犬瘟热病毒H蛋白与RIG-I互作抑制β干扰素转录活性的研究

Study on interaction between H protein of canine distemper virus and RIG-I can inhibitβ-interferon transcriptional activity

为研究犬瘟热病毒(CDV)H蛋白对β干扰素信号通路的影响,构建不同CDV毒株H蛋白真核表达质粒以及CDV H蛋白不同截短体真核表达质粒,分别与β-IFN-Luc基因报告质粒和内参质粒pRL-TK共转染细胞,用双荧光素酶报告基因试剂盒检测CDV H蛋白对仙台病毒(SeV)诱导β干扰素的影响以及RIG-I(N)、MAVS、TBK1和IKK-i等信号分子在H蛋白介导的β干扰素转录活性的作用,通过激光共聚焦试验和GST-pull down试验检测CDV H蛋白与RIG-I(N)的相互作用。结果,CDV2野毒株H蛋白能够特异地抑制SeV感染诱发的β干扰素转录活性,而且这种抑制作用与RIG-I(N)激活相关。激光共聚焦试验显示,CDV2野毒株H蛋白与RIG-I(N)在细胞质中存在共定位现象;GST-pull down试验证明了CDV2野毒株H蛋白与RIG-I(N)存在直接的相互作用;H蛋白截短表达试验证明其N端结构域(1-462 bp)是抑制β干扰素转录活性的关键结构域。本项目的成功开展为深入研究CDV H蛋白抗宿主天然免疫机制奠定了基础,对该病的预防和治疗具有重要意义。

To study the effect of canine distemper virus(CDV)H protein onβ-interferon signaling pathway,the recombinant plasmids expressing H proteins of different CDV strains and truncated H protein were constructed.The recombinant plasmids were co-transfected into the cells together withβ-IFN-Luc report gene plasmid and control p RL-TK plasmid,respectively.The effect of CDV H protein onβinterferon induced by Sendai virus(Se V)and the roles of signal molecules such as RIG-I(N),MAVS,TBK1 and IKK-i in H protein-mediated transcription activity ofβinterferon were detected by dual luciferase reporter kit.The interaction between CDV H protein and RIG-I(N)was detected by confocal laser scanning and GST pull-down assay.The results showed that H protein of CDV2 wild strain could specifically inhibit the transcription activity ofβinterferon induced by Se V infection,and the inhibition was related to the activation of RIG-I(N).The confocal laser scanning showed that H protein of CDV2 and RIG-I(N)were co-localized in the cytoplasm,and GST pull-down assay confirmed the direct interaction between H protein of CDV2 and RIG-I(N).The truncated expression of H protein showed that its N-terminal domain(1-462 bp)was the key domain to inhibit the transcription activity ofβinterferon.The successful development of this project has laid the foundation for further studying the mechanism of CDV H protein inhibiting innate immunity of host,which is of great significance for the prevention and treatment of canine distemper infection.