Entry inhibition of SARS-CoV-2 by human LRRC15

严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）是冠状病毒病19 （COVID-19）的病原体，是全球健康威胁。病毒刺突蛋白作为同源三聚体固定在病毒包膜表面，与血管紧张素转换酶2 （ACE2）结合并介导病毒进入细胞。刺突的受体结合域（RBD）直接与ACE2结合，诱导构象变化，促进病毒融合。这一融合事件将SARS-CoV-2基因组释放到细胞质中。刺突蛋白，特别是RBD，是市场上COVID疫苗的主要抗原靶点，干扰RBD和ACE2之间的界面是大多数现有治疗性抗体的作用机制，这表明RBD及其与细胞受体的结合对控制SARS-CoV-2的重要性。到目前为止，已经确定了几种促进SARS-CoV-2进入细胞的细胞因子（神经磷脂-1、硫酸肝素和c型凝集素）。然而，目前尚不清楚是否有任何细胞蛋白可以抑制病毒进入。我们新的初步数据显示，SARS-CoV-2的细胞进入受到一种新的抑制细胞蛋白，富含白细胞重复序列15 （LRRC15）的抑制。我们在细胞和无细胞模型中使用重组蛋白进行了结合实验，结果表明LRRC15与刺突的RBD直接相互作用，具有中等亲和力（KD = 43~148 nM，取决于结构域和变体）。虽然ACE2也通过RBD与突状突起相互作用，但LRRC15-RBD的相互作用并不竞争或稳定ACE2-RBD的相互作用，这表明ACE2-RBD的结合位点不重叠。进一步分析人肺单细胞RNA测序数据显示，LRRC15主要在成纤维细胞中表达，在COVID-19患者的病理性成纤维细胞中表达尤其丰富。ACE2和LRRC15在肺的相同细胞类型中不共表达。引人注目的是，LRRC15不仅在同一细胞中抑制刺突介导的病毒进入，而且在反式中也抑制邻近细胞。LRRC15在ACE2+细胞中的表达阻断了ACE2+LRRC15-细胞中突刺介导的病毒进入，提供了一种独特的抑制因子抑制病毒进入的概念。这一结果表明LRRC15在生理环境中具有保护作用。我们的中心假设是，人类LRRC15作为SARS-CoV-2的抑制进入因子，在肺中非易感病理性成纤维细胞中作为诱骗受体表达。本提案将探讨LRRC15如何通过两个特定目的抑制SARS-CoV-2进入trans。目的1。确定LRRC15进入抑制的分子机制。目标2。评估病理性成纤维细胞在体外提供抑制功能。这项研究为治疗发展和更好地了解COVID-19提供了见解。

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19), representing a global health threat. The viral spike protein, anchored on the surface of the viral envelope as homotrimers, binds to angiotensin-converting enzyme 2 (ACE2) and mediates the cellular entry of this virus. The receptor binding domain (RBD) of spike directly binds to ACE2, which induces a conformational change that facilitates virus fusion. This fusion event releases the SARS-CoV-2 genome into the cytoplasm. Spike protein, specifically the RBD, is the primary antigenic target for COVID vaccines in the market and interfering with the interface between RBD and ACE2 is the mechanism of action for the majority of existing therapeutic antibodies, indicating the importance of RBD and its binding to the cellular receptor for controlling SARS-CoV-2. Thus far, several cellular factors have been identified to facilitate cellular entry of SARS-CoV-2 (neuropilin-1, heparan sulfate, and C-type lectins). However, it is unclear whether there are any cellular proteins that inhibit viral entry. Our new preliminary data reveal that cellular entry of SARS-CoV-2 is inhibited by a novel inhibitory cellular protein, Leucin-rich repeat containing 15 (LRRC15). We performed binding assays using recombinant proteins in cells and in cell-free models that show LRRC15 directly interacts with the RBD of spike with a moderate affinity (KD = 43~148 nM, depending on domain and variant). Although ACE2 also interacts with the spike via the RBD, the interaction of LRRC15-RBD does not compete or stabilize ACE2-RBD interactions, suggesting non- overlapping binding sites. Further analysis of human lung single cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not co-expressed in the same cell types in the lung. Strikingly, LRRC15 inhibits spike-mediated viral entry not only in the same cells, but also in neighboring cells in trans. Expression of LRRC15 in ACE2+ cells blocked spike-mediated viral entry in ACE2+LRRC15- cells, providing a unique concept of viral entry inhibition by an inhibitory factor. This result suggests a protective role of LRRC15 in a physiological context. Our central hypothesis is that human LRRC15 acts as an inhibitory entry factor for SARS-CoV-2, acting in trans as a decoy receptor expressed in non-susceptible pathological fibroblasts in the lung. This proposal will explore by which LRRC15 inhibits entry of SARS-CoV-2 in trans through two specific aims. Aim 1. Determine molecular mechanisms of entry inhibition by LRRC15. Aim 2. Evaluate pathological fibroblasts providing the inhibitory function ex vivo. This study provides an insight into therapeutic development and a better understanding of COVID-19.