牛病毒性腹泻病毒p7蛋白的离子通道属性及其在病毒增殖中的作用

Viral viroporin can form selective ion channel on the membrane of host cell, which has an important function for viral replication, assembly and release. Some studies have shown that p7 protein from flaviviridae hepatitis C virus (HCV) is a viroporin, but viroporin from bovine viral diarrhea virus (BVDV), as a member of the flaviviridae, have not been reported. Our bioinformatics studies display that the predicted structure of BVDV p7 protein is extremely similar to that of HCV p7 protein, which was furher confirmed by membrane protein analysis. Moreover, viroporin inhibitors can inhibit BVDV proliferation. These preliminary results suggest that BVDV p7 protein may be a viral viroporin and it have impact on the proliferation of BVDV. In this project, we will study BVDV p7 protein by subcellular localization, oligomer formation, p7 effects on membrane permeability and so on, which will confirm BVDV p7 is a viral viroporin or not; We will use reverse genetics technology to construct BVDV virion with p7 mutation, which will be used for studying the role of p7 protein in BVDV replication, assembly and release. Our results will provide a foundation for further studying BVDV mechanism, developing new drugs and anti-BVDV vaccines.

病毒离子通道蛋白可在宿主细胞膜上形成选择性离子通道,在病毒复制、装配和释放等过程中具有重要功能。现已证实黄病毒科的丙肝病毒（HCV）p7蛋白为离子通道蛋白，但同属黄病毒科的牛病毒性腹泻病毒（BVDV）离子通道蛋白的研究还未见报道。课题组前期的生物信息学分析和膜蛋白检测实验表明BVDV p7蛋白与HCV p7蛋白结构极其相似，BVDV p7为细胞膜蛋白，而且病毒离子通道蛋白抑制剂可抑制BVDV增殖。这些前期实验提示：BVDV p7可能是病毒离子通道蛋白且对BVDV的增殖有影响。本项目拟通过研究BVDV p7蛋白的亚细胞定位，寡聚体形成、p7对细胞膜通透性的影响等特性，明确BVDV p7蛋白的离子通道属性；利用反向遗传学技术构建p7突变的BVDV感染性cDNA克隆，研究p7蛋白在BVDV复制、装配和释放中的作用，为深入研究BVDV增殖机制、研发抗BVDV的药物和新型疫苗奠定基础。

病毒离子通道蛋白可在宿主细胞膜上形成选择性离子通道,在病毒复制、装配 和释放等过程中具有重要功能。现已证实黄病毒科的丙肝病毒（HCV）p7 蛋白为离子通道蛋 白，但同属黄病毒科的牛病毒性腹泻病毒（BVDV）离子通道蛋白的研究还未见报道。本项目构建了p7 蛋白与 EGFP 融合表达载体pP7-EGFP，pP7-EGFP转染细胞后利用激光共聚焦显微镜证实 p7在细胞膜上表达；利用免疫印迹法和透射电子显微镜分析 p7为多聚体蛋白。发现 p7蛋白可以影响原核细胞和真核细胞膜的通透性，确定 p7蛋白为细胞膜通道蛋白。证实了p7关键氨基酸突变可以影响 BVDV病毒的复制和释放。同时，发现离子通道蛋白抑制剂金刚烷胺可以抑制BVDV的复制。因此， P7蛋白作为离子通道蛋白，在BVDV 复制、装配和释放中的作用，P7蛋白可以作为抗BVDV的药物和新型疫苗研发的新靶点。