Surfaceome CRISPR screening for SARS-CoV-2 virulent proteins

严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）是冠状病毒的病原体 疾病19（COVID-19）。刺突蛋白是COVID疫苗的主要抗原靶点， 刺突的受体结合域（Receptor Binding Domain，RBD）与ACE 2之间的界面是其作用机制。 RBD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。 控制SARS-CoV-2的细胞受体。目前尚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的细胞进入受到一种新的抑制性细胞因子的抑制。 富含亮氨酸的重复序列15（LRRC 15）。我们产生了一个集中的CRISPR激活 （CRISPRa）库，命名为表面组，涵盖了所有约6000个已知/预测的表面蛋白， 细胞质膜表面组CRISPRa筛选通过用荧光素酶染色细胞来进行。 重组刺突蛋白鉴定SARS-CoV-2结合因子。引人注目的是，LRRC 15抑制 刺突介导的病毒进入不仅在相同的细胞中，而且在相邻细胞中反式表达。 ACE 2+细胞中的LRRC 15阻断了ACE 2 + LRRC 15-细胞中刺突介导的病毒进入，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LRRC 15在细胞凋亡中的保护作用。 生理背景。我们的中心假设是，人LRRC 15作为一种抑制进入因子， SARS-CoV-2，作为诱饵受体在非易感的病理成纤维细胞中表达， 肺该提案将探索LRRC 15抑制SARS-CoV-2进入的机制， 通过目标1的三个子目标来实现。 我们将利用筛选平台来鉴定分泌的毒力蛋白的细胞受体， SARS-CoV-2、Orf3a、Orf7a和Orf8。重要的是，相同的筛选平台被验证为 分泌毒性蛋白的有效平台。在一个单独的筛选诺如病毒分泌毒力 蛋白（NS 1），表面组筛选成功地确定Syndecan-4作为假定的细胞受体 对于诺如病毒NS 1（Li等人，正在编写中）。在目标2中，我们将进行表面组筛选， Orf 3a、Orf 7a和Orf 8，并将发现分泌的毒力蛋白的推定细胞受体。

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.