蝙蝠SARS样冠状病毒受体识别和入侵机制研究

SARS coronavirus (SARS-CoV) is the causative agent of the severe acute respiratory syndrome that outbroke in 2002-2003. The membrane spike protein (S) is responsible for the entry into cells and intersepcies infection. The SARS-CoV receptor binding domain (RBD) in S directly recognizes and binds to receptor, angiotension converting enzyme II (ACE2). The key amino acid sites in RBD play a key role in viral infection, transmission, and pathogenesis. Bat SARS-like coronavirus WIV1 (SL-CoV WIV1) was isolated from sample collected from Chinese horseshoe bat. This isolate has a higher similarity with the SARS-CoV in genomic sequence and uses ACE2s from human, civet and Chinese horseshoe bat. Interestingly, WIV1 is different from the SARS-CoV in S protein, particularly at several key amino acid residues of the RBD. We hypothesise that there are differences in receptor binding efficiency between the WIV1 and SARS-CoV S proteins with ACE2 of different origin. In this project, we will analyse and compare the ACE2 (human, civet and bat) binding efficiency between the WIV1 and SARS-CoV S protein using pseudovirus report system and protein-protein intereaction techniques. The expected results will be helpful for further understanding the interspecies infection and pathogenesis of the SL-CoV WIV1.

SARS冠状病毒是严重急性呼吸综合症病原,其囊膜蛋白（S）在病毒的入侵和跨种感染过程中起关键作用。研究证实S蛋白受体为血管紧张素转换酶II(ACE2)，其受体结合区（RBD）的关键氨基酸位点的改变可以严重影响SARS病毒与ACE2的识别和结合，从而影响病毒的感染和跨种传播。蝙蝠SARS样冠状病毒WIV1是申请人近期从菊头蝠分离到的与SARS病毒高度同源的病毒，可以高效利用人、果子狸和中华菊头蝠的ACE2。有趣的是，WIV1与SARS病毒在RBD区已知的几个关键位点存在着显著差异，推测这些位点的变异增强了WIV1利用不同物种ACE2的效率。本项目结合前期工作，利用假病毒报告系统和蛋白质相互作用技术研究和比较WIV1和SARS病毒的S蛋白利用不同物种ACE2的效率，解析影响WIV1跨种感染的关键氨基酸位点，从受体利用和入侵的角度评估WIV1的跨种感染能力和潜在的致病性。

蝙蝠SARS样冠状病毒（SL-CoV）遗传多样性高，SARS冠状病毒（SARS-CoV）由SL-CoV演化而来。蝙蝠SL-CoV WIV1与SARS-CoV高度同源，二者均利用血管紧张素转换酶II （ACE2）为受体，但是WIV1与SARS-CoV在囊膜蛋白（S）尤其是与受体结合的区域（RBD）的几个关键位点存在着显著差异。本研究成功构建了分别稳定表达人、果子狸和蝙蝠AEC2的HeLa细胞系，用Western-blot和免疫荧光检测验证了ACE2的稳定表达；利用定点突变技术分别构建了S、RBD、ACE2关键结合位点的表达突变体；利用假病毒感染和荧光素酶分析、RBD和ACE2结合实验证实SARS-CoV BJ01株利用果子狸和人ACE2的效率远远高于蝙蝠ACE2，而WIV1利用三种不同物种ACE2的能力相近；证明S蛋白的Y452S位点突变显著提高BJ01对蝙蝠ACE2的利用能力，显示此位点变异影响种间受体结合；证明BJ01和WIV1能有效利用老鼠ACE2，且ACE2的L440P突变能显著降低RBD结合能力及假病毒入侵效率。本研究揭示了SL-CoV WIV1 和SARS-CoV BJ01的S蛋白利用不同物种ACE2的效率有差异，且S蛋白的Y452S和ACE2的L440P是两个影响种间感染的关键氨基酸位点。研究结果加深了我们对SL-CoV进化和传播的理解，为相关疫苗和药物的研发提供了新的基础数据。