猪流行性腹泻病毒变异株抑制宿主补体C3蛋白的分子机制

The outbreaks of porcine epidemic diarrhea virus (PEDV) have caused major economic losses to pig industries in the world. Host complement C3 protein plays a central role in the whole complement system activation and antiviral infection, but there is no report about the interactions and molecular mechanisms of C3 protein and PEDV infection. Previous studies have found that PEDV variant strains have the ability to evade the antiviral response by inhibiting the expression of C3 protein compared to the classical strains. In this study, the non-structural and structural protein expression vectors of different PEDV strains and recombinant strains will be constructed by using gene mutation and reverse genetics techniques to further clarify the functional proteins and key sites of PEDV variants, which inhibit C3 protein expression. Through the viral infection and functional protein expression vectors transfection, the expression and activation of important proteins in different activation pathways of C3 protein, the levels of inflammatory cytokines and the interaction between viral functional proteins with C3 and its activation pathway proteins will be detected to elucidate the signaling pathway of PEDV inhibiting the C3 protein expression. This research plans to enrich the theoretical basis of PEDV variants escaping innate immunity and its pathogenesis.

猪流行性腹泻病毒（PEDV）的暴发流行给世界养猪业造成重大经济损失。宿主补体C3蛋白在整个补体系统激活及抗病毒感染中起着核心作用，但目前关于C3蛋白与PEDV感染的相互作用关系及分子机制尚无报道。前期研究发现，与PEDV经典毒株相比，变异株具有通过抑制C3蛋白表达而逃避其抗病毒的能力。本研究拟利用基因突变和反向遗传技术，构建不同PEDV毒株非结构和结构蛋白表达载体和重组病毒，进一步明确PEDV变异株抑制C3蛋白表达的关键蛋白及其关键位点；通过病毒感染和关键蛋白表达载体转染细胞，检测C3蛋白不同激活途经中重要蛋白的表达及活化水平，炎性细胞因子水平，以及病毒关键蛋白与C3蛋白本身及其激活途径蛋白的相互作用关系，阐明PEDV变异株抑制C3蛋白的信号通路分子机制，丰富其致病机制和逃避先天免疫的理论基础。

自2010年以来，PEDV变异株的暴发给全球养猪业造成巨大的经济损失。然而，PEDV变异株逃避宿主免疫反应的策略还不完全清楚。补体成分3（C3）被认为是三种补体激活途径的核心蛋白，在预防病毒感染中起着关键作用。在本研究中，我们发现与阴性血清相比，PEDV阳性猪血清中的C3含量降低。在体外，C3显著抑制PEDV复制。变异株和经典株均能在人肝癌细胞系Huh7中诱导高水平的IL-1β。然而，与PEDV经典株相比，PEDV变异株显著降低由IL-1β诱导的C3转录和蛋白水平。对C3转录信号通路关键分子的检测显示，PEDV变异株通过抑制CCAAT/增强子结合蛋白β（C/EBP-β）的磷酸化抑制C3转录。进一步发现PEDV非结构蛋白1（NSP1）通过第50位氨基酸残基抑制C/EBP-β磷酸化。最后，我们通过重组病毒进一步验证了NSP1的第50位氨基酸对C3转录调控的关键作用。.另外，该项目还对PEDV变异株起源进行了分析，发现PEDV变异株由中国早期流行毒株JS-2004-2与早期的韩国流行株重组而来；建立了PEDV抗原定量ELISA方法，可实现对灭活疫苗样本和临床样本的抗原检测；同时阐明了宿主mortalin蛋白可通过对网格蛋白重链CLTC的降解抑制PEDV的入胞。综上，本项目加深了对PEDV病原学、致病机制和免疫逃逸的了解。