人乳头瘤病毒（HPV）是一类导致宫颈癌等疾病的双链DNA病毒，衣壳由主要蛋白L1和次要蛋白L2组成。L1可组装成以五聚体为单位的T=7二十面体结构的颗粒，是已上市HPV疫苗的免疫原；L2参与病毒的组装、入胞和免疫逃逸等事件，且含有广谱中和表位，但至今仍缺乏高分辨的L2结构信息，限制了L2的功能研究。如何获得稳定均一的样品以解析L2三维结构是HPV病毒学研究的关键科学问题。前期我们基于L1结构鉴定了L1和L2上的相互作用关键氨基酸，并发现这些位点与病毒感染细胞相关。本项目拟在L1和L2之间定点引入二硫键，锁定L1和L2的相互作用，以期获得稳定的L1-L2衣壳颗粒或五聚体，进而解析复合物中L2的三维结构，揭示L2和L1的相互作用细节、病毒颗粒组装机制和病毒感染细胞时的L2变构现象，基于结构进行L2参与病毒感染细胞事件的功能研究和广谱疫苗设计，从而丰富HPV的病毒学理论和疫苗设计思路。

Human papilloma virus (HPV) is double-strand DNA virus tightly associated with the disease of cervical cancer, its capsid is comprised of major structure protein L1 and minor protein L2. L1 is capable of self-assembly into pentamer-based T=7 icosahedral virus-like particles, which is the sole immunogen for the commercial HPV vaccine; L2 involves in virus infection procedure including virion assembly, virus entry, immune escape and so on, some wide-spectrum immune epitopes have been mapped on L2, however, the dearth of L2 structure limits the extensive investigation on HPV virology. Thus, the critical scientific enquire was raised for HPV field as how to acquire well-behaved L2 sample for structure determination. In our preliminary study, we have identified key amino acids in both L1 and L2 that are essential for L1:L2 interaction and the virus infectivity. In the grant proposal, we seek to lock the L1:L2 interaction via zero-length crosslinking of disulfide bonds that engineered on these interacting sites between L1 and L2. Then the disulfide linking L1:L2 complex will be generated in either pseudovirus or pentamer forms by mammalian cell, baculovirus-infected insect cell or bacterial expression systems. The purified complex samples are expected to meet the need of cryo-EM reconstruction or protein crystallography aiming to solve the L2 structure out. The unprecedent L2 structure may elucidate the interaction mystery of L2 and L1, the assembly mechanism of HPV virion and the allosteric modality of L2 while virus attaching to host cells. The functional study on L2 involved in the virus-host interaction and rational design of pan-HPV vaccine will be implemented based on the L2 structure. The outcome of this proposed grant will boost the knowledge of HPV virology and expand the mindset for vaccine design.