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（SGP）在病毒感染宿主中发挥关键作用，最终导致COVID-19疾病。病毒颗粒表面的SGP通过其受体结合域（RBD）与人血管紧张素转换酶2（ACE 2）结合而附着于宿主细胞。越来越多的证据表明，宿主细胞硫酸乙酰肝素蛋白聚糖（HSPG）作为共受体促进病毒感染，并可能影响宿主的易感性。然而，HSPG影响病毒-宿主细胞结合和进入的机制在很大程度上是未知的。HSPG具有强阴离子糖胺聚糖（GAG）链。虽然已经在SGP上鉴定了三种基本的GAG结合基序（GAG-BM），但是先前的研究集中在RBD中的GAG-BM。他们已经表征了肝素（HSPG的简化模型）与该位点的结合，并且已经表明SGP、ACE 2和肝素可以形成三元复合物。然而，SARS-CoV 1和SARS-CoV 2之间的GAG-BM及其周围环境的差异相当适度。因此，我们提出，需要其他因素来解释SARS-CoV 2的高传染性。值得注意的是，除了在以前的病毒株中的两个保守的GAG-BM之外，还在SARS-CoV 2 SGP的S1/S2区域中鉴定了一种新的碱基插入。该基本插入具有切割位点基序，其被宿主弗林蛋白酶靶向以在病毒-宿主细胞融合之前切割SGP。我们推测基本S1/S2位点可能在与HSPGs相互作用和促进SARS-CoV 2宿主细胞感染中起关键作用。 在meCocan项目中，我们提出了一个多学科的方法，采用计算和实验方法，研究（i）S1/S2基本基序在SARS CoV 2 SPG-HSPG结合中的作用;（ii）HSPG对SGP构象和SGP-ACE 2相互作用以及病毒-宿主细胞感染的机制和变构效应，以及（iii）S1/S2基本基序之间的关系，SPG-HSPG相互作用和宿主易感性。具体目标是执行：1）分子动力学模拟，以获得具有长（> 30 mer）肝素链的开放和闭合糖基化SGP胞外域以及与肝素和ACE 2的三元复合物的经验证的模型;2）表面等离子体共振分析，以测量糖基化野生型和突变SGP的结合（S1/S2碱性位点中和）与肝素和/或ACE 2的结合，以确定S1/S2位点在HSPG结合中的作用; 3）活细胞结合试验，以评估野生型和突变型SGP结合不同表达HSPG的细胞的能力，以研究它们在宿主易感性中的作用。19领域通过提供新的见解冠状病毒感染的机制，新的治疗目标的SARS-CoV 2抗病毒化合物的开发，并为宿主细胞感染抑制剂的设计基础。

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.