Effects Of TRIM 52&67 On The Replication Of Japanese Encephalitis Virus And Molecular Mechnisms Of Their Regulation On NF-?B Activation

The innate immune system is a universal and crucial form for host defensing against infection,which also plays an important role on developing the adaptive immune system.Tripartite motif-containing(TRIM)proteins constitute a super family with a conserved RBCC construct.More than 70 TRIM members have been identified in human being.TRIMs participate in various cellular processes and play an important role in host antiviral function.TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity,or indirect by promoting host innate immunity.Such as,TRIM25 induces the K63-linked ubiquitination of RIG-I CARD doamin,which is important for RIG-I-mediated interferon production to elicit host antiviral innate immunity,TRIM56 and TRIM32 targeted MITA for K63-linked ubiquitination to facilitate the interaction of MITA and TBK1,which is important for host innate immunity against both DNA and RNA virus.TRIM22 inhibits IAV and HCV replication by targeted NP protein of IAV,and NS5 A of HCV for degradation.Currently,many TRIMs have been reported confer direct antiviral activity.Malfavon-Borj et al.suggested that TRIM52 contains an expanded RING domain,which might indicate that it serves as a novel antiviral TRIM protein.However,TRIM52 has no effect on HIV replication.Thus,there is a need to study whether TRIM52 impacts on replication of other kinds of viruses.Previous studies have reported that TRIM67 is selectively expressed in the cerebellum,which modulates Ras signaling pathway by degradation of 80K-H,resulted in neural differentiation and neuritogenesis.TRIM67 also has been reported that it direct activated IFN? and ISRE promoter activity.However,the role of TRIM67 in NF-kB signaling pathway is still unknown.Therefore,in this study,we investigated the role of TRIM proteins in JEV replication and NF-kB signaling pathway.Moreover,we investigated the antiviral activity of TRIM52 and TRIM67 in JEV infection,and their regulation on NF-kB signaling pathway.1.TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2AJapanese encephalitis virus(JEV),a member of family Flaviviridae,is a neurotropic flavivirus that causes Japanese encephalitis(JE).JEV is one of the most important causative agents of viral encephalitis in humans,and this disease leads to high fatality rates.Although effective vaccines are available,no effective antiviral therapy for JE has been developed.Hence,the study on host anti-JEV is significant.The members of tripartite-motif containing(TRIM)protein participate in various cellular processes and play an important role in host antiviral function.TRIM proteins exert their antiviral activity either direct by degrading viral proteins through their E3 ligase activity,or indirect by promoting host innate immunity.This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus(JEV)infection.Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293 T cells.In addition,JEV nonstructural protein 2A(NS2A)is a protein that interacts with TRIM52.Their interaction degraded NS2 A in a proteasome-dependent manner via the E3 ligase activity of TRIM52.Thus,TRIM52 is a novel antiviral TRIM protein,and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2 A.2.Overexpression of E3 ubiquitin ligase TRIM52 activates the nuclear factor-kappa B signaling pathwayEmerging evidences suggest that TRIM family proteins play a crucial role in regulation of NF-?B signaling pathway.TRIM52 is a novel noncanonical antiviral TRIM gene with unique expanded RING domain.We know less about the biological function of TRIM52.Here we demonstrated TRIM52 involved in the activation of NF-?B.We found that overexpression of TRIM52 specifically activates NF-?B signal pathway.TRIM52 overexpression could significantly induce the expression of TNF? and IL-6.We also found that the RING domain of TRIM52 is essential for it activating NF-?B signal.Further study showed that overexpression of TRIM52 has no effect on the protein level of I?B? and phosphorylated p65 protein.We also found that pro-inflammatory cytokine TNF? and IL-6 could induce TRIM52 expression.Thus,these data suggested that TRIM52 was a positive regulator of NF-?B pathway.3.Tripartite motif 67(TRIM67)inhibits TNF?-triggered NF-?B activation through binding ?Tr CPThe transcription factor NF-?B plays an important role in modulation of the inflammatory pathway,which was associated with inflammatory diseases,neurodegeneration,apoptosis,immune responses,and cancer.There is increasing evidence indicated that TRIM proteins play a crucial role in regulation of NF-?B signaling pathways.In this study,we demonstrated that TRIM67 is a negative regulator of TNF?-triggered NF-?B signaling pathway.Overexpression of TRIM67 significantly repressed TNF?-induced NF-?B activation and expression of TNF? and IL-6.RING-B-box domain of TRIM67 is required for inhibiting TNF?-triggered NF-?B activation.Finally,we identified ?Tr CP as an interaction protein of TRIM67.We also found that the C-terminus of ?Tr CP and RING-B-box domain of TRIM67 contributed to the interaction between ?Tr CP and TRIM67.Thus,our findings revealed that TRIM67 serves as a negative regulator of TNF?-triggered NF-?B activation by binding ?Tr CP.