The Effect And Mechanism Of NLRP3 Inflammasome Inhibition By SARS-CoV-2 M Protein

Coronavirus Disease 2019(COVID-19)is caused by Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).It has become the largest global pandemic,seriously endangering people’s health and social economy.However,the pathogenic mechanism of SARS-CoV-2 is still unclear,and revealing its pathogenic mechanism is important for the prevention and control of COVID-19.SARS-CoV-2 belongs to the family Coronaviridae,the genus βcoronavirus,and is a class of single-stranded positive-stranded RNA viruses with envelopes with a genome size of about 29.7 kb,including the 5’UTR,non-structural protein-coding region,structural protein-coding region,accessory protein-coding region and 3’UTR.It encodes for 16 non-structural proteins,4 structural proteins,and about 9 accessory proteins.The protein encoded by SARS-CoV-2 is not only involved in viral invasion,but also involved in the replication of viral genome,protein translation and packaging of mature viral particles,and plays an important role in innate immune signaling pathways.SARS-CoV-2 infection generally involves viral invasion and replication,dysregulated immune response,and multisystem organ damage.When viruses infect host cells,it can play a regulatory role on the NLRP3 inflammasome.The activation of inflammasome induced by viral infection has two sides.On the one hand,proper activation can activate downstream mechanisms,thus inhibiting viral replication,and on the other hand,excessive activation can trigger excessive inflammatory response.The NLRP3 inflammasome is the most widely studied classical inflammasome,which is an important part of the natural immune system and plays an important role in the resistance to viral infection and inducing inflammatory response.It has been reported that SARS-CoV-2 infection leads to the activation of NLRP3 inflammasome,inducing the massive production of inflammatory cytokines such as IL-1β,promoting the formation of cytokine storm,thus inducing acute respiratory distress syndrome(ARDS),acute pulmonary edema(ALI),cardiovascular diseases such as myocarditis,and nervous system diseases.As we all know,NLRP3 inflammasome consists of the NLRP3 receptor,the ASC,and pro-caspase-1.NLRP3 inflammasome activation can be generally divided into two steps:initiation and activation.In the initiation phase,pathogen-associated molecular pattern(PAMP)or damage-associated molecular pattern(DAMP)interacts with Toll-like receptor(TLR),leading to the initiation of nuclear transcription factor κB(NF-κB)to induce the expression of interleukin gene and NLRP3.During the activation phase,NLRP3 receptors first undergo deubiquitination and self-aggregation,recruiting ASC and oligomerization,binding pro-caspase-1 to form large inflammatory complexes,leading to self-cleavage of pro-capase-1.Activated caspase-1 induces lysis of IL-1β and IL-18 precursors,and mature cytokines are released outside the cell,triggering the inflammatory immune response,while activated caspase-1 binds and cleaves Gasdermin D(GSDMD)to induce pyroptosis and inflammatory response.In this study,on the basis of the original SARS-CoV-2 encoded protein particles in the laboratory,we systematically screened for the effect of the novel coronavirus encoded protein on the NLRP3 inflammasome activation.First,we constructed the in vitro NLRP3 inflammasome system in 293T cells by co-transfected expression plasmids encoding NLRP3,ASC,pro-caspase-1 and pro-IL-1β.Then,we detected the effect of the novel coronavirus-encoded protein on the NLRP3 inflammasome,and it was found that multiple proteins encoded by the virus could regulate the NLRP3 inflammasome.Further by verification,we found that the structural proteins N and S could promote the activation of NLRP3 inflammasome,and M,ORF7,ORF8 and NSP14 could significantly inhibit the activation of NLRP3 inflammasome.The mechanism of structural proteins N and S for NLRP3 inflammasome activation has been reported,but the mechanism of viral inflammasome inhibition is not clear,so we subsequently focused the inhibitory effect of M protein on NLRP3 inflammasome and elucidated the molecular mechanism of its effect.First,the result of co-immunoprecipitation showed that M protein could interact with NLRP3,and the immunofluorescence results indicated that M protein colocalized with NLRP3.Colocalization of M protein with NLRP3 inhibited Nig-mediated NLRP3 vesicle formation,suggesting that M protein may inhibit Nig-mediated NLRP3 activation.This study initially revealed the mechanism of SARS-CoV-2 infection to inhibit the NLRP3 inflammasome,providing a basis for elucidating the pathogenic mechanism of SARS-CoV-2 infection.