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 (SARS-CoV-2) 是冠状病毒病 19 (COVID-19) 的病原体，对全球健康构成威胁。病毒刺突蛋白以同源三聚体形式锚定在病毒包膜表面，与血管紧张素转换酶 2 (ACE2) 结合并介导该病毒进入细胞。 Spike 的受体结合域 (RBD) 直接与 ACE2 结合，从而诱导构象变化，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，是市场上新冠疫苗的主要抗原靶标，干扰 RBD 和 ACE2 之间的界面是大多数现有治疗抗体的作用机制，这表明 RBD 及其与细胞受体结合对于控制 SARS-CoV-2 的重要性。迄今为止，已经确定了几种促进 SARS-CoV-2 进入细胞的细胞因子（neuropilin-1、硫酸乙酰肝素和 C 型凝集素）。然而，尚不清楚是否存在任何抑制病毒进入的细胞蛋白。我们的新初步数据表明，SARS-CoV-2 的细胞进入受到一种新型抑制性细胞蛋白——富含亮氨酸重复序列 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测定，结果显示 LRRC15 以中等亲和力（KD = 43~148 nM，取决于结构域和变体）直接与刺突的 RBD 相互作用。尽管 ACE2 也通过 RBD 与刺突相互作用，但 LRRC15-RBD 的相互作用不会竞争或稳定 ACE2-RBD 相互作用，表明结合位点不重叠。对人肺单细胞 RNA 测序数据集的进一步分析表明，LRRC15 的表达主要在成纤维细胞中检测到，尤其在 COVID-19 患者的病理成纤维细胞中富集。 ACE2 和 LRRC15 不在肺中的相同细胞类型中共表达。引人注目的是，LRRC15 不仅抑制刺突介导的病毒进入同一细胞，而且还抑制反式邻近细胞的进入。 ACE2+ 细胞中 LRRC15 的表达阻断了 ACE2+LRRC15- 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。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 的抑制性进入因子，反式作为在肺部不敏感的病理成纤维细胞中表达的诱饵受体。该提案将探讨 LRRC15 通过两个具体目标抑制 SARS-CoV-2 反式进入。目标 1. 确定 LRRC15 进入抑制的分子机制。目标 2. 评估提供离体抑制功能的病理成纤维细胞。这项研究提供了对治疗开发的深入见解和对 COVID-19 的更好理解。