中东呼吸综合征冠状病毒刺突蛋白N-端结构域及S1亚基的结构与功能研究

Understanding the viral entry process of Middle East respiratory syndrome coronavirus (MERS-CoV) mediated by the surface spike (S) protein is a key in the pathogenesis study. During the maturation of coronaviruses, the S protein is, in most cases, cleaved by the host furin-like proteases into S1 and S2 subunits, which function to mediate receptor-recognition and membrane fusion, respectively. Our previous work on MERS-CoV focused on the receptor binding domain (RBD) locating at the S1 C-terminus and the heptad repeats residing in S2. The atomic structures of the RBD/receptor complex and the S2 fusion core were individually solved, which paves the way for a better understanding of the viral entry process. However, the N-terminal domain (NTD) of MERS-CoV S1, as a natural and an important component of the S protein, remains to be investigated for the structural and functional features. In addition, the steric arrangement of NTD and RBD and the interaction mode between S1 and S2 are also key issues for the MERS-CoV entry study, which are currently elusive. Therefore, in this proposal, we would use the baculovirus expression system to prepare both MERS-CoV NTD and S1 proteins. The proteins would then be purified and subjected to crystallization screenings. We would solve the high-resolution protein structures to elucidate their structural features. In addition, we would further work on the functions of MERS-CoV NTD by investigating its potential sugar-binding activity, its immunogenicity, etc. Finally, we would also try to swap the functional domains (eg, NTD, RBD, S1, S2) in S between different coronaviruses and test the domain-swapped recombinant pseudoviruses for the receptor binding specificity and the cell infection capacity, aiming to gain some information on coronaviral S proteins from a perspective of seeing them as integral entities.

深入了解中东呼吸综合征冠状病毒（MERS-CoV）刺突（S）蛋白所介导的病毒侵入过程对于认识其致病机制至关重要。在冠状病毒成熟过程中，S蛋白通常会被切割成S1和S2两个亚基，分别介导受体识别和膜融合。我们先前的研究工作集中在MERS-CoV S1亚基C-端的受体结合区（RBD）以及S2亚基的七肽重复区，但S1亚基N-端结构域（NTD）的结构与功能，NTD与RBD的排布方式，以及S1与S2的互作模式等目前仍未有研究。这些问题已经成为全面、深入了解病毒S蛋白所介导的侵入过程的关键。因此，本项目拟通过杆状病毒表达系统制备高纯度的MERS-CoV NTD与S1蛋白；利用结构生物学手段解析其结构以揭示其各自的结构特征；同时对NTD的免疫原性和潜在功能进行深入研究。我们还将尝试把S蛋白的功能结构域在不同的冠状病毒间互换，通过测定其受体选择性和感染能力来获得S蛋白的整体结构信息。

中东呼吸综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）的暴发和持续传播已成为威胁世界公共卫生安全的严重隐患之一。由病毒表面刺突（spike, S）蛋白所介导的病毒侵入过程是决定MERS-CoV致病性和跨种传播潜能的关键因素。先前对MERS-CoV S蛋白的研究主要集中在S蛋白中位于S1亚基C-端的受体结合区以及S2亚基的七肽重复区，但S1亚基N-端结构域（NTD）的结构与功能仍有待深入研究。因此，本项目拟利用蛋白质晶体衍射学手段解析MERS-CoV NTD的晶体结构，利用多糖结合实验和免疫原性测定等方法探索MERS-CoV NTD的潜在功能，并进一步尝试通过结构生物学方法和S蛋白功能结构域互换等揭示MERS-CoV S蛋白的整体结构特征。按照项目预设目标，我们成功制备了高纯度的MERS-CoV NTD蛋白、获得了高质量的蛋白晶体，并解析了高分辨率（1.5埃）的晶体结构。由此，成功揭示了MERS-CoV NTD的结构特征。开展了MERS-CoV NTD与唾液酸分子和mucin糖蛋白的体外互作实验研究，证明MERS-CoV与大多数冠状病毒（如MHV，HKU1，SARS-CoV等）相似，在进化中失去了利用S蛋白结合糖分子的能力。制备了MERS-CoV NTD的小鼠免疫多抗，通过基于假病毒的中和活性研究，证明MERS-CoV NTD不能高效诱导机体产生中和抗体。进一步利用冷冻电镜技术，解析了MERS-CoV S蛋白的电镜结构。由此，我们得以对MERS-CoV NTD的结构和功能有了较深入和全面的认识，也对MERS-CoV NTD/RBD功能结构域和S1/S2亚基的空间排布方式、以及S蛋白的整体结构特征等给出了重要提示。