Entry inhibition of SARS-CoV-2 by human LRRC15

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号(新冠肺炎)的病原体，对全球健康构成威胁。病毒刺突蛋白作为同源三聚体锚定在病毒被膜表面，与血管紧张素转换酶2(ACE2)结合，介导病毒进入细胞。SPRKE的受体结合域(RBD)直接与ACE2结合，引起构象变化，促进病毒融合。这种融合事件将SARS-CoV-2基因组释放到细胞质中。Spike蛋白，特别是RBD是市场上COVID疫苗的主要抗原靶蛋白，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，表明RBD及其与细胞受体的结合在控制SARS-CoV-2方面的重要性。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(神经粘连蛋白-1、硫酸肝素和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的细胞进入被一种新的抑制性细胞蛋白-富含亮氨酸重复序列15(LRRC15)所抑制。我们在细胞和无细胞模型中使用重组蛋白进行了结合分析，表明LRRC15以中等亲和力(Kd=43~148 nM，取决于结构域和变体)与SPEKE的RBD直接相互作用。虽然ACE2也通过RBD与尖峰相互作用，但LRRC15-RBD的相互作用不竞争或稳定ACE2-RBD的相互作用，表明结合位点不重叠。进一步分析人肺单细胞核糖核酸测序数据集表明，LRRC15主要在成纤维细胞中检测到表达，尤其在新冠肺炎患者的病理性成纤维细胞中表达。ACE2和LRRC15在肺中不同类型的细胞中共表达。值得注意的是，LRRC15不仅在相同的细胞中抑制尖峰信号介导的病毒进入，而且在邻近的反式细胞中也是如此。LRRC15在ACE2细胞中的表达阻断了尖峰信号介导的病毒进入ACE2 LRRC15-细胞，提供了一个独特的概念，即通过抑制因子来抑制病毒进入。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，在反式作用中作为诱饵受体在肺不敏感的病理性成纤维细胞中表达。这项建议将探讨LRRC15通过两个特定目的抑制SARS-CoV-2反式病毒进入的机制。目的1.确定LRRC15抑制进入的分子机制。目的2.体外评价病理性成纤维细胞的抑制作用。这项研究提供了对治疗发展的洞察和对新冠肺炎的更好理解。

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.