MP 12, but not with the deletion mutant.
That does not suppress host transcription. Here the cells were transfected with in vitro synthesized mRNA instead of infection with the live virus. The red fluorescent signal in the nuclei indicates that compared to unresected cells, cells transfected with mRNA encoding green fluorescent protein did not change Transcriptional activity only transfection with mRNA encoding the Rift Valley fever virus.
Virulence factor N SS results in decreased red fluorescence. When quantified, the flow cytometry data obtained can be represented as scatter plots with the fluorescent signal for EU incorporation into nascent RNA plotted on the Y axis, and that for the anti rift Valley fever virus staining plotted on the X axis. The quadrant gates were set so that the majority of mock infected cells was situated in the upper left quadrant.
The majority of acty DRE cells was situated in the lower left quadrant and the majority of infected cells was situated in the right half of the plot. When the cells were infected with MP 1280 1.5%of total cells, or 93%of anti rift valley fever virus positive cells showed reduced transcriptional activity. In contrast, when cells were infected with R MP 12, clone 13 type 91.6%of total cells, or 97%of anti rift valley fever virus positive cells showed transcriptional activity, which was comparable to that of M infected cells.
These data can also be depicted in the form of a histogram for fluorescence, RNA labeling with EU incorporation. Following this other methods like intracellular immuno staining can be performed in order to visualize expression of viral protein in these cells. After watching this video, you should have a good understanding of how to utilize click chemistry to visualize transcriptional activity in virus infected cells, either by fluorescent microscopy or by flow cytometry.
Don't forget, working with infectious virus can be extremely hazardous, so use precautions such as wearing appropriate PPE and avoiding inhalation of aerosols.
