Experiment Summary

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging.

Fig. 1 Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection.

Materials and Reagents

1. Cell culture flasks, 75 cm²
2. Cell culture dishes, 100/20 mm
3. 2 ml reaction tubes, 3 ml sterile syringes
4. Syringe filters 0.2 μm hydrophilic polyethersulfone, 32 mm diameter
5. 10 ml sterile syringes
6. Syringe filters 0.45 μm hydrophilic cellulose acetate, 28 mm diameter
7. 15 ml conical tubes, 0.5 ml reaction tubes
8. Hexadimethrine bromide
9. 48-well cell culture plates, 1.5 ml reaction tubes, 12-well cell culture plates, 1.5 ml light protection reaction tubes
10. Cell culture flasks, 25 cm²
11. 6-well cell culture plates, 96-well black cell culture plates, 24-well cell culture plates
12. HEK 293T cells
13. Plasmids: pLenti-FlaviA-GFP-puro, pLenti-CMV-FlaviA-mNeptune-puro, pMD2.G, psPAX2
14. LB agar plates with 100 µg/ml ampicillin
15. LB broth (Miller)
16. 100 mg/ml ampicillin solution
17. Plasmid mini kit
18. DMEM, high glucose, GlutaMAXTM, pyruvate
19. Antibiotic-antimycotic 100×
20. Fetal bovine serum (FBS) qualified, heat inactivated
21. Polyethylenimine (PEI), linear, MW 25000, transfection grade
22. UltraPureTM DNase/RNase-free distilled water
23. Hydrochloric acid, 36.5-38.0%
24. BHK-21 [C-13]
25. MEM, GlutaMAXTM supplement
26. Sodium pyruvate, 100 mM
27. PBS, pH 7.4
28. TrypLETM express enzyme (1×), no phenol red
29. Puromycin dihydrochloride from Streptomyces alboniger
30. Clinical isolate DENV-2/CR/13538/2007
31. Vaccine strain YFV/US/17D/1937
32. FluoroBriteTM DMEM
33. GlutaMAXTM supplement
34. Carboxymethylcellulose sodium salt
35. Sodium bicarbonate
36. Complete DMEM
37. PEI solution (1 mg/ml)
38. Polybrene solution (10 mg/ml)
39. Complete MEM
40. PBS 1% FBS
41. Puromycin solution (10 mg/ml)
42. FluoroBriteTM DMEM 2% FBS
43. Plaque media 2% FBS

Equipment

1. Biological safety cabinet
2. CO₂ incubator
3. 4 °C refrigerator
4. -20 °C freezer
5. -80 °C freezer
6. Water bath
7. Centrifuge
8. Microcentrifuge
9. Pipettes
10. Pipet filler
11. Hemocytometer
12. Shaking incubator
13. ThermoMixer^® C block
14. Vortex mixer
15. Ultraviolet crosslinker
16. NanoDropTM 2000 spectrophotometer
17. Steam sterilizer
18. Water purification system
19. Inverted microscope
20. Flow cytometer
21. Cell sorter
22. Automated fluorescence microscope

Procedure

A. Lentiviral vectors assembly and titration

1. Prepare plasmid stocks.
2. Manually seed 6,000,000 HEK 293T cells in 100/20 mm cell culture dishes with 8 ml of DMEM 10% FBS and incubate overnight at 37 °C and 5% CO₂ to reach 70-80% confluency.
3. Using a pipette, replace the medium by removing and discarding all the DMEM 10% FBS in the dishes (~8 ml) and adding 4 ml of fresh DMEM 2% FBS.
4. Prepare the transfection mix as follows:

Suspension A: 45 µl PEI 1 mg/ml (Recipe 2) + 955 µl unsupplemented DMEM (without FBS and antibiotic-antimycotic).

Suspension B: 6 µg pLenti-FlaviA-GFP-puro or pLenti-CMV-FlaviA-mNeptune-puro + 6 µg pMD2.G + 6 µg psPAX2 and bring to 1,000 µl with unsupplemented DMEM.

Mix suspensions A with B and incubate for 15 min at room temperature.

5. Add the 2 ml transfection mix to the cells (dropwise) and incubate overnight at 37 °C and 5% CO₂.

6. Replace the medium with 6 ml of fresh DMEM 2% FBS and incubate for 48 h at 37 °C and 5% CO₂.

7. Harvest culture supernatants and filter them (0.45-μm pore size) to eliminate cells and debris. Use filters with low protein adherence, like cellulose acetate or polyethersulfone (PES). Do not use nitrocellulose filters as they could bind the lentiviral particles.

8. Prepare 500 µl aliquots and store at -80 °C.

9. Determination of titers by flow cytometry.

B. Reporter cell lines production and selection

Production

1. As described above, seed 100,000 BHK-21 cells per well in a 12-well cell culture plate with 1 ml/well of MEM 2% FBS and incubate overnight at 37 °C and 5% CO₂. Prepare an extra well for the control cells.
2. Thaw in a water bath (37 °C) one of the 500 µl stock aliquots of lentiviral particles carrying genetic constructs codifying for either the FlaviA-GFP or the FlaviA-mNeptune reporters and add polybrene at a final concentration of 5 µg/ml.
3. Based on the previously calculated biological titer in TU/ml, prepare 500 µl/well of lentiviral inoculum at a multiplicity of infection (MOI) of 1 (1 TU per cell). Incubate for 15 min at room temperature.
4. Replace the medium of the cells with the 500 µl/well of lentiviral inoculum. Add 500 µl/well of MEM 2% FBS + 5 µg/ml polybrene to the control cells.
5. Centrifuge at 300 × g for 2 h at 25 °C for viral adsorption.
6. Replace the inoculum with 1 ml/well of MEM 2% FBS and incubate for 48 h at 37 °C and 5% CO₂.
7. Based on the basal background of the reporter proteins, monitor the effectiveness of the transduction by fluorescence microscopy in the green/GFP (FlaviA-GFP) and far-red/Cy5 (FlaviA-mNeptune) channels.

Selection

1. For the antibiotic selection of transduced cells, replace the medium with 750 µl/well of MEM 10% FBS containing 8 µg/ml of puromycin and incubate overnight at 37 °C and 5% CO₂. Apply the same treatment to the control cells.
2. Replace the medium of both transduced and control cells with 1 ml/well of MEM 10% FBS containing 4 µg/ml of puromycin. Incubate at 37 °C and 5% CO₂ until 100% mortality of the control cells is evidenced by light microscopy (commonly 24-48 h).
3. Replace the medium of the selected cells with 1 ml/well of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO₂ until reaching confluency of 80-90%.
4. Passage the selected cells to 25 cm culture flasks with 5 ml of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO₂ until reaching a confluency of 80-90%.
5. For the FACS selection, discard the medium, wash the cells once with 1 ml of PBS, add 500 µl of TrypLETM express (no phenol red), incubate for 5 min at 37 °C, and resuspend the cells with 1 ml of PBS 1% FBS.
6. Count the cells with a hemocytometer and prepare 2 ml of cell suspensions at 1,000,000 cells/ml in PBS 1% FBS.
7. Aspirate the cell suspensions into the cell sorter. For the FlaviA-GFP reporter isolate at least two cell subpopulations with different but homogeneous levels of the reporter's basal background using the FL1 detector (488 nm laser - 530/30 nm filter) (Figure 2). Apply the same procedure to the FlaviA-mNeptune reporter but using the FL4 detector (640 nm laser - 675/25 nm filter).

Fig. 2 FACS analysis of BHK-21/FlaviA-GFP stable cells.

8. Seed the isolated cell suppopulations in different wells of a 48-well cell culture plate with 1 ml/well of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO₂ until reaching a confluency of 80-90%.

9. Passage the selected cells to a 6-well cell culture plate with 3 ml of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO₂ until reaching a confluency of 80-90%.

10. Test the different isolated cell suppopulations by a live-cell imaging flavivirus infection kinetics according to the protocol described in Procedure C.

C. Infection kinetics in reporter cells by live-cell imaging

1. Seed 15,000 BHK-21/FlaviA-GFP stable cells per well in a µClear black 96-well plate with 100 µl/well of MEM 2% FBS and incubate overnight at 37 °C and 5% CO₂.
2. Based on the calculated titer of the flaviviral seed in plaque forming units (PFU)/ml.
3. Replace the medium of the cells with 50 µl/well of either the infectious or the UV-inactivated flaviviral inoculum and incubate for 2 h at 37 °C and 5% CO₂ for viral adsorption. Using your hands, shake the plate for 5 s every 15 min.
4. Replace the inoculum with 150 µl/well of FluoroBriteTM DMEM 2% FBS and incubate for the desired time of your kinetics (e.g., 120 h) at 37 °C and 5% CO₂ into the automated fluorescence microscope. Add 200 µl/well of PBS to the surrounding wells to avoid desiccation.
5. Using your microscope's software (e.g., Gen5 Image+), program the image acquisition with the 4× or the 20× objective in the green/GFP channel at the desired post-infection times (Fig. 2A).

Fig. 3 DENV-2 infection kinetics in reporter BHK-21 cells by live-cell imaging.

D. Data analysis