Surfaceome CRISPR screening for SARS-CoV-2 virulent proteins

严重急性呼吸综合征冠状病毒2型是冠状病毒的病原体 疾病19(新冠肺炎)。刺突蛋白是冠状病毒疫苗和干扰的主要抗原靶 在SPEKE的受体结合域(RBD)和ACE2之间的界面是 对大多数现有治疗性抗体的作用，表明RBD及其结合的重要性 与控制SARS-CoV-2的细胞受体有关。目前还不清楚是否有任何内在的细胞 抑制SARS-CoV-2病毒进入的蛋白质。 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所抑制 蛋白质，富含亮氨酸的重复序列，包含15(LRRC15)。我们生成了一个聚焦的CRISPR激活 (CRISPRa)文库，名为Surfaceome，涵盖所有~6000个已知/预测的表面蛋白 细胞质膜。表面蛋白CRISPRA的筛选是通过对细胞进行染色进行的 重组S蛋白鉴定SARS-CoV-2结合因子。引人注目的是，LRRC15抑制了 刺激物介导的病毒不仅在相同的细胞内，而且在邻近的细胞中也以反式。的表达 ACE2细胞中的LRRC15阻断了尖峰信号介导的病毒进入ACE2 LRRC15-细胞，提供了一种独特的 通过抑制因子抑制病毒进入的概念。这一结果表明LRRC15在脑内具有保护作用。 一种生理背景。我们的中心假设是人类LRRC15作为一种抑制进入因子 SARS-CoV-2作为诱骗受体在非易感病理性成纤维细胞中的表达 肺部。本提案将探讨LRRC15抑制SARS-CoV-2进入小鼠体内的机制 贯穿目标1的三个分目标。 我们将利用该筛选平台来识别分泌的毒力蛋白的细胞受体。 SARS-CoV-2、Orf3a、Orf7a和Orf8。重要的是，相同的筛查平台被验证为 分泌毒力蛋白的有效平台。在对诺沃克病毒分泌毒力的单独筛查中 蛋白(NS1)，表面组筛选成功鉴定Syndecan-4为一种可能的细胞受体 用于诺沃克病毒NS1(Li等人，正在准备中)。在目标2中，我们将进行表面筛查 Orf3a、Orf7a和Orf8，并将发现分泌的毒力蛋白的假定细胞受体。

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.