meCocan - Towards a mechanistic understanding of the interaction of SARS-CoV-2 spike glycoprotein and host heparan sulphate proteoglycans

SARS-CoV-2刺突糖蛋白在宿主感染病毒的过程中起着关键作用，最终导致新冠肺炎病。病毒粒子表面的SGP通过将其受体结合域(RBD)与人血管紧张素转换酶2(ACE2)结合而与宿主细胞结合。越来越多的证据表明，宿主细胞硫酸乙酰肝素蛋白多糖(HSPGs)作为辅助受体促进了病毒感染，并可能影响宿主的易感性。然而，HSPGs影响病毒-宿主细胞结合和进入的机制在很大程度上尚不清楚。HSPG具有强阴离子糖胺聚糖链。虽然在SGP上已经确定了三个基本的Gag结合基序(Gag-BM)，但以往的研究主要集中在RBD中的Gag-BM。他们已经表征了肝素(HSPG的简化模型)与这个位点的结合，并表明SGP、ACE2和肝素可以形成三元复合体。然而，SARS-CoV1和SARS-CoV2在这种Gag-BM及其周围环境上的差异相当小。因此，我们认为需要其他因素来解释SARS-CoV2的高传染性。值得注意的是，除了先前病毒株中的两个保守的Gag-BMS外，在SARS-CoV2 SGP的S1/S2区域还发现了一个新的碱基插入。这一基本插入具有一个切割位点基序，该基序在病毒与宿主细胞融合之前被宿主Furin蛋白酶靶向切割Sgp。我们推测，基本的S1/S2位点可能在与HSPGs相互作用和促进SARS-CoV2宿主细胞感染方面发挥关键作用。在meCocan项目中，我们提出了一个多学科的方法，利用计算和实验方法，研究(I)S1/S2基本基序在SARS CoV2 SPG-HSPG结合中的作用；(Ii)HSPG对SGP构象和SGP-ACE2相互作用的机制和变构效应，从而病毒-宿主细胞感染；以及(Iii)S1/S2基本基序、SPG-HSPG相互作用和宿主易感性之间的关系。其具体目的是执行：1)分子动力学模拟，以获得开放和闭合糖基化的SGP胞外结构域与长(-gt；30mer)肝素链以及在与肝素和ACE2的三元复合体中的验证模型；2)表面等离子体共振分析，以测量糖基化的野生型和突变型SGP(S1/S2碱性位点中和)与肝素和/或ACE2的结合，以确定S1/S2位点在HSPGs结合中的作用；3)活细胞结合实验评估野生型和突变型SGp与不同表达HSPGs的细胞的结合能力，以了解它们在宿主易感性中的作用。meCocan项目的结果有望为冠状病毒感染机制的研究提供新的见解，为SARS-CoV2抗病毒化合物的开发提供新的治疗靶点，并为设计宿主细胞感染的抑制剂奠定基础。

The SARS-CoV-2 spike glycoprotein (SGP) plays a key role in host infection by the virus, which ultimately leads to the COVID-19 disease. The SGP on the virion surface attaches to host cells by the binding its receptor-binding domain (RBD) to human angiotensin converting enzyme 2 (ACE2). There is increasing evidence that viral infection is facilitated by host cell heparan sulphate proteoglycans (HSPGs) that act as co-receptors and may influence host susceptibility. However, the mechanism by which HSPGs affect virus-host cell binding and entry is largely unknown. The HSPGs possess strongly anionic glycosaminoglycan (GAG) chains. Although three basic GAG binding motifs (GAG-BM) have been identified on the SGP, prior studies have focused on the GAG-BM in the RBD. They have characterized the binding of heparin, a simplified model for HSPGs, to this site, and have shown that the SGP, ACE2 and heparin can form a ternary complex. However, the differences in this GAG-BM and its surroundings between SARS-CoV1 and SARS-CoV2 are quite modest. Therefore, we propose that other factors are needed to explain the high infectivity of SARS-CoV2. Notably, in addition to the two conserved GAG-BMs in previous viral strains, a novel basic insertion has been identified in the S1/S2 region of SARS-CoV2 SGP. This basic insertion has a cleavage site motif that is targeted by the host furin protease to cleave the SGP prior to virus-host cell fusion. We hypothesize that the basic S1/S2 site may play a critical role in interacting with HSPGs and facilitating SARS-CoV2 host cell infection. In the meCocan project, we propose a multidisciplinary approach, employing computational and experimental methods, to investigate (i) the role of the S1/S2 basic motif in SARS CoV2 SPG-HSPG binding; (ii) the mechanistic and allosteric effects of HSPGs on SGP conformation and SGP-ACE2 interactions, and therefore virus-host cell infection, and (iii) the relation between the S1/S2 basic motif, SPG-HSPG interaction and host susceptibility. The specific aims are to perform:1) molecular dynamics simulations to derive validated models of the open and closed glycosylated SGP ectodomain with long (>30mer) heparin chains and in the ternary complex with heparins and ACE2;2) surface plasmon resonance analysis to measure the binding of glycosylated wild-type and mutant SGP (S1/S2 basic site neutralized) to heparin and/or ACE2 to determine the role of the S1/S2 site in the binding of HSPGs;3) binding assays on living cells to evaluate the ability of wild-type and mutant SGP to bind cells that differently express HSPGs to investigate their role in host susceptibility.The results of the meCocan project are expected to impact molecular and translational medicine in the COVID-19 field by providing new insights into the mechanisms of coronavirus infection, new therapeutic targets for the development of SARS-CoV2 antiviral compounds, and a foundation for the design of inhibitors of host-cell infection.