Study On The Antiviral Mechanism Of EV71 Escape Inflammasome AIM2 Mediated By 2A Protease

Enterovirus 71(EV71)is one of the main pathogens causing hand,foot,and mouth disease(HFMD)in infants and young children.In addition,EV71 infection can also cause central nervous system diseases such as aseptic meningitis,acute flapper paralysis,and neurogenic pulmonary edema.At present,the specific mechanism of innate immunity triggered by EV71 infection has been widely studied,but the virus also escapes to damage the body accordingly.Therefore,it is essential to further study the relationship between innate immunity and viral immune escape.The aim of this study is to investigate how EV71 escapes the antiviral effect of AIM2 inflammasome through autoprotease 2A.First,RD and Hela cells were infected with EV71,and AIM2 expression at protein and m RNA levels was detected by Western-Blot and q PCR,respectively,to clarify the activation of AIM2 after EV71 infection.Western Blot,q PCR and ELISA were used to detect the activation of AIM2 inflammasome downstream genes Caspase-1 and IL-1β to confirm AIM2 inflammasome activation after EV71 infection.The expression of VP1 and AIM2 protein was detected by immunofluorescence assay.The results showed that the expression of AIM2 protein increased 6h after infection and decreased 24 h after infection.To explore the role of AIM2 after EV71 infection,we used virus infection of primary neuronal cells isolated from AIM2 knockout neonatal mice and wild-type neonatal mice.We found that AIM2 knockout cells had a higher viral load in the supernatant of infected neuronal cells compared with wild-type neuronal cells.The replication ability of EV71 decreased,which indicated that AIM2 played a protective role in the body after EV71 infection.Surprisingly,when we examined AIM2 protein after EV71 infection at the protein level,we found an additional cleaved band,about 20 k D in size,After treatment with inhibitors such as Z-VAD-YMK,MG132 and NH4 CL,AIM2 cleavage was found to be Caspase-,proteasomal-and Caspase pathway-dependent.Therefore,when EV71 protease 3C and2 A were co-transfected into 293 T cells in vitro with AIM2 plasmid,we found that AIM2 cleavage was not dependent on caspase pathway.The cleavage of AIM2 is mediated by2 A protease.The protease activity of 2A-mutant was found to be necessary for the cleavage of AIM2 by co-transfection with AIM2 plasmid.We constructed three AIM2 mutants G148A,G172 A,and G217 A by cutting the size of the band and combining the2 A protease action site.We cotransfected each of the three mutants with the 2A plasmid and found that the mutated AIM2 was still cleaved by 2A.This suggests that 2A protease cleaves AIM2 protein at G172-K173 and G217-F218.In conclusion,our findings suggest that EV71 infection activates AIM2 inflammasome and its downstream pathways,which in turn facilitates viral replication by cleavage of AIM2 protein by autoprotease 2A.These findings provide new insights into the interaction between EV71 and innate immunity.