EphA2介导EB病毒感染鼻咽上皮细胞

Epstein-Barr virus (EBV) is the earliest discovered tumor virus, and implicated as an etiological factor in nasopharyngeal carcinoma (NPC). Due to the lack of representative cell model that are highly susceptible to cell-free EBV infection, the mechanisms of EBV infection in nasopharyngeal epithelial cells remain elusive. We observed EGF significantly promoted EBV infection of nasopharyngeal epithelial cells. And then we found EphA2 was one of the most significantly EGF-upregulated genes by microarray assay. We preliminarily identified EphA2 was a potential receptor for EBV infection of nasopharyngeal epithelial cells by RNA interference and overexpression. To verify it, we will employ co-immunoprecipitation (co-IP), antibody neutralization and co-localization to further confirm whether EphA2 is a receptor for EBV infection of nasopharyngeal epithelial cells, and to identify EBV glycoprotein interaction with EphA2. Then we will explore the mechanisms involved in EphA2-mediated EBV infection of nasopharyngeal epithelial cells by domain negative EphA2, RNA interference, endocytic and pathway inhibitors, rescue assays, co-localization and TnT Transcription/Translation System (TNT) in combination with co-IP. Last but not least, we will investigate whether EphA2-targeting inhibitors disrupt EBV infection by blocking assays. Taken together, this project has important significance for elucidating the mechanism of EBV infection in nasopharyngeal epithelial cells and developing antagonists against EBV infection.

EB病毒是最早发现的人类肿瘤病毒，与鼻咽癌的发生发展密切相关。由于缺乏cell-free EB病毒高效感染的上皮细胞模型，其感染鼻咽上皮细胞的机制仍不清楚。我们在前期研究中发现，EGF显著增加EB病毒感染鼻咽上皮细胞；进一步采用人类全基因组表达谱芯片、siRNA干扰和质粒过表达等方法，初步鉴定EphA2是EB病毒感染鼻咽上皮细胞潜在的受体。本项目将在前期结果基础上，拟采用免疫共沉淀、抗体中和及共定位等方法，进一步确认EphA2是否是EB病毒感染鼻咽上皮细胞的受体，并鉴定与其相互作用的糖蛋白；同时运用胞内段缺失型EphA2、siRNA干扰、抑制剂、回复实验、共定位及体外转录翻译系统等技术，初步阐明EphA2介导EB病毒感染的机制；通过阻断实验，评价EphA2作为靶点在抗EB病毒感染中的作用。因此，本项目的实施在EB病毒感染鼻咽上皮细胞机制的研究和抗EB病毒感染药物的开发中都具有重要意义。

EBV感染全球90%以上成年人。研究表明EBV与伯基特淋巴瘤、鼻咽癌和10%的胃癌发病密切相关。因此，阐明EBV感染上皮细胞机制对于鼻咽癌和胃癌的预防尤为重要。本项目旨在鉴定EBV感染上皮细胞的关键受体，初步阐明EBV感染上皮细胞的分子机制，并最终探索抵抗EBV感染上皮细胞的手段。我们在前期研究中发现表皮生长因子促进EBV感染鼻咽上皮细胞。本项目在此基础上，采用基因表达谱芯片和RNA沉默技术初步鉴定EphA2是EBV感染上皮细胞的宿主因子。Crispr/cas9技术敲除EphA2显著降低EBV感染上皮细胞，而过表达EphA2则增强EBV感染。EphA2中和抗体、配体EphrinA1及抑制剂2,5-dimethylpyrrolyl benzoic acid显著阻断EBV感染。机制研究表明，EphA2直接结合EBV糖蛋白gB和gH/gL并促进EBV內吞和融合。进一步研究发现EphA2胞外段的EBD和FNR结构域对其介导EBV感染极为关键，而胞内段结构域则可有可无。本项目成功鉴定了EBV感染上皮细胞的首个功能受体，揭示了EphA2介导EBV进入的分子机制，并发现了数个阻断EBV感染的抑制剂。因此，该研究从基础医学和转化医学层面，完善了EBV进入上皮细胞的机制，并为EBV感染的干预提供实验依据。