牛病毒性腹泻病毒利用DDIT3降解MAVS的机制研究

Bovine viral diarrhea virus (BVDV) is the causative agent of of bovine viral diarrhea-mucosal disease, and its relationship with MAVS has not been reported. We have demonstrated that DDIT3 was significantly up-regulated during BVDV infection; and over-expression DDIT3 could degrade MAVS to facilitate BVDV replication through proteasome pathway. In this study, high-throughput sequencing technology will be used to screen differentially expressed E3 ubiquitin ligase after DDIT3 over-expression, and then mRNA and protein level of candidate genes involved in promoting MAVS degradation will be confirmed through regulating the expression of DDIT3. In addition, Candidate genes over-expression or knockdown cell lines will be used to determine the protein level of MAVS during BVDV infection, and the mechanism of MAVS degradation promoted by DDIT3 will be elucidated. What's more, viral proteins involving in upregulating DDIT3 will be screened, and then signal transduction pathway of viral promoting viral gene up-regulating DDIT3 will be confirmed. Collectively, this research will reveal the mechanisms of MAVS degradation promoted by DDIT3 during BVDV infection and provide target molecules for the development of anti-BVDV drugs.

牛病毒性腹泻病毒（BVDV）是牛病毒性腹泻-黏膜病的病原，其与MAVS之间的关系尚未见报道。本团队研究发现，感染BVDV后DDIT3表达显著上调；过表达DDIT3可通过蛋白酶体途径降解MAVS，促进BVDV复制。本研究拟通过高通量测序技术筛选过表达DDIT3后差异表达的宿主E3泛素连接酶，利用RT-qPCR及Western blot实验验证候选基因；构建候选E3泛素连接酶的过表达或干扰细胞系，分析BVDV感染后MAVS的变化情况，发现DDIT3促进MAVS降解的分子机制；筛选调控DDIT3上调表达的病毒基因，检测并分析病毒基因调控DDIT3的信号通路，发现BVDV上调DDIT3表达的分子机制。本研究拟从宿主DDIT3与MAVS的关系出发，率先发现DDIT3与天然免疫反应的关系，揭示BVDV利用DDIT3降解MAVS促进自身复制的分子机制，以期为抗BVDV药物的研发提供靶标分子。

牛病毒性腹泻病毒（BVDV）是牛病毒性腹泻-黏膜病的病原，给养牛业造成了巨大的经济损失。深入探讨病毒与宿主之间的相互作用及其分子机制，进而为抗病毒药物的研发提供靶标分子，为综合防控BVD-MD奠定基础。本团队利用高通量测序技术筛选到在感染BVDV后表达显著上调的基因DDIT3,并进一步通过RT-qPCR及Western blot加以验证。过表达或干扰DDIT3后测定其对病毒复制的影响，证实DDIT3为BVDV复制的正调控宿主因子。进一步研究发现，DDIT3通过抑制干扰素的表达进而促进病毒复制。机制研究发现DDIT3通过NF-КB信号通路促进去泛素化酶OTUD1的上调表达。过表达的OTUD1与E3连接酶Smurf1相互作用通过对Smurf1去泛素化作用促进其稳定，进而提高Smurf1的蛋白表达量。高表达的Smurf1通过泛素蛋白酶体途径促进MAVS降解，从而阻断I型干扰素信号通路。动物实验证实DDIT3敲除小鼠在感染BVDV后病理损伤程度明显减轻，且病毒在DDIT3敲除小鼠心、肝、肺等脏器中的载量显著低于野生型小鼠。本研究从宿主DDIT3与MAVS的关系出发，率先发现DDIT3与天然免疫反应的关系，揭示BVDV利用DDIT3降解MAVS促进自身复制的分子机制，以期为抗BVDV药物的研发提供靶标分子。