An accurate eukaryotic plasma membrane assay for coronavirus binding

该项目的重点是开发一种基于表面的ACE 2膜传感器，该传感器将提供冠状病毒细胞表面结合的高度逼真的模型，并适合于高通量筛选。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S蛋白与ACE 2（血管紧张素转换酶-2）（一种1型跨膜蛋白）相互作用。一旦病毒附着到膜表面，S蛋白被细胞表面蛋白酶（TMPRSS 2）修饰以形成插入膜中并促进病毒转导的融合肽。目前的S蛋白/ACE 2相互作用研究主要集中在利用ACE 2的重组可溶性构建体，因此不能真正代表体内过程的发生。利用我们的专业知识与表面为基础的支持双层，我们将制造一个精确的膜模拟的真核细胞膜含有全长ACE 2的传感器表面。将使用中子反射法和石英晶体微量天平（QCM）验证表面组装和病毒组分结合。然后，该系统将进一步开发，以包括已知参与ACE 2/冠状病毒相互作用的其他组分（例如TMPRSS 2和B 0AT 1），以提供冠状病毒膜表面相互作用的真实模型。该系统将直接转化为适合高通量筛选的技术（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，并可用作鉴定冠状病毒-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.