Influenza A Virus NS1 Protein Inhibits Host Antiviral Innate Immune Response Through Inducing Expression Of A20 And MCPIP1

The antiviral innate immune response is a prerequisite for the host's first line of defense against viral invasion and activation of subsequent acquired immunity,and is the focus of competition between the virus and the host,and its outcome determines the survival of the virus and the direction of the viral disease.The virus has the ability to antagonize the host's antiviral natural immune response through a diverse mechanism during evolution.Influenza A Virus(IAV)still poses a serious threat to the health and safety of humans and susceptible animals.Studies have shown that the non-structural protein 1(NS1)encoded by the IAV genome is widely involved in antagonizing the host's antiviral innate immune response and largely determines the strength of IAV virulence,but the molecular mechanism has not yet completely clarified.Zinc finger protein A20 is a ubiquitin-modifying enzyme protein encoded by TNF alpha induced protein 3(TNFAIP3)gene in the cytoplasm.It has ubiquitination and deubiquitination double enzyme activities and participates in the regulation of the body.Immune and inflammatory responses,such as the negative regulation of retinoic acid inducible-gene I(RIG-I)-mediated activation of Interferon regulatory factor 3(IRF3).MCP-1 induced protein-1(MCPIP1)is a protein in which MCP1 induces expression as a transcriptional activator,and it may induce expression of an apoptotic gene leading to cardiomyocyte death.The existing research reports should show that MCPIP1 RNase activity can directly degrade the mRNA of cytokines such as IL-6,IL-12 and IL-2.This study found that IAV NS1 protein can effectively induce the expression of host cell A20 and MCPIP1.The NS1 protein of different IAV strains can induce the expression of A20 in A549 cells,and the increased expression level is positively correlated with the virulence of the virus.Overexpression of A20 in A549 cells significantly inhibited IAV-induced IRF3 and IFN promoter activity,resulting in downregulation of IFN-? and IFN-stimulated genes(ISGs)mRNA.In contrast,silencing A20 expression significantly enhanced IRF3-mediated antiviral immune responses.In addition,A20 overexpression in A549 cells significantly promoted IAV replication,whereas inhibition of endogenous A20 expression by siRNA inhibited viral replication.Proteins such as IAV NS1 and PB-F2 can induce MCPIP1 expression,especially NS1.Functional studies have shown that MCPIP1 can negatively regulate IAV-induced RIGI-dependent natural antiviral immune responses and exhibits a function of promoting viral replication in the middle and late stages of IAV infection in A549 cells.Overexpression of MCPIP1 in A549 cells inhibited the expression of RIG-I mRNA and protein,while siRNA inhibited the expression of endogenous MCPIP1,which enhanced the expression of RIG-I.Further determination of RIG-I mRNA half-life and luciferase assays confirmed that MCPIP1 can accelerate the degradation of RIG-I mRNA.Experiments with mutants transfected with different functional domains of MCPIP1 indicate that MCPIP1 degrades RIG-I mRNA through its RNase activity.Taken together,this study demonstrates a novel mechanism by which IAV NS1 antagonizes the expression of host A20 and MCPIP1 to antagonize the antiviral innate immune response and ultimately contribute to viral replication.