Analysis of Virus-Induced Host Transcriptional Suppression Using Flow Cytometry

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 flow cytometry to measure virus-induced suppression of host-cell transcription. Mammalian cells are infected with a virus that expresses a nuclear virulence protein disrupting the host transcription factor complex, while control cells remain transcriptionally active. Incorporation of 5-ethynyl uridine labels newly synthesized mRNA, enabling quantification of transcriptional activity in infected and control cells through dual fluorescence detection.

Protocol

1. Infect Cells with MP-12 and Label Nascent RNA with 5-ethynyluridine (EU)

1. Seed 293 cells into a 6-well tissue culture plate in growth 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 an m.o.i. 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 ActD at step 1.4. 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 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 h.p.i., 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 h.p.i., wash cells once with PBS and harvest by trypsinization of cells. Wash harvested cells three times with PBS containing 1 mM EDTA (PBS-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 a 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% PFA and incubate for 30 min at RT.
7. Wash once with 1 ml PBS-EDTA per well. Proceed immediately to step 6.

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 resuspending in 0.5 ml of 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. If cells do not sediment properly during this step, increase the centrifugation time to 5 min. Sedimentation behavior will improve once cells are suspended in the FACS buffer. .

2. Wash once with 1 ml PBS.

NOTE: Do not use EDTA in this step as this will chelate the Cu²⁺ ions necessary for the click reaction.

3. Resuspend cells in 200 μl click staining solution (100 mM Tris pH 8.5, 1 mM CuSO4, 200nM azide labeled with fluorescent dye [excitation/emission maximum: 650/665 nm], 100 mM ascorbic acid). Incubate for 1 hr at RT in the dark.

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. If cells clump together during this step, resuspend by pipetting up and down several times. Be sure to select a fluorophore that can be detected by your flow cytometer. Also prepare one sample of infected cells which are not subjected to the click staining procedure; these will be needed for calibration of the flow cytometer. Either use half of the infected cells or include an additional infected well at step 1.2.

Wash twice with FACS buffer (PBS containing 1 mM EDTA and 0.5% (w/v) BSA).

3. Immunofluorescence to Detect Expression of Viral Proteins

1. Resuspend cells in 200 μl of primary antibody (anti-RVFV) diluted in FACS buffer (1:10,000) and incubate for 1 hr at RT in the dark.

NOTE: Alternatively, this step can be performed O/N at 4 °C in the dark.

NOTE: Also prepare one sample of uninfected cells which were subjected to the click staining procedure but will not be immunostained, these will be needed for calibration of the flow cytometer. Either use half of the mock infected cells or include an additional uninfected well at step 1.2.

NOTE: When adapting this protocol for use with a different virus, determine the optimal dilution for your primary antibody first.

2. Wash cells twice in the FACS buffer.
3. Resuspend cells in 200 μl of secondary antibody (anti-mouse IgG conjugated to fluorescent dye [excitation/emission maximum: 495/519 nm]) diluted in FACS buffer (1:1,000) and incubate for 1 hr at RT in the dark.
4. Wash cells once in the FACS buffer.

NOTE: At this point, cells can be stored O/N at 4 °C in the dark.

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 coverslips	Fisherbrand	12-545-82
5 ml polystyrene tubes	BD Biosciences	352058
Actinomycin D (ActD)	Sigma	9415	dissolve in DMSO for 10 mM stock
Alexa Fluor 488 goat anti-mouse IgG	Invitrogen	A-11029
Bovine Serum Albumin (BSA)	Santa Cruz	sc-2323
CuSO₄	Sigma	C-8027	dissolve in H2O for 100 mM stock
DAPI	Sigma	D-9542	dissolve in H2O for 5 mg/ml stock
DMEM	Invitrogen	11965092
FBS	Invitrogen	16000044
fluorescent azide (Alexa Fluor 594-coupled)	Invitrogen	A10270
fluorescent azide (Alexa Fluor 657-coupled)	Invitrogen	A10277
Fluoromount-G	Southern Biotech	0100-01
L-ascorbic acid	Sigma	A-5960	dissolve in H2O for 500 mM
paper filter	VWRbrand	28333-087
paraformaldehyde	Sigma	158127
Penicillin-Streptomycin	Invitrogen	15140122
poly-L-lysine (MW ≥ 70000)	Sigma	P1274
Triton X-100	Sigma	T-8787
Trizma base	Sigma	T-1503	dissolve in H2O for 1.5 M stock, pH 8.5
Trypsin-EDTA	Invitrogen	25200056
Fluorescence Microscope			e.g. Olympus IX71
Flow Cytometer			e.g. BD Biosciences LSRII Fortessa