The Mechanism Of Enterovirus 71 Type Inhibiting GSDMD-mediated Cell Pyroptosis

Hand-foot-mouth disease(HFMD)is a common disease among children and it can be caused by multiple virus.It has been reported tha the main pathogen is the Coxsackie virus A16(CV-A16)and Enterovirus 71 type(EV71)belong to the Enterovirus genus.Unlike CV-A16,EV71 infection is more likely to result in neurological complications and even death in severe cases.The first time EV71 was isolated in California,outbreak of EV71 appeared in many regions of the world one after another.The public health problem of how to prevent EV71 and other virus infection to cause HFMD is an urgent need to be solved.At present,there are not specific drugs applied to clinical treatment.Therefore,intense research of the pathogenesis of EV71 is needed in search of the effective antiviral drugs.Once EV71 attacked the host,it can be recognized by the specific pattern recognition receptor.The production of interferon and inflammatory factors resulted from the cell signal transduction pathways downstream,forming the host denfense against the virus infection.Meanwhile it can escaped the response of host innate immunity,which is significant in the pathogenesis of EV71 infection.Inflammsomes are vital component of the innate immunity,equally important in the defense against virus infection.Previous studies have indicated that NLRP3 inflammasome can be activated by EV71 infection.The viral protease 2A and 3C can cleave NLRP3 inflammasome,antagonizing the activity of inflammasome,finally controlled the release of inflammatory factor IL-1β.Recent reports have found GSDMD,the NLRP3 inflammasome pathway downstream,can be cleaved by caspase-1 and its N-terminal fragment 1-275aa induce cell pyroptosis.So,we guessed whether the infection with EV71 can regulate GSDMD?We have showed that infection of EV71 in RD,Hela and THP-1 cells,can decrease the expression of GSDMD,what is more,viral protease 3C can cleave GSDMD depending on its protease activity.However,3C could not cleave GSDMD with the mutation of the key site of protease 3C.We constructed the mutation of the potential cleavage site within GSDMD.Q193-G194 pair is a major cleavage site of 3C within GSDMD,thus the N-terminal fragment 1-193 and the C-terminal fragment 1-193 emerged.A functional study of the fragments by 3C has indicated that the fragment of 1-193 could not induce cell pyroptosis or affect virus replication,but the fragment 1-275aa cleaved by caspase-1 induced cell pyroptosis and inhibited virus replication.The key site of 1-275aa-induced cell pyroptosis have not been determined,then we constructed a variety of mutants and confirmed that T239 and F240 amino acids are key sites for 1-275-induced pyroptosis.In summary,we demonstrated that the infection of EV71 could decrease the expression of GSDMD,and EV71 3C could directly cleave GSDMD.The cleavage fragment of GSDMD by 3C could not induce cell pyroptosis,further explained the relationship between virus infection and cell death,and provided different theoretical foundation to clarify the pathogenesis of EV71.