An accurate eukaryotic plasma membrane assay for coronavirus binding

本项目重点开发一种基于表面的ACE2膜传感器，该传感器将提供冠状病毒细胞表面结合的高度真实模型，并适用于高通量筛选。SARS-CoV-2通过转导途径进入哺乳动物细胞，其第一阶段是病毒表面的刺突(S)蛋白与1型跨膜蛋白ACE2（血管紧张素转换酶-2）相互作用。一旦病毒附着在膜表面，S蛋白就会被细胞表面蛋白酶（TMPRSS2）修饰，形成一种融合肽，这种融合肽插入膜中，促进病毒转导。目前S蛋白/ACE2相互作用的研究主要集中在利用ACE2的重组可溶性结构，因此不能真正代表体内发生的过程。利用我们在表面支撑双层方面的专业知识，我们将在传感器表面上制造含有全长ACE2的真核膜的精确模拟膜。中子反射仪将与石英晶体微天平（QCM）一起用于验证表面组装和病毒成分结合。然后将进一步开发该系统，以包括已知参与ACE2/冠状病毒相互作用的其他成分（例如TMPRSS2和B0AT1），以提供冠状病毒膜表面相互作用的现实模型。该系统将直接转化为适用于高通量筛选的技术（QCM和表面等离子体共振）。这将有助于科学界研究冠状病毒膜结合，并可作为鉴定冠状病毒膜相互作用抑制剂的诊断工具。

This project focuses on the development of a surface based ACE2 membrane sensor that will provide a highly realistic model of coronavirus cell surface binding and be amenable to high throughput screening. SARS-CoV-2 enters mammalian cells by a transduction pathway whose first stage is the interaction of its spike (S) protein on the viral surface with ACE2 (angiotensin converting enzyme-2), a type-1 transmembrane protein. Once the virus attaches to the membrane surface the S protein is modified by a cell surface protease (TMPRSS2) to form a fusion peptide which inserts into the membrane and facilitates viral transduction. Current S protein/ACE2 interaction studies have primarily focused on utilising a recombinant soluble construct of ACE2 and thus do not truly represent the in vivo processes occurring. Using our expertise with surface based supported bilayers we will fabricate an accurate membrane mimetic of the eukaryotic membrane containing full length ACE2 on a sensor surface. Neutron Reflectometry together with quartz crystal microbalance (QCM) will be used to validate the surface assemblage and viral component binding. The system will then be further developed to include other components known to be involved in ACE2/Coronavirus interaction (e.g. TMPRSS2 & B0AT1), to provide a realistic model of coronavirus membrane surface interaction. This system will be directly translatable to techniques amenable to high throughput screening (QCM and surface plasmon resonance). This will aid the scientific community in studying coronavirus membrane binding and can be used as a diagnostic tool for the identification of inhibitors of coronavirus-membrane interactions.