| The global pandemic of SARS-CoV-2 in 2020 has brought unprecedented public health and safety problems to the world,and has caused a tremendous impact on the world economy.A comprehensive understanding of the infection characteristics of the virus,especially the biological characteristics of the pathogen itself and its interaction with the host cellular immune system,is of great significance to reveal the pathogenic mechanism of SARS-CoV-2.Now many studies and clinical reports have shown that SARS-CoV-2 infection can inhibit innate immunity and subsequent adaptive immune mobilization.In this process,the interaction between virus and host cells produces a series of molecular events,including viral genome replication,the regulation of cellular innate immune molecules by structural and non-structural proteins,which will affect the immune system.Previous studies on SARS-CoV suggest that the membrane protein(M)and nucleocapsid protein(N)in the structural protein of the virus can inhibit the innate immune energy.At present,many literatures have reported that the M protein of SARS-CoV-2 also has the effect of innate immunosuppression,but there are few reports on N protein.The purpose of this study is to further explore the effect of N protein of SARS-CoV-2 on innate immunity in the process of infection.In this paper,we first studied the characteristics of infection and proliferation of SARS-CoV-2 in 16HBE cells in vitro.We found that the early proliferation of SARS-CoV-2 on 16HBE cells was weak.We detected the expression of ISG15,CXCL-10,NF-κB,IFN-a and IFN-β by Real-timePCR and the phosphorylation of IRF3,TBK1 and NF-κB by Western Blot.It was found that the RLRs signal pathway would be suppressed with the progress of virus infection.After that,we overexpressed different SARS-CoV-2 virus protein components in HEK293T cells and detected their effects on the production of IFN-β.The results showed that N protein,as a member of SARS-CoV-2 structural protein,could inhibit the production of IFN-β.Using co-immunoprecipitation assay,we found that the N protein of SARS-CoV-2 could interact with TRIM25 to inhibit the activation of RLRs signal pathway.At the same time,we found that G3BP2 protein could interact with TRIM25 and N protein at the same time.In further experiments,we found that the emergence of N protein can promote the interaction between G3BP2 and TRIM25.In order to further explore the role of G3BP2 in RLRs signaling pathway,we overexpressed G3BP2 in A549 cells.Through the changes of nucleic acid level of related genes and phosphorylation level of related proteins,it was found that the activation of RLRs signal pathway was inhibited,and overexpressed G3BP2 could interact with intracellular TRIM25.We speculated that G3BP2 might inhibit the activation of RLRs signal pathway by interacting with TRIM25,so we used siRNA to knock down G3BP2.the results showed that when G3BP2 was knocked down,it could promote the activation of RLRs signal pathway,and the phosphorylation levels of TBK1,IRF3 and NF-κB were significantly higher than those of control cells,but the production of IFN-β seemed to be inhibited by overactivation of RLRs.Therefore,we speculate that G3BP2 may play other important roles in the activation and regulation of RLRs signal pathway.In further infection experiments in rhesus monkeys,we found that the number of NK cells in rhesus monkeys infected with SARS-CoV-2 was inhibited,which indicated that SARS-CoV-2 could indeed inhibit the activation of innate immunity.Immunofluorescence experiments were carried out on nasopharynx and lung tissue sections of infected rhesus monkeys,and we also found that viral N protein was related to promoting the recruitment and binding of G3BP2 and TRIM25.These results suggest that further elucidation of the mechanism of N protein inhibiting the activation of RLRs pathway may play a guiding role in understanding the pathogenesis of SARS-CoV-2 pulmonary infection and the role of viral nucleocapsid protein in immune escape. |
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