This method will help to determine and confirm whether a protein in influenza virus infected cells undergoes degradation or cleavage by caspases and identify the caspase cleavage sites. This method requires only basic molecular and cellular biology laboratory settings and research skills and do not require any specialized equipment or software training. This method can be adapted to achieve similar objectives involving infection with other animal viruses or microbes, and exposure to external stimuli, like toxins and chemicals.
To begin, seed MDCK or A549 cells in a 12-well cell culture plate. Incubate the cells overnight at 37 degrees Celsius under a 5%carbon dioxide atmosphere. On the next day, remove the old culture medium from the cells and wash the cells in each well twice with one milliliter of serum-free MEM.
Then, infect the cells by adding 400 microliters of influenza A virus inoculum, and place the plate for incubation for one hour. After incubation, remove the virus inoculum and wash the cells with MEM. Add one milliliter of serum-free MEM to each well and incubate the cells as shown in the previous step.
After 24 hours, harvest the cells by scraping them with a one-milliliter syringe plunger and transfer them to a 1.5 milliliter tube. Centrifuge the tube at 12, 000G for two minutes at room temperature and collect the supernatant. Wash the cell pellet with 250 microliters of phosphate-buffered saline and centrifuge.
Remove the supernatant and add 100 microliters of cell lysis buffer. Mix by vortex. Heat the tube at 98 degrees Celsius for 10 minutes to completely lyse the cells.
Then, resolve equal amounts of protein from uninfected and infected samples by standard SDS polyacrylamide gel electrophoresis. After electrophoresis, transfer the protein gel to a nitrocellulose or PVDF membrane and perform Western blotting. Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.
In 100 microliters of appropriate medium, like Opti-MEM, dilute 100 nanomolars of control or caspase siRNA, or appropriate volume of transfection reagent. Incubate for five minutes at room temperature. Mix 100 microliters of each siRNA solution with 100 microliters of transfection reagent solution and incubate for 20 to 45 minutes at room temperature.
Split and add 100, 000 MDCK or A549 cells in 800 microliters of the complete growth medium. Add 200 microliters of siRNA transfection reagent complex and add this suspension to a 12-well culture plate. Incubate the cells at 37 degrees Celsius under a 5%carbon dioxide atmosphere for 72 hours.
Infect the cells with influenza A virus, but without inhibitors. Perform Western blotting, and compare the protein levels as shown previously. Locate the caspase cleavage motifs XXXD and DXXD on the polypeptide.
Mutate the P1 aspartic acid, two glutamic acid, or alanine by site-directed mutagenesis and confirm by DNA sequencing. In 100 microliters of an appropriate medium, dilute two micrograms of wild-type or mutant plasma DNA or recommended volume of the transfection reagent and incubate the tubes for five minutes at room temperature. Mix 100 microliters of plasma DNA solution with 100 microliters of the transfection reagent solution and incubate for 20 to 45 minutes at room temperature.
In the meantime, split and add 300, 000 MDCK or A549 cells to 800 microliters of complete growth medium. Add 200 microliters of DNA transfection reagent complex and add this suspension to a 12-well culture plate. Incubate the plate at 37 degrees Celsius under a 5%carbon dioxide atmosphere.
After 48 hours, infect the cells with influenza A virus without inhibitors. Perform Western blotting, and compare the protein levels as shown previously. Using Western blotting, the degradation of host cortactin by influenza-induced host caspases was evaluated.
Treatment of the influenza virus infected cells with a caspase-3 inhibitor resulted in the rescue of cortactin and viral NP degradation. Quantification of the intensity of cortactin bands and its normalization with the corresponding actin band showed that the level of cortactin in caspase-3 inhibitor treated infected cells was higher compared to untreated infected cells. In caspase-6 or caspase-7 depleted cells.
No significant recovery in cortactin polypeptide levels was observed, whereas in caspase-3 depleted cells, cortactin recovered from the influenza virus induced degradation. Quantification and comparison of polypeptide levels showed that the cortactin polypeptide level in infected cells with depleted caspase-3 expression was higher than cells with normal caspase-3 expression. The mutation of aspartic acid at position 116 to glutamic acid rendered the cortactin polypeptide resistant to influenza virus induced degradation.
Quantification and comparison of polypeptide levels showed that the level of mutant cortactin polypeptide in infected cells was higher than that of wild-type cortactin polypeptide. It is required to generate tens of mutants and perform repeated Western blotting to identify the caspase cleavage site in the polypeptide. An in vitro biochemical assay containing purified caspase and target polypeptide could be developed to confirm that the protein of interest is a direct gas-based substrate.
This protocol will help to understand the significance, the degradation or cleavage of target protein in the system that is being studied, such as virus infection or a disease.
