Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells

Overview

The video demonstrates a method to assess caspase-mediated protein cleavage in virus-infected cells. Post-viral infection, caspases get activated and degrade various host cellular proteins and viral nucleoproteins while specific caspase inhibitors prevent this degradation, as confirmed by western blotting.

Protocol

1. Assessing the cleavage or degradation of proteins in Influenza A virus, IAV-infected cells by caspases

1. Seed 3 x 10⁵ Madin-Darby Canine Kidney (MDCK) or A549 cells per well in a 12-well cell culture plate.       
   NOTE: If using MDCK cells, ensure that the antibody against the protein of interest recognizes its canine species.
2. Seed the wells in pairs, i.e., a control pair-one well for the uninfected-mock sample and one for the infected-mock sample, a test pair-one well for uninfected-inhibitor A sample and one for the infected-inhibitor A sample. Increase the number of pairs with each additional inhibitor.
3. Incubate the cells at 37 °C under a 5% CO₂ atmosphere overnight.
4. The next day, infect the cells with IAV (an H1N1 subtype or another subtype).
   1. For this, prepare the virus inoculum by diluting virus stock in 400 µL of serum-free minimum essential medium (MEM) (see Table of Materials) at a multiplicity of infection (MOI) of 0.5-3.0 plaque-forming units (pfu)/cell (a factor of the doubling of cell number when calculating the MOI).
      NOTE: For infecting MDCK cells, supplement the virus inoculum with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin at a final concentration of 1 µg/mL.
5. Remove the old culture medium from the cells (step 1.3) and wash the cells with 1 mL/well of serum-free MEM 2x.
6. Add 400 µL of virus inoculum (step 1.4.1) to the cells and incubate them for 1 h at 35 °C under a 5% carbon dioxide (CO₂) atmosphere.
7. In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., ammonium chloride, NH₄Cl), or a proteasome inhibitor (e.g., MG132) (see Table of Materials) at a final concentration of 40 µM, 20 mM, or 10 µM, respectively, in serum-free MEM (see Table of Materials). The latter two inhibitors serve as a control. Dilute an equal volume of the solvent (if other than water) used to reconstitute one of the inhibitors in serum-free medium as a mock.
8. Remove the virus inoculum and wash the cells as in step 1.5 (1x).
9. Add the serum-free MEM, 1 mL/well, mock or supplemented with an inhibitor, to the cells and incubate the cells as in step 1.6. This time point is considered as 0 h infection.
10. After 24 h, harvest the cells by scraping them with a 1 mL syringe plunger (rubber side) and transfer them into a 1.5 mL polypropylene tube.
11. Centrifuge the tube at 12,000 x g for 1-2 min at room temperature. Collect the supernatant using a pipette.         
    NOTE: This supernatant could be discarded or used for a plaque assay to measure the titer of the released virus progeny.
12. Wash the cell pellet with 250 µL of phosphate-buffered saline (PBS) by re-centrifugation as in step 1.11.
13. Remove the supernatant and lyse the cells by adding 80-100 µL of cell lysis buffer (50 mM Tris (hydroxymethyl) aminomethane (THAM) hydrochloride (Tris-HCl), pH 7.4, 150 mM sodium chloride (NaCl), 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail, see Table of Materials) and vortexing.
14. Heat the tube at 98 °C for 10 min to completely lyse and prepare the total cell lysate. Store the lysates at 4 °C for performing steps 1.15-1.17 the next day, but, for best results, finish these steps on the same day.
15. Estimate the protein amount in each sample using a bicinchoninic acid (BCA) tableassay kit (see Table of Materials).
16. Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE) along with molecular weight markers.
17. Transfer the protein to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane (see Table of Materials).
    NOTE: PVDF membrane may give a high background in some western blot imagers; check for compatibility.
18. Perform western blotting to detect the protein of interest using the method described elsewhere.
19. Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.   
    NOTE: If the protein level has recovered in the caspase inhibitor-treated infected sample lane, then the protein is cleaved or degraded by caspases. Otherwise, it is degraded by either lysosome or proteasome.
20. Quantify the protein recovery by measuring its band intensity in each lane from at least three replicates of the same experiment and normalizing it with the corresponding loading control band.
21. Use any contemporary imager and associated software (see Table of Materials) to image the western blots and quantify the protein recovery.

Materials

Name	Company	Catalog Number	Comments
A549 cells	ATCC	CRM-CCL-185	Human, epithelial, lung
Ammonium chloride	Sigma-Aldrich	A9434
Caspase 3 Inhibitor	Sigma-Aldrich	264156-M	Also known as 'InSolution Caspase-3 Inhibitor II - Calbiochem'
Complete, Mini Protease Inhibitor Cocktail	Roche	11836153001
Goat anti-NP antibody			Gift from Richard Webby (St Jude Children's Research Hospital, Memphis, USA) to MH
MDCK cells	ATCC	CCL-34	Dog, epithelial, kidney
MG132	Sigma-Aldrich	M7449
Minimum Essential Medium (MEM)	ThermoFisher Scientific	11095080	Add L-glutamine, antibiotics or other supplements as required
Odyssey Fc imager with Image Studio Lite software 5.2	LI-COR		Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software.
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23225
Protran Premium nitrocellulose membrane	Cytiva (Fomerly GE Healthcare)	10600003
Rabbit anti-actin antibody	Abcam	ab8227
Rabbit anti-cortactin antibody	Cell Signaling	3502
SeeBlue Pre-stained Protein Standard	ThermoFisher Scientific	LC5625
Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100	Merck
Trypsin, TPCK-Treated	Sigma-Aldrich	4370285