外泌体 ESCRT 转运途径在 BVDV 持续感染中的作用及机制研究

BVDV感染可引起牛外周血白细胞减少，从而抑制宿主的免疫应答，造成牛持续性感染。BVDV究竟通过什么方式造成宿主免疫抑制还不清楚。研究发现，哺乳动物细胞能产生介导细胞间通讯的外泌体，在病毒感染中发挥重要作用。我们在前期研究中发现外泌体标记蛋白CD63、CD81的表达促进BVDV感染；通过对纯化外泌体色谱-质谱分析，发现ESCRT与 CD63、CD81、ALIX、AFR6、TSG101等蛋白高度相关。因此，本研究拟从ESCRT转运途径影响BVDV感染入手，利用分子生物学、生物化学和病毒学等方法鉴定 ESCRT在BVDV感染中的作用；利用免疫共沉淀、免疫印迹和激光共聚焦技术分析ESCRT与其相关互作蛋白的关系；利用荧光素酶基因报告系统实时动态分析ESCRT对互作通路蛋白的表达调控，明确ESCRT相关蛋白诱导BVDV持续感染的机制，从而为新型疫苗和特异性抗病毒药物的研制提供理论依据和技术支撑。

Bovine Viral Diarrhea is an extremely complex disease which is caused by Bovine Viral Diarrhea Virus (BVDV). The infection can cause bovine peripheral leukopenia, dominated by lymphopenia, thereby inhibiting the host immune response and resulting in persistent infection in cattle. So far, it is unclear how BVDV cause immunosuppression of host. It has been reported that most mammalian cells can produce exosome that mediates cell-to-cell communication. The infection of virus could change the composition of proteins and RNA of exosomes in the host cells, incorporate the viral proteins into the exosomes, and play its role in immunosuppression and pathogenic responses. In our previous study, we found that the expression of exosome protein CD63,CD81 can promote BVDV infection. Purified exosome was analyzed by the chromatochrome-mass spectrometry technology and ESCRT proteins were found to communicate with CD63, CD81, CD9, ALIX, TSG101, PD-L1 and other proteins .Therefore, It is important to identify the role of ESCRT protein transport pathway in BVDV infection through flow cytometry, CRISPR/Cas9 gene editing technology, biochemistry and virology methods. immunoprecipitation, western blot and laser confocal techniques will be used to analyze the relationship between ESCRT proteins and its related interaction proteins.The luciferase report gene system will be used to elucidate protein of exosomes in regulating the protein expression of the interacting pathway and exclicit the molecular mechanism of ESCRT proteins in BVDV persistent infection by the live cell real-time dynamic imaging system. This study will provide theoretical basis and technical support for the development of new vaccines and antiviral drugs against BVDV.