Entry inhibition of SARS-CoV-2 by human LRRC15

人 LRRC15 对 SARS-CoV-2 进入的抑制

• 批准号: 10575888
• 负责人: Sanghyun Lee
• 金额: $ 21.73万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-12 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10575888
• 关键词: 2019-nCoV; ACE2; Affinity; Binding; Binding Sites; Biological Assay; C-Type Lectins; COVID-19; COVID-19 patient; COVID-19 susceptibility; CRISPR-mediated transcriptional activation; Cell membrane; Cells; Cytoplasm; DAXX gene; Data; Data Set; Epithelial Cells; Event; Fibroblasts; Genes; Heparitin Sulfate; Human; Interferons; Leucine-Rich Repeat; Libraries; Lung; Marketing; Mediating; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Mutagenesis; Names; Neuropilin-1; Pathologic; Pattern Recognition; Physiological; Proteins; Recombinant Proteins; Recombinants; Reporting; Role; SARS-CoV-2 entry inhibitor; SARS-CoV-2 genome; SARS-CoV-2 infection; SARS-CoV-2 variant; Signal Transduction; Stains; Stromal Cells; Surface; System; Therapeutic antibodies; Vaccines; Variant; Viral; Virus; Work; cell type; coronavirus disease; cytokine; domain mapping; effectiveness evaluation; global health; insight; member; novel; receptor; receptor binding; screening; single-cell RNA sequencing; therapeutic development; tumor; vaccine development; viral entry inhibitor

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.

严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）是冠状病毒病19（COVID-19）的病原体，代表着全球健康威胁。作为同源三聚体锚定在病毒包膜表面的病毒刺突蛋白与血管紧张素转换酶2（ACE 2）结合并介导该病毒进入细胞。刺突的受体结合结构域（RBD）直接结合ACE 2，其诱导有利于病毒融合的构象变化。这种融合事件将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疫苗的主要抗原靶标，并且干扰RBD和ACE 2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于控制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（神经毡蛋白-1、硫酸乙酰肝素和C型凝集素）。然而，目前还不清楚是否有任何细胞蛋白质抑制病毒进入。我们的新的初步数据显示，SARS-CoV-2的细胞进入被一种新的抑制性细胞蛋白LRRC 15抑制。我们在细胞和无细胞模型中使用重组蛋白进行结合测定，显示LRRC 15以中等亲和力（KD = 43~148 nM，取决于结构域和变体）与刺突的RBD直接相互作用。虽然ACE 2也通过RBD与刺突相互作用，但LRRC 15-RBD的相互作用不竞争或稳定ACE 2-RBD相互作用，表明非重叠结合位点。对人肺单细胞RNA测序数据集的进一步分析揭示，LRRC 15的表达主要在成纤维细胞中检测到，并且在COVID-19患者的病理性成纤维细胞中特别富集。ACE 2和LRRC 15在肺中的相同细胞类型中不共表达。引人注目的是，LRRC 15不仅在相同的细胞中抑制刺突介导的病毒进入，而且在邻近的细胞中反式表达LRRC 15在ACE 2+细胞中的表达阻断了在ACE 2 + LRRC 15-细胞中刺突介导的病毒进入，提供了通过抑制因子抑制病毒进入的独特概念。该结果表明LRRC 15在生理环境中的保护作用。我们的中心假设是人LRRC 15作为SARS-CoV-2的抑制性进入因子，反式作为诱饵受体在肺中的非易感病理成纤维细胞中表达。该提案将探索LRRC 15通过两个特定目标抑制SARS-CoV-2的反式进入。目标1.通过LRRC 15确定进入抑制的分子机制。目标二。评价病理性成纤维细胞提供离体抑制功能。这项研究提供了对治疗发展的深入了解和对COVID-19的更好理解。