Mechanism Of SARS-CoV-2 M Protein Inhibiting The Production Of Type ? Interferon Through Ubiquitination-induced TBK1 Degradation

Background:Since the outbreak of novel coronavirus pneumonia(COVID-19)in 2019,it has caused a large number of infections and deaths worldwide,posing a great threat to global health.After the virus invades host cells,it will induce a series of natural antiviral immune responses in the host,of which the most important are the non-specific cytokine antiviral responses mediated by type I and type III interferons.The RLRs signaling pathway mediates the innate immune response of most RNA viruses(including SARS-CoV),and ubiquitination plays an important role in the regulation of RLRs-mediated signaling pathways.But in the process of interacting with the host's immune system,the coronavirus has established a series of mechanisms that suppress the host's natural immune response and enhance self-replication.The M protein of SARS-CoV-2 is a glycosylated transmembrane protein that is essential for viral particle assembly,membrane fusion,and neutralizing antibodies,and has been shown to antagonize interferon production,but its The mechanism by which immune escape is exerted is unclear.In order to further reveal the immune escape mechanism of SARS-CoV-2,this study deeply explored the interferon antagonistic function of SARS-CoV-2 M protein,and revealed the mechanism of its antagonism of interferon.Lay the theoretical foundation.Method:In this study,the dual luciferase reporter gene detection and RT-PCR experimental methods were used to first determine the phenotype of M protein to antagonize the production of interferon;PCR and other experimental methods,screened out the key transduction signal proteins involved in the immune escape process of M protein anti-interferon production;by adding various degradation pathway inhibitors and experimental methods of co-immunoprecipitation,to explore the positive and negative regulation of key signal molecules.How this process is regulated in a complex network,and its domains that play a role in immune evasion.Research result:In this study,we found that the SARS-CoV-2 M protein was confirmed to inhibit the expression of IFN? and the interferon-stimulated gene ISRE induced by RIG-I,MDA5,IKK? and TBK1,and at the same time,inhibit the phosphorylation of IRF3 caused by TBK1,Dimerization and nuclear translocation,can also interact with MDA5,TRAF3,IKK?,and TBK1,and induce TBK1 degradation through K48-linked ubiquitination,further inhibiting the formation of the TRAF3-TANK-TBK1-IKK?complex,leading to I IFN production is inhibited.Conclusion:1.M protein was confirmed to inhibit the expression of IFN? and interferonstimulated gene ISRE induced by RIG-I,MDA5,IKK? and TBK1.2.M protein inhibits IRF3 phosphorylation,dimerization and nuclear translocation activated by TBK1.3.M protein can interact with MDA5,TRAF3,IKK?,and TBK1,and induce the degradation of TBK1 through K48-linked ubiquitination,further inhibiting the formation of TRAF3-TANK-TBK1-IKK? complex,resulting in the production of type I interferon.inhibition.