Cloning And Immune Functional Characterization Of MAVS,TRAF5 And Their Splicing Variants In Large Yellow Croaker Larimichthys Crocea

As an adaptor protein of retinoic acid inducible gene I(RIG-I)-like receptors(RLRs),mitochondrial antiviral signaling protein(MAVS)plays an important role in host antiviral immune signaling pathways in vertebrates.Notably,with the findings of MAVS splicing variants in teleost fish,the role of MAVS and its splicing variants in host immune response is attracting widespread interest.TNF receptor-associated factor 5(TRAF5),which can interact with MAVS,plays an important role in MAVS mediated antiviral immune signal pathway.In the present study,the MAVS,TRAF5 and their splicing variants of large yellow croaker were cloned and identified.The expression patterns of them in various tissues/organs of healthy fish and that stimulated with different pathogen-associated molecular patterns(PAMPs)were detected by q RT-PCR.Additionally,the subcellular localizations of MAVS,TRAF5 and their splicing variants were revealed by using fluorescent tracer plasmids.And the immune functions as well as signaling pathways that involved were preliminarily determined by dual-luciferase reporter assays.In the present study,the typical form of MAVS and its splicing variant were cloned and identified,named as Lc-MAVS＿tv1 and Lc-MAVS＿tv2,respectively.Lc-MAVS＿tv1 protein is composed of caspase activation and recruitment domains(CARD),roline-rich region(PRR)and trans-membrane(TM)domain,whereas Lc-MAVS＿tv2 lacks the TM domain.q RT-PCR analysis revealed that Lc-MAVS＿tv1 was broadly expressed in the examined organs/tissues and showed extremely higher level than that of Lc-MAVS＿tv2.Additionally,the expression levels of LcMAVS＿tv1 and Lc-MAVS＿tv2 in gills,spleen and head kidney were significantly up-regulated under the stimulation of poly I:C,LPS,PGN,and Pseudomonas plecoglossicida,suggesting the roles of Lc-MAVS＿tv1 and Lc-MAVS＿tv2 in the host immune response.Lc-MAVS＿tv1 was identified as a mitochondrion localized protein whereas Lc-MAVS＿tv2 exhibited an entire cytosolic distribution.Both Lc-MAVS＿tv1 and Lc-MAVS＿tv2 overexpression could significantly induced nuclear transcription factor-κB(NF-κB)promoter activation,with Lc-MAVS＿tv2 induced a relative higher level of NF-κB promoter activation.Lc-MAVS＿tv1 but not Lc-MAVS＿tv2 could induce the activation of interferon regulatory factor 3(IRF3)promoter.Conspicuously,the coexpression of Lc-MAVS＿tv1 and Lc-MAVS＿tv2 significantly enhanced the activation of NF-κB and IRF3 promoter.Both Lc-MAVS＿tv1 and Lc-MAVS＿tv2 have a synergistic effect with TRAF3/5/6 on the induction of NF-κB promoter.However,Lc-MAVS＿tv2 can significantly inhibit the induction of TRAF3/5/6 on IRF3 promoter activation,indicating that Lc-MAVS＿tv2 may negatively regulate the activation of IRF3 signaling pathway mediated by TRAF3/5/6.Additionally,the typical form of TRAF5 as well as its splicing variant were also cloned and identified,named Lc-TRAF5＿tv1 and Lc-TRAF5＿tv2,respectively.Lc-TRAF5＿tv1 protein is composed of RING Finger domain,Zine Finger domain,coiled coid domain and MATH domain,while Lc-TRAF5＿tv2 contains only RING Finger domain.Lc-TRAF5＿tv1 was broadly expressed in the examined organs/tissues and showed extremely higher level than that of Lc-TRAF5＿tv2,and the expression levels of Lc-TRAF5＿tv1 and Lc-TRAF5＿tv2 in gills,intestine and head kidney were significantly up-regulated under poly I:C,LPS,PGN,and Pseudomonas plecoglossicida stimulation,indicating the roles of Lc-TRAF5＿tv1 and Lc-TRAF5＿tv2 involved in the host immune response.Subcellular localization analysis showed that Lc-TRAF5＿tv1 was evenly distributed in the cytoplasm,whereas Lc-TRAF5＿tv2 was also identified as a cytoplasmic protein but with apparently brilliant spots around the nucleus.Both Lc-TRAF5＿tv1 and Lc-TRAF5＿tv2overexpression could significantly induced NF-κB and IRF3 promoter activation,with LcTRAF5＿tv1 induced a higher level compared to that of Lc-TRAF5＿tv2 in NF-κB promoter activation.Conspicuously,Lc-TRAF5＿tv2 can significantly inhibit the induction activity of LcTRAF5＿tv1 on NF-κB promoter,suggesting that Lc-TRAF5＿tv2 may negatively regulate the activation of NF-κB signaling pathway mediated by the typical TRAF5 in large yellow coraker.