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

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.