Surfaceome CRISPR screening for SARS-CoV-2 virulent proteins

表面组 CRISPR 筛选 SARS-CoV-2 毒力蛋白

• 批准号: 10892771
• 负责人: Sanghyun Lee
• 金额: $ 16.87万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10892771
• 关键词: 2019-nCoV; ACE2; Binding; COVID-19; COVID-19 screening; CRISPR screen; CRISPR-mediated transcriptional activation; Cell membrane; Cells; Centers of Research Excellence; Computational Biology; Data; Fibroblasts; Human; Libraries; Lung; Mediating; Membrane Proteins; Names; Norovirus; Pathologic; Physiological; Preparation; Proteins; Recombinants; Role; SARS-CoV-2 entry inhibitor; Stains; Therapeutic antibodies; Vaccines; Viral; Virulence; Virulent; coronavirus disease; human disease; novel; receptor; receptor binding; screening; syndecan-4; viral entry inhibitor

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19). Spike protein is the primary antigenic target for COVID vaccines and interfering with the interface between RBD (Receptor Binding Domain) of spike 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. It is unclear whether there are any intrinsic cellular proteins that inhibit viral entry of SARS-CoV-2. 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 generated a focused CRISPR activation (CRISPRa) library, named surfaceome, that covers all ~6000 known/predicted surface proteins on the cellular plasma membrane. The surfaceome CRISPRa screening was performed by staining cells with a recombinant spike protein to identify SARS-CoV-2 binding factors. 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 the mechanism by which LRRC15 inhibits entry of SARS-CoV-2 in trans through three subaims of Aim 1. We will leverage the screening platform to identify cellular receptors for secreted virulence proteins of SARS-CoV-2, Orf3a, Orf7a, and Orf8. Importantly, the same screening platform is validated to be a efficient platform for secreted virulent proteins. In a separate screening for norovirus secreted virulence protein (NS1), the surfaceome screening successfully identified Syndecan-4 as a putative cellular receptor for norovirus NS1 (Li et al., In preparation). In the Aim 2, we will perform a surfaceome screening for Orf3a, Orf7a, and Orf8 and will discover putative cellular receptors for the secreted virulence proteins.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是冠状病毒的病原体 疾病 19 (COVID-19)。刺突蛋白是新冠疫苗和干扰物的主要抗原靶点 刺突蛋白的RBD（受体结合域）与ACE2之间的界面是 对大多数现有治疗抗体的作用，表明 RBD 及其结合的重要性 控制 SARS-CoV-2 的细胞受体。目前尚不清楚是否存在任何内在的细胞 抑制 SARS-CoV-2 病毒进入的蛋白质。 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的抑制 蛋白质，富含亮氨酸重复序列，含有 15 (LRRC15)。我们生成了集中的 CRISPR 激活 (CRISPRa) 文库，名为表面组，涵盖了所有约 6000 种已知/预测的表面蛋白 细胞质膜。表面组 CRISPRa 筛选是通过用 重组刺突蛋白用于鉴定 SARS-CoV-2 结合因子。引人注目的是，LRRC15 抑制 刺突介导的病毒不仅进入同一细胞，而且还反式进入邻近细胞。表达 ACE2+ 细胞中的 LRRC15 阻断了 ACE2+LRRC15- 细胞中刺突介导的病毒进入，从而提供了独特的 抑制因子抑制病毒进入的概念。这一结果表明 LRRC15 在 生理背景。我们的中心假设是人类 LRRC15 作为抑制性进入因子 SARS-CoV-2，以反式作为诱饵受体，在非易感病理成纤维细胞中表达 肺。该提案将探讨 LRRC15 抑制 SARS-CoV-2 进入体内的机制。 贯穿目标 1 的三个子目标。 我们将利用筛选平台来识别分泌毒力蛋白的细胞受体 SARS-CoV-2、Orf3a、Orf7a 和 Orf8。重要的是，相同的筛选平台被验证为 分泌有毒蛋白的有效平台。单独筛选诺如病毒分泌毒力 蛋白 (NS1)，表面组筛选成功将 Syndecan-4 鉴定为假定的细胞受体 诺如病毒 NS1（Li 等人，准备中）。在目标 2 中，我们将进行表面组筛选 Orf3a、Orf7a 和 Orf8 将发现分泌毒力蛋白的假定细胞受体。