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.

严重的急性呼吸综合症冠状病毒2（SARS-COV-2）是冠状病毒19（Covid-19）的病因，代表了全球健康威胁。病毒尖峰蛋白固定在病毒包膜的表面上，作为同构二聚体，与血管紧张素转化酶2（ACE2）结合并介导该病毒的细胞进入。 SPIKE的受体结合结构域（RBD）直接与ACE2结合，这诱导了促进病毒融合的构象变化。这个融合事件将SARS-COV-2基因组释放到细胞质中。尖峰蛋白，特别是RBD，是在市场中互联疫苗的主要抗原靶标，并且干扰RBD和ACE2之间的界面是大多数现有治疗性抗体的作用机理，表明RBD的重要性及其对控制细胞受体的影响，以控制SARS-SARS-COV-2。到目前为止，已经确定了几种细胞因子，以促进SARS-COV-2（Neuropilin-1，硫酸乙酰肝素和C型凝集素）的细胞进入。但是，目前尚不清楚是否有任何细胞蛋白抑制病毒的进入。我们的新初步数据表明，SARS-COV-2的细胞进入被新型的抑制性细胞蛋白，含15的含白细胞素的重复（LRRC15）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，这些模型显示LRRC15直接与具有中等亲和力的Spike RBD相互作用（KD = 43〜148 nm，取决于域和变体）。尽管ACE2也通过RBD与SPIKE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，这表明非重叠的结合位点。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，尤其富含Covid-19患者的病理成纤维细胞。 ACE2和LRRC15在肺中的相同细胞类型中未共表达。引人注目的是，LRRC15不仅在同一细胞中，而且在反式细胞中抑制了尖峰介导的病毒进入。 LRC15在ACE2+细胞中的表达阻断了ACE2+ LRRC15-细胞中的尖峰介导的病毒进入，从而提供了抑制因子抑制病毒进入抑制的独特概念。该结果表明LRC15在生理环境中具有保护作用。我们的中心假设是，人LRC15充当SARS-COV-2的抑制性进入因子，该因子在反式的诱饵受体中作用在肺中不敏感的病理成纤维细胞中表达。该提案将探讨LRC15通过两个特定目标抑制SARS-COV-2进入Trans的进入。 AIM 1。确定LRRC15进入抑制的分子机制。 AIM 2。评估病理成纤维细胞提供抑制作用的离体。这项研究提供了对治疗发展的见识，并对Covid-19有了更好的了解。