EB病毒核衣壳装配与稳定的结构基础研究

EBV infection is closely associated with the development of nasopharyngeal carcinoma (NPC), yet there is no commercial antiviral drugs or prophylactic vaccines against EBV. The assembly of EBV nucleocapsid is the crucial step in the process of infectious EBV virion formation; therefore, revealing the mechanism of EBV nucleocapsid assembly should inform the rational design of anti-viral drugs. However, there are no high-resolution structures of EBV available. The proposed study is based on our previous success for setting up the cell culture system for EBV preparation using CNE2 cells-an NPC cell line, and the EBV infection model in vitro. We obtained both the B-capsid and C-capsid from the cell culture medium and determined their sub-nanometer resolution structures using cryo-electron microscopy (cryo-EM). Structural comparison between them shows that the C-capsid has stronger tegument protein densities associated with the penton and its surrounding triplexes, indicating the tegument proteins play roles in stabilizing EBV nucleocapsid. We will optimize the procedure of virus growth and purification to get EBV sample preparation with high quality, and then determine the near atomic structure of EBV nucleocapsid using our state-of-the-art 300kV Titan Krios electron microscope with developed image process strategies of cryo-EM data. This structure will provide us the long-sought-after structural information not only for better understanding the mechanism of EBV capsid assembly, genome package and capsid stabilization, but also would facilitate the development of antiviral agents.

EBV感染与鼻咽癌的发生密切相关，目前还没有抗EBV感染的治疗药物及预防性疫苗。EBV核衣壳的装配与稳定是感染性病毒粒子形成的关键，阐明其组装机制将为抗病毒药物设计提供指导，但目前尚无高分辨率EBV核衣壳结构的报道。我们在本项目前期工作中利用鼻咽癌细胞CNE2初步建立了EBV生产体系，并且在病毒培养液中能观察到B型衣壳和C型衣壳（包裹病毒基因组）的存在，通过对两种衣壳初步的冷冻电镜结构分析发现，EBV衣壳相关间层蛋白同时与衣壳五聚体及周围的三联体蛋白相互作用，且C型衣壳具有比B型衣壳更强的间层蛋白结构密度，表明间层蛋白在核衣壳结构稳定过程中发挥作用。后续我们将进一步优化病毒培养与纯化条件以及冷冻电镜数据处理方法以解析EBV核衣壳高分辨率结构，描述EBV核衣壳的基本结构特征和衣壳上各个蛋白间的相互作用，阐明EBV核衣壳装配过程的重要分子细节，从而为抗EBV感染药物的设计提供重要的结构依据。

Epstein-Barr virus（EBV）属于gamma亚家族疱疹病毒，EBV感染与鼻咽癌的发生密切相关，目前还没有抗EBV感染的治疗药物。EBV核衣壳的装配与稳定是感染性病毒粒子形成的关键，以旨在干扰病毒衣壳组装和成熟的小分子化合物将有望成为抗疱疹病毒感染的有效武器，然而由于天然样品制备存在挑战，针对EBV的结构学研究和靶向性药物分子设计长期滞后。本研究首先基于对疱疹病毒生活史的深入理解，利用整合有EBV基因组的B95-8细胞株优化出一种新颖的EBV病毒培养方法，并成功制备出高质量的EBV样品。在此基础上，我们利用冷冻电镜技术成功解析出EBV核衣壳二十面体结构以及功能组分portal和（capsid-associated tegument complex, CATC）高分辨率原位结构，并搭建首个EBV核衣壳全原子模型。该研究全方位描绘出EBV衣壳蛋白之间、衣壳相联的间层蛋白CATC与衣壳蛋白、衣壳蛋白与portal以及portal与病毒DNA多层次的相互作用细节，从而为针对阻断病毒衣壳成熟的抗病毒药物开发提供了关键结构基础。此外，我们还进一步发现不同疱疹病毒衣壳五聚体（penton）的结构柔性以及CATC在衣壳不同顶点（vertex）上的结合数量和作用形式均存在差异，且这些差异与病毒基因组大小紧密相关。基于此，我们提出了一种基于“压力调控”的EBV衣壳装配以及病毒基因组包装、稳定和释放机制，本研究也为靶向病毒复制过程的抗病毒药物研发提供了重要理论支持。