冠状病毒膜融合肽的研究

Enveloped viruses including coronavirus (CoV) initiate infection through fusion of the viral and cellular membrane, and viral fusion peptides (FP) play an essential role in this process. Coronaviruses are important human respiratory pathogens that may lead to severe pneumonia, and currently there are no effective drug treatment and vaccine available. Coronavirus entry is mediated by the interaction between viral spike (S) glycoprotein and its host receptor. Despite having made significant advances in understanding of how viral S proteins mediate virus entry, the location and properties of the FPs in the S proteins of different coronaviruses have not yet been determined. Through bioinformatics analyses of S proteins of different CoVs, we recently identified a common region as a putative FP (pFP) that shares the characteristics of FPs of Class-I viral fusion proteins, despite lack of amino acid sequence identity. Our primary mutagenesis analyses on pFP of the S protein of Middle East Respiratory Syndrome coronavirus (MERS-CoV) indicate that this region is essential for viral and cellular membrane fusion. In this study, we proposed to systemically characterize pFPs of S proteins of MERS-CoV, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), human coronavirus 229E, and mouse hepatitis virus (MHV), and determine if these pFPs are the functional fusion peptides by combination of different approaches from molecular virology, biochemistry, and biophysics. In addition, we also plan to use liquid and solid state NMR to solve their structures in lipids. These discoveries will make significant contribution to our understanding of how the S protein evolved and mediates fusion, and provide a potential new target for drug development.

包膜病毒入侵宿主细胞时，需要融合病毒包膜和细胞膜，而病毒包膜蛋白上的一段核心序列（膜融合肽）在此过程中起着关键作用。冠状病毒带有包膜，是一类重要呼吸道病毒，其中SARS和MERS病毒是引起人类严重急性呼吸综合征和中东呼吸综合症的感染病因。长期的研究发现，冠状病毒的感染依赖于病毒刺突（S）蛋白和其受体的相互作用，然而其关键的膜融合肽在S蛋白上的序列、位置以及作用机制仍然不清。在我们的前期研究中，通过应用生物信息学手段我们对不同冠状病毒S蛋白的膜融合肽区域进行了预测，进而在MERS病毒中应用氨基酸突变与膜融合实验分析，初步证实了这一区域对于膜融合的关键作用。本项目将在此基础上进一步对MERS、SARS、229E和小鼠肝炎病毒的该区域进行深入的病毒学、生物化学与生物物理学研究，以期确认其膜融合肽的核心序列，进而解析其三级结构，为阐明冠状病毒膜融合肽介导膜融合的作用机制提供关键的证据。

膜融合肽是病毒膜融合蛋白的必不可少的关键元件，而冠状病毒膜融合肽的核心序列仍然未知。本项目利用生物信息学的分析，找到了潜在冠状病毒膜融合肽的序列，随后通过病毒学、生物化学和生物物理学工具和手段在MERS病毒，SARS病毒和小鼠肝炎病毒的S蛋白上证实了该序列为冠状病毒的膜融合肽，并进一步结合液相和固相核磁方法研究了胆固醇对MERS病毒膜融合肽在脂类中构象变化的影响，阐明了它们介导膜融合的机制，为进一步阐明S蛋白介导的细胞进入机制以及研究新型抑制冠状病毒药物和疫苗打下良好基础。