NS2蛋白在PPV诱导猪胎盘滋养层细胞自噬性死亡过程中的作用及调控机制

Porcine parvovirus (PPV) causes porcine placental trophoblastic cells (PTCs) death, which is closely related to the abortion of sows. Our previous research has found that most of the PTCs demise through autophagic death in the sow placental tissue infected with PPV. Further research showed that PPV NS2 protein plays a key role in the process of autophagic death induced by PPV in PTCs, however, the exact roles and regulatory mechanism are still unclear. In this project, we will first identify the key signaling pathway regulating PPV-induced autophagic death. And then we will investigate the exact roles of NS2 protein in autophagic death induced by PPV, constructing the eukaryotic expression plasmid of NS2 gene and NS2 gene deletion PPV strain to transfer/infect PTCs to detect the effect of NS2 on autophagic death of PTCs. Furthermore, we will screen the key interacting host proteins of NS2 protein and define the interaction domains and key amino acids, then establish a NS2 binding protein knockout cell line and a mutant strain of PPV that lacks the binding activity of the key NS2 binding protein. Finally, to compare the differences between the mutant strain and NS2 deficient PPV strain and wild-type PPV strain in activating autophagic death pathways and inducing tissue lesions in vivo and vitro, and confirm the roles and mechanism of the interaction of NS2 and its binding proteins in the development of PPV-induced autophagic death. The results of this study will reveal the exact roles and mechanism of PPV NS2 and its binding protein in the regulation of autophagic death of PTCs induced by PPV, and provide a theoretical basis for further elucidating the pathogenesis of PPV.

猪细小病毒（PPV）致胎盘滋养层细胞（PTCs）病变与母猪流产密切相关。前期研究发现，在PPV感染的猪胎盘组织中，大多数PTCs的死亡是以自噬性死亡方式进行的，PPV非结构蛋白NS2在这一过程中发挥了关键作用，但其具体作用方式及调控机制尚不清楚。本项目首先确定调控PPV诱导细胞自噬性死亡的信号通路；然后构建NS2真核表达质粒和NS2缺失型PPV毒株，转染/感染PTCs后检测对细胞自噬性死亡的影响；进一步筛选NS2的宿主互作蛋白并确定相互作用结构域和关键氨基酸位点，建立敲除互作蛋白的PTCs和缺失NS2关键互作蛋白结合活性的PPV突变毒株；最后通过细胞和动物试验，比较突变毒株与野生型毒株之间活化细胞自噬性死亡通路及致组织病变方面的差异，证实NS2及其互作蛋白的调控作用和机制。研究结果将揭示NS2及其互作蛋白在PPV诱导PTCs自噬性死亡过程中的作用及机制，为阐明PPV致病机制提供理论依据。

猪细小病毒（PPV）致胎盘滋养层细胞（PTCs）病变与母猪流产密切相关。前期研究发现，在PPV感染的猪胎盘组织中，大多数PTCs的死亡是以自噬性死亡方式进行的，PPV非结构蛋白NS2在这一过程中发挥了关键作用，但其具体作用方式及调控机制尚不清楚。本项目首先确定调控PPV诱导细胞自噬性死亡的信号通路；然后构建NS2真核表达质粒和NS2缺失型PPV毒株，转染/感染PTCs后检测对细胞自噬性死亡的影响；进一步筛选NS2的宿主互作蛋白并确定相互作用结构域和关键氨基酸位点，建立敲除互作蛋白的PTCs和缺失NS2关键互作蛋白结合活性的PPV突变毒株；最后通过细胞和动物试验，比较突变毒株与野生型毒株之间活化细胞自噬性死亡通路及致组织病变方面的差异，证实NS2及其互作蛋白的调控作用和机制。研究结果将揭示NS2及其互作蛋白在PPV诱导PTCs自噬性死亡过程中的作用及机制，为阐明PPV致病机制提供理论依据。