Mechanism Study Of TNF Receptor-associated Factor 3 On Regulating Influenza A Virus Replication

Background:Influenza viruses,especially Influenza A virus(IAV),which can cause global pandemics,existinng in nature for a long time.Due to the rapid mutation rate of IAV antigen and the oning challenge of resistance to existing antiviral drugs,it is urgent to develop new and effective prevention and treatment methods for anti-influenza therapy.When the virus infects the host,the immune system of host will be activated instantly through a variety of mechanisms,among which innate immunity is the first line of defense against the invasion of exogenous pathogens.With the in-depth study of innate immunity,a series of innate immune regulatory factors have been discovered to have antiviral function,which attracted widespread attention.Recently,more and more studies have shown that innate immune response or innate immune regulatory factors play an important role in the antiviral process of host,which provide new targets for antiviral therapy.Tumor necrosis factor receptor-associated factor 3(TRAF3)is one of the important host factors in antiviral innate immune response.Several studies have found that TRAF3 can regulate virus infection through the activation of downstream signaling pathways.Besides,TRAF3 can also interact with viral proteins,which directly interfering with virus replication.Due to the common characteristic of innate immune responses to different viral infections,TRAF3 may have the potential to be developed as a new antiviral target.Objectives:This study aims to clarify the effect and mechanism of TRAF3 on regulating influenza A virus replication,and elucidate the role of TRAF3 and its key domains in the innate immune response stimulated by influenza A virus.Methods:1.Western blotting,qRT-PCR,plaque formation experiment,mini-replicon assay were measured to verify the regulation of IAV replication by knockdown(RNA interference,RNAi)and overexpression of TRAF3.2.Western blotting and qRT-PCR were used to illuminate the effect of IAV infection on the intracellular expression of TRAF3.3.Western blotting,qRT-PCR,and promoter activation assay were performed to detect the effect of TRAF3 on the signal pathway activation and cytokines production triggered by IAV infection.4.Western blotting was used to measured the effect of TRAF3 overexpression on IRF3 phosphorylation.5.A549 stable cell lines with deletion of different domains in TRAF3 were constructed to verify the function of key domains of TRAF3 on regulating IAV infection.Results:1.Plaques formation,gene and protein expression levels after IAV infection were increased in TRAF3 knockdown experiment.On the contrary,these indexes were decreased in the condition of TRAF3 overexpression.2.IAV infection could degrade intracellular protein expression of TRAF3,while the degradation is not related to ubiquitination degradation pathway and autophagylysosomal degradation pathway.AT406,the smac mimics(cIAP antagonists)could inhibit TRAF3 degradation,resulting in the reduction of influenza virus protein expression and increase of type-I IFNs gene expression.3.TRAF3 knockdown by RNAi experiment decreased the expression of downstream antiviral cytokines and IFN-β mRNA,and weakened the activation of IFN-β promoter.Furthermore,TRAF3 overexpression could promote the phosphorylation of IRF3.4.The deletion of the RING domain did not affect the anti-influenza effects mediated by TRAF3,while the deletion of the remaining domains(Zinc Finger,Coiled-coil and MATH domains)weakened the anti-influenza effects of TRAF3.Conclusion:1.TRAF3 could regulate IAV infection intracellularly.2.IAV infection could degrade expression of TRAF3 protein.Inhibiting TRAF3 degradation could decrease expression levels of influenza proteins and increase type-ⅠIFNs.3.The regulatory effect of TRAF3 on IAV infection is associate with the type-Ⅰ IFNs signaling pathway.4.The Zinc Finger,Coiled-coil,and MATH domains of TRAF3 are important in its regulation.