坦布苏病毒非结构蛋白与鸭STING互作抑制I型干扰素表达的分子机制

Tembusu virus is a member of Flaviviridae that mainly infects waterfowls. However, its pathogenesis is not fully understood. Our previously study found that the non-structural protein NS1 and NS4B of Tembusu virus and the virus itself could suppress the activation and transcription of IRF7 and IFN-beta promoters by duck STING (Stimulator of the interferon gene). Therefore, this study intends to understand the molecular mechanism of the interaction between Tembusu virus non-structural protein and duck STING that suppressing the expression of type I interferon. Firstly, the transcription and expression of duck STING and its downstream signaling molecules in DEF (Duck embryo fibroblast) will be determined by qPCR and WB (Western blot). Subsequently, whether the NS1 and NS4B proteins could suppress the transcription and expression of duck STING and its downstream signaling molecules will be confirmed by overexpression or knockdown of duck STING in DEF or co-expressed with NS1, NS4B proteins and their truncates. At last, whether the NS1, NS4B proteins and their truncates could interact or intracellular co-localize with duck STING will be analyzed by immunoprecipitation and confocal laser scanning microscopy. Finally, the molecular mechanism of interaction between Tembusu virus non-structural protein and duck STING suppressing the expression of type I interferon will be elucidated. This study will be useful for further elucidating the molecular mechanism of duck Tembusu virus infection.

坦布苏病毒是黄病毒科成员之一。该病毒主要感染水禽，但其致病机制尚不明确。前期研究发现，坦布苏病毒及其非结构蛋白NS1和NS4B均能够抑制鸭STING对IRF7和IFN-β启动子的激活及转录，从而抑制宿主细胞I型干扰素的表达。因此，本项目拟研究坦布苏病毒非结构蛋白与鸭STING互作抑制I型干扰素表达的分子机制。首先通过qPCR、WB等方法确定病毒对STING及其下游分子转录和表达的影响。随后过表达或敲减STING后，分别共表达NS1、NS4B蛋白及其截短体，确定NS1和NS4B蛋白能否抑制鸭STING及其下游分子的转录和表达。最后利用免疫共沉淀和激光共聚焦等技术解析NS1和NS4B蛋白全长及其截短体与鸭STING能否互作、互作的区域及其细胞内定位。最终，阐明坦布苏病毒非结构蛋白与鸭STING互作抑制I型干扰素表达的分子机制，这将为进一步了解鸭坦布苏病毒感染的分子机理提供研究数据。

坦布苏病毒是黄病毒科成员之一，主要感染禽，其致病机制尚不明确。前期研究发现坦布苏病毒能抑制RIG-I介导的Ⅰ型干扰素表达。通过比较研究，发现在DEF细胞中，2型坦布苏病毒JM株在感染后期显著抑制Ⅰ型干扰素mRNA转录水平，而3型坦布苏病毒GX株在感染后期仍保持较高Ⅰ型干扰素mRNA转录水平。通过双荧光质粒报告系统发现TMUV-JM NS1蛋白是抑制Ⅰ型干扰素表达的关键非结构蛋白。为了进一步确定TMUV-JM NS1蛋白抑制IFN-β启动子激活的关键区域，我们构建了TMUV-JM NS1蛋白和TMUV-GX NS1蛋白重组蛋白，鉴定出NS1蛋白255-352aa区域为抑制RIG-Ⅰ介导Ⅰ型干扰素通路的功能区域。通过双荧光质粒报告系统发现TMUV-JM NS1蛋白抑制Ⅰ型干扰素表达的作用节点分子为TBK1。通过激光共聚焦、co-IP、WB发现TMUV-JM NS1蛋白与TBK1不存在直接相互作用，而是间接降低TBK1磷酸化水平从而降低Ⅰ型干扰素表达水平。利用反向遗传操作系统构建了2型坦布苏病毒NS1蛋白重组毒株TMUV-JM/GXNS1，验证TMUV-JM NS1蛋白是病毒抑制Ⅰ型干扰素表达的关键分子。本项目成果为不同亚型坦布苏病毒对鸭的致病性差异研究提供研究方向，也为进一步研究坦布苏病毒拮抗宿主天然免疫提供借鉴。