Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Summary

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Abstract

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Introduction

Influenza A virus (IAV) is the prototypic member of the Orthomyxoviridae family and is known to cause global epidemics and unpredictable pandemics. IAV causes human respiratory disease, influenza, commonly known as "flu". The flu is an acute disease that results in the induction of host pro- and anti-inflammatory innate immune responses and the death of epithelial cells in the human respiratory tract. Both processes are governed by a phenomenon called programmed cell death¹. The signaling for programmed cell death is induced as soon as various pathogen recognition receptors sense the incoming virus particles in host cells. This leads to the programming of the death of infected cells and signaling to the neighboring healthy cells by three interconnected pathways called pyroptosis, apoptosis, and necroptosis-recently coined as one process, PANoptosis¹.

PANoptosis involves the proteolytic processing of many host and viral proteins from induction to execution. Such processing of proteins is primarily spearheaded by a family of cysteine proteases called caspases^1,2. Up to 18 caspases (from caspase 1 to caspase 18) are known³. Most caspases are expressed as pro-caspases and activated by undergoing their own proteolytic processing either by autocatalysis or other caspases⁴ in response to a stimulus like a virus infection. The PANoptosis of IAV-infected cells was thought to be a host defense mechanism, but IAV has evolved ways to evade and exploit it to facilitate its replication^1,2,5,6. One of them is to antagonize the host factors via caspase-mediated cleavage or degradation that are either inherently antiviral or interfere with one of the steps of the IAV life cycle. To this end, host factors, cortactin, HDAC4, and HDAC6 have been discovered to undergo caspase-mediated cleavage or degradation in IAV-infected epithelial cells^7,8,9. The HDAC4 and HDAC6 are anti-IAV factors^8,10, and cortactin interferes with IAV replication at a later stage of infection, potentially during viral assembly and budding¹¹.

In addition, various caspases are also activated, which, in turn, cleave multiple proteins to activate the host inflammatory response during IAV infection^1,2. Furthermore, nucleoprotein (NP), ion-channel M2 protein of IAV^12,13,14, and various proteins of other viruses^3,15,16 also undergo caspase-mediated cleavage during infection, which influences viral pathogenesis. Therefore, there is a continuous need to study caspase-mediated cleavage or degradation of host and viral proteins during IAV and other virus infections to understand the molecular basis of viral pathogenesis. Herein, the methods are presented to (1) assess the cleavage or degradation of such proteins by caspases, (2) identify those caspases, and (3) locate the cleavage sites.

Protocol

Regulatory approvals were obtained from the University of Otago Institutional Biological Safety Committee to work with the IAV and mammalian cells. Madin-Darby Canine Kidney (MDCK) or human lung alveolar epithelial A549 cells and IAV H1N1 subtypes were used for the present study. IAV was grown in chicken eggs, as described elsewhere¹⁷. Sterile and aseptic conditions were used to work with mammalian cells, and a Biosafety Level 2 (or Physical Containment 2) facility and Class II biosafety cabinet were used to work with IAV subtypes.

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

1. Seed 3 x 10⁵ 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-and 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 (see references^7,8).
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^7,11 (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% CO₂ atmosphere.
7. In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., 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 MEM⁷ (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 assay⁸ 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-HCl, pH 7.4, 150 mM 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 BCA assay kit (see Table of Materials).
16. Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE)¹¹ along with molecular weight markers¹¹.
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 elsewhere¹¹.
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.

2. Confirmation of caspase-mediated cleavage or degradation of proteins in IAV-infected cells by RNA interference

1. Design or obtain a pre-design small-interfering RNA (siRNA) targeting either canine or human caspase 3, caspase 6, and caspase 7 (executioner caspases¹) and a non-targeting control siRNA (see Table of Materials).
2. Deliver each siRNA to MDCK or A549 cells by reverse transfection^8,11.
3. For this, dilute in separate tubes 100 nM of control siRNA or caspase siRNA or a recommended volume of transfection reagent (see Table of Materials) in 100 µL of appropriate medium (like OptiMEM, see Table of Materials), and incubate for 5 min at room temperature.
4. Prepare each dilution in duplicate.
   NOTE: This duplicate includes one replicate for analyzing the knockdown of caspase expression by RT-qPCR or western blotting (if antibodies to caspases are available) and another for assessing the recovery of the protein of interest by western blotting.
5. Mix 100 µL of each siRNA solution with 100 µL of transfection reagent solution (step 2.3) and incubate for 20-45 min at room temperature to form the siRNA-transfection reagent complex.
6. In the meantime, split and add 1 x 10⁵ MDCK or A549 cells in 800 µL of the complete growth medium, mix with 200 µL of siRNA-transfection reagent complex (step 2.5), and add 1 mL of the suspension to a well of a 12-well culture plate. This dilution brings the final concentration of control siRNA or caspase siRNA to 10 nM.
7. Incubate the cells at 37 °C under a 5% CO₂ atmosphere for 72 h.
8. Harvest the cells from one replicate as in step 1.10, and process them to validate or confirm the knockdown of the expression of caspase 3, caspase 6, and caspase 7 either by RT-qPCR or western blotting, respectively, using standard methods and protocols^8,11.
9. Infect the cells in the other replicate with IAV as described in steps 1.4-1.9 (without inhibitors).
10. After 24 h, harvest, process, and analyze the cells by western blotting and measure and quantify the protein recovery as described in steps 1.10-1.20.

3. Site-directed mutagenesis for locating the caspase cleavage site(s) in the polypeptide

1. Clone the gene-encoding protein of interest in a mammalian expression plasmid¹¹ or obtain it from a research lab or commercial source (see Table of Materials).
   NOTE: It is preferred if the gene is cloned with an epitope tag or as GFP fusion to distinguish the ectopically expressed polypeptide from the endogenously expressed one during western blotting.
2. Using caspase substrate databases⁴ or protein alignment tools, locate the consensus caspase cleavage motifs, XXXD and DXXD, on the polypeptide. Almost all caspase cleavage motifs possess the aspartic acid at the cleavage site (P1)^3,4.
3. Mutate the putative P1 aspartic acid to glutamic acid or a non-polar amino acid (alanine, valine) by site-directed mutagenesis followed by DNA sequencing¹¹ using standard methods and protocols.
4. Deliver the wild-type (WT) and mutant plasmid DNA to MDCK or A549 cells by reverse transfection¹¹.
   1. For this, dilute 2 µg of plasmid DNA or a recommended volume of the transfection reagent in 100 µL of an appropriate medium (see step 2.3 and Table of Materials) in separate tubes, and incubate for 5 min at room temperature.
   2. Mix 100 µL of each plasmid DNA solution with 100 µL of the transfection reagent solution and incubate for 20-45 min at room temperature to form the DNA-transfection reagent complex.
5. In the meantime, split and add 3 x 10⁵ MDCK or A549 cells to 800 µL of the complete growth medium, mix with 200 µL of DNA-transfection reagent complex, and add the 1 mL suspension to a well of a 12-well culture plate.
6. Incubate the cells at 37 °C under a 5% CO₂ atmosphere.
7. After 48 h, infect the cells with IAV as described in steps 1.4-1.9 (without adding the inhibitors).
8. After 24 h, harvest, process, and analyze the cells by western blotting (if applicable, using the antibody to an epitope tag or GFP). Then, measure and quantify the protein recovery as described in steps 1.10-1.20.

Results

Treatment with caspase 3 inhibitor
It has been discovered that host cortactin, HDAC4, and HDAC6 polypeptides undergo degradation in response to IAV infection in both canine (MDCK) and human (A549, NHBE) cells^7,8,9. By using the above approaches, it was uncovered that IAV-induced host caspases, particularly caspase 3, cause their degradation^7,8,<...

Discussion

It is established that viruses tailor the host factors and pathways to their benefit. In turn, the host cells resist that by employing various strategies. One of those strategies is PANoptosis, which host cells use as an antiviral strategy against virus infections. However, viruses like IAV have evolved their own strategies to counter PANoptosis and exploit it to their advantage^1,3,6. This interplay involves the cleavage of vari...

Materials

List of materials used in this article

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
Lipofectamine 2000 Transfection Reagent	ThermoFisher Scientific	31985062
Lipofectamine RNAiMAX Transfection Reagent	ThermoFisher Scientific	13778150
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
MISSION siRNA Universal Negative Control #1	Sigma-Aldrich	SIC001
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
Plasmid expressing human cortactin-GFP fusion	Addgene	50728	Gift from Kenneth Yamada to Addgene
Pre-designed small interferring RNA (siRNA) to caspase 3	Sigma-Aldrich	NM_004346	siRNA ID: SASI_Hs01_00139105
Pre-designed small interferring RNA to caspase 6	Sigma-Aldrich	NM_001226	siRNA ID: SASI_Hs01_00019062
Pre-designed small interferring RNA to caspase 7	Sigma-Aldrich	NM_001227	siRNA ID: SASI_Hs01_00128361
Pre-designed SYBR Green RT-qPCR Primer pairs	Sigma-Aldrich	KSPQ12012	Primer Pair IDs: H_CASP3_1; H_CASP6_1; H_CASP7_1
Protran Premium nitrocellulose membrane	Cytiva (Fomerly GE Healthcare)	10600003
Rabbit anti-actin antibody	Abcam	ab8227
Rabbit anti-cortactin antibody	Cell Signaling	3502
Rabbit anti-GFP antibody	Takara	632592
SeeBlue Pre-stained Protein Standard	ThermoFisher Scientific	LC5625
Transfection medium, Opti-MEM	ThermoFisher Scientific	11668019
Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100	Merck
Trypsin, TPCK-Treated	Sigma-Aldrich	4370285