PHEV入侵神经细胞胞吞途径及其胞内运输的分子机制

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent virus that invades the central nervous system, where nerve cells are a target for viral replication. Most viruses achieve early infection of host cells by endocytosis, which can not only make the virus into cells, but also transport the virus to the replication site. Previous studies have found that PHEV can invade the nerve cells by endocytosis, but the mechanism of PHEV entering into nerve cells is unclear. In this study, the morphological changes and the dynamic characteristics of PHEV invasion of nerve cells will be analyzed through the ultrastructural observation, invasion dynamics and binding kinetics tests based on neural cells model of PHEV infection. Then, the endocytic pathway of PHEV invasion of the neural cells will be studied using inhibitor blocking test, co-localization analysis of virus and endocytosis markers, exogenous expression of dominant negative mutants, and small interfering RNA techniques. Furthermore, the dynamic relationship between PHEV transport and the cytoskeleton remodeling of the host cells will be analyzed by fluorescence labeled live cell tracer test and subcellular localization, in order to identify the host cell transport system and regulatory mechanism of PHEV in the process of intracellular transport in nerve cells. In conclusion, we will deeply investigate the endocytosis mechanism of PHEV infection of nerve cells from the two aspects: the initiation of viral invasion and intracellular transport. The expected results not only lay the foundation for revealing the molecular basis of neurotropism and pathogenicity of PHEV, but also provide a new idea for the design of specific targeting antiviral drugs for the key sites of PHEV infection and replication.

病毒可通过多种方式入侵宿主细胞，其中的胞吞作用既可以介导病毒内化，也可将病毒运输至复制位点。前期研究发现，PHEV可通过胞吞方式入侵神经细胞，但其机制尚不清楚。为此，本项目拟利用已建立的PHEV感染神经细胞模型，从病毒侵入起始和胞内运输两个环节对PHEV入侵神经细胞的胞吞途径及其运输机制进行研究。首先，通过超微结构观察、侵入动力学和结合动力学试验，系统地分析PHEV侵入神经细胞的形态学变化和动力学特征；然后，利用抑制剂阻断、共定位分析、表达显性抑制突变体等方法，深入探讨PHEV入侵神经细胞的胞吞途径类型及所依赖的分子基础；进而，利用活细胞示踪、亚细胞结构定位以及相关信号通路激活检测等方法，解析PHEV经内吞小体转运与细胞骨架重塑之间的动态关系，明确PHEV胞内运输所依赖的宿主固有运输系统。预期研究成果不仅有利于揭示PHEV感染神经细胞的分子机制，而且也将为新型抗病毒药物的设计提供理论依据。

病毒对靶细胞的侵入是病毒生命周期中的始动环节，也是影响病毒宿主范围、组织嗜性和致病性的决定因素之一。从病毒入侵细胞的分子机制角度开展相关工作，是针对特异性靶点设计抗病毒策略的一个重要基础。本研究针对嗜神经性β冠状病毒——猪血凝性脑脊髓炎冠状病毒（PHEV）侵入及胞内转运机制进行系统研究，分析PHEV侵入神经细胞的形态学变化和动力学特征，挖掘PHEV入侵神经细胞的胞吞途径类型及所依赖的分子基础，解析PHEV经内吞小体转运与细胞骨架重塑之间的动态关系，明确PHEV胞内运输所依赖的宿主固有运输系统。研究结果证实，PHEV通过劫持神经细胞网格蛋白介导的内吞作用（CME）侵入神经细胞，该过程需要在低pH环境下多种宿主蛋白的共同参与，包括发动蛋白、Rab5、Rab7、胆固醇、EGFR通路底物15等。本项目研究工作，首次揭示PHEV侵入神经细胞的重要途径和分子基础，为全面认识冠状病毒的基本生命周期特征及新型抗病毒药物靶标筛选提供了关键的数据支持。