Visualizing Transcriptional Suppression in Virus-Infected Cells

Abstract

Source: Kalveram, B. et al. Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis. J. Vis. Exp. (2013)

This video demonstrates the use of click chemistry and fluorescence microscopy to visualize nascent RNA transcription in mammalian cells. It highlights how viral infection suppresses host transcription by comparing fluorescent signals between infected and control cells.

Protocol

1. Infect cells with Rift Valley fever virus (RVFV) strain MP-12 and label nascent RNA with 5-ethynyl uridine (EU)

   1. Seed human embryonic kidney 293 (HEK293) cells into a 6-well tissue culture plate in growth medium (Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin). Incubate in a humidified incubator at 37 °C and 5% CO₂ until cells have reached 80 - 100% confluency.
   2. Infect cells with MP-12 at a multiplicity of infection (MOI) of 3. Include at least two uninfected wells: one of the two wells will serve as the uninfected control, the other will be treated with actinomycin D (ActD). Remove the growth medium and dilute the virus stock so that the total volume added to each well is 400 μL. Incubate for 1 hr in a humidified incubator at 37 °C and 5% CO₂.
   3. Remove the inoculum and add 2 mL fresh growth medium per well. Incubate at 37 °C for 12 hr.

   NOTE: If adapting this protocol for use with another virus, it is advisable to perform a time course experiment to determine the optimal time point for labeling and harvesting. Set up this time course so that the infection times are staggered and all samples can be labeled, harvested, and stained at the same time.

   4. At 12 hours post-infection (hpi), replace the growth medium with a medium containing 0.5 mM EU. To one of the mock-infected wells, also add 5 μg/mL ActD. This will serve as the control for transcriptional suppression, as ActD inhibits DNA-dependent RNA synthesis. Return cells to the incubator.
   5. At 13 hpi, wash cells once with phosphate-buffered saline (PBS) and harvest them by trypsinization. Wash harvested cells three times with PBS containing 1 mM ethylenediaminetetraacetic acid (EDTA).

   NOTE: During this and subsequent steps, do not centrifuge cells faster than 500-1,000 x g to prevent cell breakage. Centrifuge cells for 1-2 min to sediment.

   NOTE: If surface immunostaining is planned following the click reaction, harvest using a rubber policeman or disposable cell scraper instead.

   6. Fix cells by resuspending them in 1 mL of 4% paraformaldehyde (PFA) and incubate for 30 min at room temperature (RT).
   7. Wash once with 1 mL PBS-EDTA per well. Proceed immediately to step 2.

   NOTE: It is important to immediately proceed with the click reaction, as the RNA degenerates if kept at this stage for prolonged periods of time. Similarly, if performing a time course experiment, be sure to label, fix, and stain all samples at the same time.

2. Click Reaction to Detect Labeled RNA

   1. Permeabilize cells by adding 1 ml 0.2% Triton X-100 in PBS. Incubate for 10 min at RT.

   NOTE: All solutions used in steps 2.1 to 2.3 need to be nuclease free.

   2. Wash three times with 1 ml PBS per well.
   3. Remove coverslips from 12-well plate and transfer into humid chamber. Cover each coverslip with 50 - 100 μl click staining solution (100 mM Tris pH 8.5, 1 mM CuSO₄, 20 μM fluorescent azide [excitation/emission maximum: 590/617 nm], 100 mM ascorbic acid). Incubate for 1 hr at RT in the dark.

   NOTE: To assemble the humid chamber, line the bottom of a cell culture or petri dish with a 10 cm diameter with plastic paraffin film. Line the inside of the edge of the dish with damp paper towels. Place the coverslips onto the plastic paraffin film.

   NOTE: Be careful not to let the coverslips dry out during this or any other step in the protocol. It is best to add the staining solution to each coverslip directly after it has been placed in the humid chamber.

   NOTE: Prepare the click staining solution fresh immediately prior to this step. Stock solutions of 1.5 M Tris pH 8.5, 100 mM CuSO₄, and 500 mM ascorbic acid may be stored at 4 °C. However, discard any ascorbic acid stock solution that has turned yellow.

   NOTE: Be sure to select a fluorophore that matches the filters on your fluorescent microscope.

   4. Remove the coverslips from the humid chamber and place into a fresh 12-well plate and wash three times with 1 ml PBS per well.
   5. Mount coverslips by placing one drop (approximately 15 μl) of Fluoromount-G onto a microscope slide. Dip the coverslip into H₂O, remove excess H₂O by touching the side against a paper towel, and place cell-side down into the drop of Fluoromount-G. Air dry for 5 min.

   NOTE: If imaging on an inverted microscope, allow Fluoromount-G to solidify at 4 °C overnight or affix the coverslips to the microscope slide by sealing the edge with transparent nail polish.

   NOTE: Slides can be imaged immediately or stored at 4 °C in the dark for up to a week.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-Ethynyl uridine	Berry Associates	PY 7563	Dissolve in DMSO for 100 mM stock
12-mm Round coverslip	Fisherbrand	12-545-82
Actinomycin D (ActD)	Sigma	9415	Dissolve in DMSO for 10 mM stock
Fluorescence Microscope			e.g. Olympus IX71
CuSO₄	Sigma	C-8027	Dissolve in H₂O for 100 mM stock
DMEM (Dulbecco's modified eagle medium)	Invitrogen	11965092
FBS (Foetal bovine serum)	Invitrogen	16000044
Fluorescent azide (Alexa Fluor 594-coupled)	Invitrogen	A10270
Fluoromount-G	Southern Biotech	0100-01
L-ascorbic acid	Sigma	A-5960	Dissolve in H₂O for 500 mM
Paraformaldehyde	Sigma	158127
Penicillin-Streptomycin	Invitrogen	15140122
Triton X-100	Sigma	T-8787
Trypsin-EDTA	Invitrogen	25200056
Trizma base	Sigma	T-1503	dissolve in H₂O for 1.5 M stock, pH 8.5