Xu Ya-Ping, Zhou Yi-Lian, Xiao Yi, Gu Wen-Bin, Li Bo, Cheng Yuan-Xin, Li Bing-Wu, Chen Da-Yong, Zhao Xiao-Feng, Dong Wei-Ren, Shu Miao-An
College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China.
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 310036, China.
Dev Comp Immunol. 2021 Jun;119:104015. doi: 10.1016/j.dci.2021.104015. Epub 2021 Jan 16.
Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-β promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-β promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-β promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.
肿瘤坏死因子受体相关因子3（TRAF3）是连接上游受体信号和下游抗病毒信号通路的关键转导蛋白。以往的研究大多阐明了TRAF3在哺乳动物、鸟类和鱼类中的功能，但对两栖动物中TRAF3的特性和功能了解甚少。在本研究中，对中国大鲵Andrias davidianus中两个TRAF3基因AdTRAF3A和AdTRAF3B进行了分子和功能鉴定。AdTRAF3A和AdTRAF3B的完整开放阅读框（ORF）长度分别为1698 bp和1743 bp，分别编码565和580个氨基酸。AdTRAF3A和AdTRAF3B推导的蛋白质均包含一个环状结构域、两个TRAF型锌指结构、一个卷曲螺旋结构和一个MATH结构域。系统发育分析表明，AdTRAF3蛋白与其他已知的TRAF3蛋白聚集在一起。基因表达分析表明，AdTRAF3s广泛分布于所有检测组织中，分布模式相似。血液或脾脏中的AdTRAF3s对大鲵虹彩病毒（GSIV）和聚肌苷酸:聚胞苷酸（poly (I:C)）诱导呈阳性反应，但表现出不同的反应模式。当白细胞用DNA病毒和病毒RNA类似物处理时，沉默AdTRAF3A/B显著抑制了IFN信号通路相关基因的表达。此外，AdTRAF3A的过表达可能诱导IFN-β启动子的激活，AdTRAF3A的锌指、卷曲螺旋和MATH结构域对于IFN-β启动子的激活至关重要。然而，AdTRAF3B的过表达显著抑制了IFN-β启动子活性，当环状结构域或MATH结构域缺失时，其抑制作用增强。此外，AdTRAF3A而非AdTRAF3B显著诱导NF-κB激活，这意味着AdTRAF3A可能在IFN和NF-κB信号通路中均起增强子的作用。综上所述，我们的结果表明，这两个TRAF3基因在中国大鲵的先天抗病毒免疫中发挥着不同的关键调节作用。
