The Molecular Mechanism Of Pyroptosis By Activating GSDME In DHAV-1-infected DEF Cells

Duck hepatitis A virus type 1(DHAV-1)is a member of the genus Avihepatovirus in the Picornaviridae family.Duck viral hepatitis(DVH)rapidly spreads infection in young ducklings aged 1 to 3 weeks with a lethal rate of 90%.DVH outbreak in China is mainly caused by duck hepatitis A virus type 1(DHAV-1)and duck hepatitis A virus type 3(DHAV-3).The cytokine storm induced by DHAV-1 infection can then cause significant liver damage which is the direct cause of ducklings’ death.Thus,the disease has caused serious production and economic losses in duck industry.Pyroptosis,a newly discovered process of proinflammatory programmed cell death,is featured by pore formation in the plasma membrane and extracellular release of pro-inflammatory factors.When host cells are infected by pathogenic microorganisms,pathogen-associated molecular patterns(PAMPs)are recognized by pattern recognition receptors(PRRs),and then the adapter protein ASC and pro-caspase-1are assembled to form the inflammasome,resulting in the cleavage and activation of caspase-1.Then,activated caspase-1 triggers the maturation of the pro-inflammatory cytokines IL-1βand IL-18.On the other hand,the activated caspase-1 cleaves Gasdermin,relieves its selfinhibition and releases active N-terminal residues,and then destroys the cell membrane to cause pyroptosis.For DHAV-1,pyroptosis not only leads to cell death but also expands the inflammatory response and induces tissue damage,which is the direct cause of host death.Unlike mammals and humans,the duck genome only encodes Gasdermin A(GSDMA)and Gasdermin E(GSDME),this suggests that the signaling pathway of pyroptosis in ducks is unique.Then,what is the cell signaling pathway of DHAV-1-induced pyroptosis? This study used the GSDME as an entry point to investigate the molecular mechanism of DHAV-1-induced pyroptosis in duck embryo fibroblasts(DEFs).1.DHAV-1 induces pyroptosis by activating caspase-3 and cleavage of GSDMEThis study first analyzed whether DHAV-1 infection causes cell pyroptosis.electron microscopy was performed on DEF cells infected with DHAV-1.The results showed that cell membrane rupture,with typical pyroptosis morphological features;Subsequently,gene expression levels of pyroptosis-related genes were detected,and it was found that DHAV-1infection upregulated caspase-3,GSDME,NLRP3,IL-1β and IL-18 in a dose-dependent manner;After infection with DHAV-1,cell samples were collected to detect ATP synthesis and LDH release in the cell supernatant.The results showed that the release of LDH increased with the increase of DHAV-1 infection concentration,while the ATP synthesis level showed a gradually decreasing trend.Based on the above results,it is speculated that DHAV-1 infection oinduced cell pyroptosis.DEF cells were infected with DHAV-1 and endogenous GSDME cleavage was detected;Overexpressing the GSDME eukaryotic expression plasmid in DEF cells,the GSDME cleavage can be detected.As a key protein mediating cell pyroptosis,the cleavage of GSDME demonstrates the occurrence of cell pyroptosis.These results confirm that DHAV-1 infection mediates cell pyroptosis,and GSDME plays a key role in DHAV-1-induced pyroptosis.2A1 and 3C proteins are important proteolytic enzymes of DHAV-1,which can cleave host proteins and play a crucial role in virus-host interactions.Therefore,DHAV-1 non-structural proteins 2A1 and 3C were used as research subjects to detect the cleavage of GSDME.Recombinant plasmids of 2A1/3C and GSDME were overexpressed in DEF cells,respectively,no cleavage product of GSDME was not found,and there was no significant change in the release level of LDH.The results proved that 2A1 and 3C of DHAV-1 cannot cleave GSDME.Current research has shown that activated caspase-3 can induce cleavage of GSDME,and thereby lead to pyroptosis.Therefore,in order to analyze whether the cell pyroptosis is related to caspase-3,the expression of endogenous caspase-3 was first detected after DHAV-1 infection.It was found that DHAV-1 infection in DEF cells resulted in the activation of endogenous caspase-3,which was time-dependent and dose-dependent.This suggests that caspase-3 may also be involved in inducing GSDME cleavage.Next,we used RNAi technology to construct si-caspase-3 and si-GSDME to silence the expression of caspase-3 and GSDME in DEF cells.After DHAV-1 infection,LDH and ATP detection,flow cytometry,and cell morphology observation showed that caspase-3 silencing alone did not cause cleavage of GSDME.Furthermore,LDH,ATP,and flow cytometry detection showed the degree of pyroptosis was significantly reduced.Pyroptosis was suppressed when cells were treated with si-GSDME;When simultaneously silencing GSDME and caspase-3 genes,the degree of pyroptosis further decreases.The above results confirm that DHAV-1 induced activation of caspase-3 and cleavage of GSDME in DEF cells,pyroptosis induced by DHAV-1 relies on caspase-3 and GSDME.2.Screening of the viral proteins responsible for cell pyroptosisAt present,research on viral proteins of DHAV-1 regulate cell pyroptosis is still blank.Therefore,expression plasmid of all structural protein genes and non-structural protein genes of DHAV-1 were constructed,and viral proteins involved in cell pyroptosis were screened through the dual-luciferase reporter gene assay.These plasmids were co-transfected with caspase-3 luciferase reporter plasmid(p GL-3-caspase-3)and internal reference granules(p RL-TK)to DEF cells,to detect the luciferase activity.The results showed that DHAV-12A2 significantly upregulated caspase-3 activation,and led to the cleavage of endogenous GSDME,showing a dose-dependence;Furthermore,a series of truncation mutant plasmids of DHAV-1 2A2 gene were constructed to detect the luciferase activity.This indicates that DHAV-1 2A2 protein participates in GSDME-mediated pyroptosis by activating caspase-3,and the site of action of 2A2 protein is located between amino acid residues 1-130.DHAV-12A2 protein is used as bait protein,high-performance liquid chromatography-mass spectrometry(HPLC-MS/MS)technology was used to screen host proteins in DEF cells.The Flag-2A2 recombinant expression plasmid was transfected into DEF cells by a liposomemediated method for Co-IP analysis,and HPLC-MS/MS technology was used to screen host proteins that interact with 2A2 protein.Studies have found that activated MAVS can bind to NLRP3,leading to the activation of caspase-1.Activation of NLRP3 inflammasomes requires MAVS and ASC,suggesting that MAVS may be related to cell pyroptosis.Therefore,we selected MAVS as the next research object to delve into the mechanism of the interaction between MAVS and DHAV-1 2A2 protein.The recombinant plasmid Flag-2A2 was co-transfected with MAVS-Myc into BHK-21 cells by a liposome-mediated method for Co-IP assay.The results showed that the MAVSMyc protein was successfully captured in the experimental group,indicating the interaction between 2A2 protein and MAVS protein in vivo;Subsequently,the MAVS-Myc was transfected into BHK-21 cells,and the fusion proteins GST-2A2 was generated through prokaryotic expression for GST pull-down assay.The successful capture of MAVS Myc protein further confirms the interaction between 2A2 and MAVS protein in vitro.To analyze the key regions of interaction between MAVS and 2A2 protein,the recombinant expression plasmid MAVS-Myc was co-transfected with 2A2 truncation mutant plasmids into BHK-21 cells for Co-IP analysis.The results showed that the interaction region between 2A2 and duck MAVS protein is located at amino acid positions 1-130 of 2A2.Then a series of MAVS truncation mutant plasmids were constructed.The recombinant expression plasmid Flag-2A2 was co-transfected with MAVS1-Myc,MAVS2-Myc,and MAVS3-Myc into BHK-21 cells for Co-IP analysis.It was found that the interaction region between 2A2 and duck MAVS protein is located at the transmembrane domain of MAVS.The above experimental results fully confirm that DHAV-1 non-structural protein 2A2 interacts with duck MAVS.3.Interaction between MAVS and NLRP3 mediates DHAV-1-induced pyroptosisTo investigate the effect of MAVS on NLRP3 inflammasome,truncation mutant plasmids MAVS-ΔTM and NLRP3-ΔLRR were constructed,verified the interaction between MAVS and NLRP3 through Co-IP.The results showed that MAVS-ΔTM cannot bind to NLRP3,indicating that the mitochondrial localization of MAVS is crucial for the interaction between MAVS and NLRP3.The interaction between MAVS and NLRP3 is located on the outer membrane of mitochondria rather than in the cytoplasm;NLRP3-ΔLRR can bind to MAVS,indicating that the region of interaction between MAVS and NLRP3 is located in the PYDNACHT domain of NLRP3.Subsequently,si-MAVS and si-NLRP3 were constructed to inhibit the expression of MAVS and NLRP3.After DHAV-1 infection,DEF cells were collected for detection of endogenous caspase-3 and GSDME cleavage,and the expression level of cytokine IL-18 and IL-1β in the cell supernatant was detected.It was found that knocking down the expression of MAVS or NLRP3 significantly inhibited the cleavage of caspase-3 and GSDME,and the release level of IL-18 and IL-1β significantly decreased.This indicates that MAVS and NLRP3 are crucial for the activation of caspase-3 and the cleavage of GSDME during DHAV-1 infection.DHAV-1 infection leads to MAVS-and NLRP3-dependent caspase-3 activation,and the "inflammatory factor storm" caused by DHAV-1infection is also related to the activation of NLRP3 inflammasomes.The above results all demonstrate that MAVS participates in cell pyroptosis caused by DHAV-1 infection by binding to NLRP3.MAVS and NLRP3 are crucial for the initiation of pyroptosis,and the release of inflammatory cytokines is also related to the activation of the NLRP3 inflammasome.This study suggests that DHAV-1 infection promoted the interaction between DHAV-12A2 and MAVS,and further promotes the interaction between MAVS and NLRP3,thereby activating caspase-3.The activated caspase-3 not only cleaves GSDME to initiate pyroptosis but also promotes the release of IL-1β and IL-18,and then triggers the inflammatory response.