Mechanism Study On Innate Immune Evasion Mediated By The Effector Domain Of NS1 Protein Of Influenza A Virus

Influenza A virus(IAV)infection induces rapidly host innate immune response.IAV triggers the activation of signaling pathways that are dependent on host pattern recognition receptors(PRRs)including toll like receptors(TLRs)and RIG-I like receptors(RLRs),which leads to the secretion of type I interferons and other cytokines.Type I IFNs stimulate hundreds of genes expression that are collectively known as IFNstimulated genes(ISGs)in cells,which induce an antiviral state.IAV develops a variety of strategies to evade the host immune response during co-evolution with the host.Nonstructural protein 1(NS1)is a well-known anti-IFN factor encoded by IAV.NS1 can targets ds RNA,RIG-I and E3 ligases TRIM25 and Riplet,antiviral ISGs,and host cell m RNA synthesis to inhibit the host IFN response.In recent years,many advances have been made in NS1 antagonistic mechanisms,but there are still many questions that have no accurate answers and deserve further study.In this study,we segmented the functional domain of NS1 protein of the H5N1/HM influenza virus to explore the role of the N-terminal RNA-binding domain(RBD)and the C-terminal effector domain in inhibiting RIG-I signaling pathway.1.The H5N1 NS1 protein inhibits the RIG-I-mediated activation of IFN-? via its Cterminal effector domainFour truncated H5N1 NS1 proteins,including NS1/1-73,NS1/74-225,NS1/1-125,and NS1/126-225,were generated to investigate the effect on the IFN-? promoter activity.We found that wt NS1,NS1/74-225 and NS1/126-225,but not NS1/1-73 and NS1/1-125,significantly decreased the IFN-? reporter activities driven by RIG-I or RIG-I(N).Conversely,wt NS1 and all truncated peptides had inhibitory effects on IFN-? reporter activities induced by viral infection or transfection with ds RNA.This suggests that NS1-RBD is sufficient to inhibit the activation of IFN-? only in the presence of ds RNA and NS-ED inhibits IFN response in an RNA binding-independent manner.Subsequently,we addressed the affect of NS1/126-225 on the phosphorylation,dimerization,nuclear localization of endogenous IRF3 and the secretion of IFN-? mediated by RIG-I(N),further confirm the inhibitory effect of NS1/126-225 on IFN-? production.2.NS1/126-225 binds TRAF3 to block the formation of MAVS-TRAF3 complexWe speculate that the NS1/126-225 protein can intimidate one component of the RLR pathway.By using luciferase reporter assay,Real-Time PCR and ELISA assay,we proved that NS1/126-225 inhibits IFN-? signaling pathway at the level between MAVS and TBK1.Co-IP experiments showed that NS1/126-225 interacted with TRAF3,as well as wt NS1.We also conformed the association between NS1 and endogenous TRAF3 when H5N1/HM infection with A549 cells,HEK293 T cell and primary mouse macrophages.However,the interaction of wt NS1 and TRAF3 is strain-specific.In cells with silenced TRAF3,after transfection of RIG-I(N),the inhibitory effect of NS1/126-225 on IRF3 phosphorylation and IFN-? transcription was markedly attenuated,confirming that TRAF3 is essential for NS1/126-225 to down-regulate IFN-?.Mechanistically,NS1/126-225 binds to the TRAF domain of TRAF3 and dissociates the MAVS-TRAF3 complex.In addition,NS1/126-225 increases the recruitment of IKK? to MAVS following Se V infection,ultimately shutting down the RIG-I signal transduction and cellular antiviral responses.3.NS1/126-225 suppresses TRAF3 K63-linked ubiquitinationNS1/126-225 also inhibits the K63-linked ubiquitination of TRAF3.It is likely that NS1/126-225 works through recruiting a deubiquitinase to cleave the TRAF3 ubiquitin chain or blocking E3 ligase bind to TRAF3 since it has been shown that NS1/126-225 does not have the ability to regulate protein ubiquitination.The DEAD-box RNA helicase DDX3 was reported to regulate TRAF3 K63-linked ubiquitination and interact with NS1.In our study,knockdown of DDX3 by si RNA,NS1/126-225 still inhibits the K63-linked ubiquitination of TRAF3,whereas viral infection did not reduce TRAF3 K63-linked ubiquitination when compared to control cells,suggesting that NS1 utilize DDX3 to inhibit the K63-linked ubiquitination of TRAF3.4.NS1 mutant HM/Mut virus induces elevated levels of IFN-?A recombinant influenza virus expressing an NS1 protein lacking the effector domain(126 to 225 aa deletion,designated HM-Mut)result in attenuation of viral growth in A549 cells and the induction of high level of IFN-? and ISGs transcripts compared to the wildtype virus.Moreover,we demonstrated that N-terminally truncated PR8 NS1 protein t NS1 interacted with TRAF3 and inhibited the production of IFN-? and ISGs.The ability of interferon antagonistic and replication of mutant virus PR8/79.81 I was not significantly different from that of wild-type virus in the TRAF3 knockdown cells,which is used to perfect the mechanism we provide.Our study is the first time to elucidate the mechanism of how the c-terminally effector domain(ED)of NS1 protein evades host innate immune response.Our findings offer an important insight into the interplay between the influenza virus and host innate immunity and also provide a potential target for the development of antiviral drugs.