PDF(Pubmed)
Abstract：
Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.
摘要：
病毒受体决定了病毒的组织嗜性,与病毒感染引起的临床结局有一定的关系,这对于识别病毒受体，了解病毒的感染机制和开发进入抑制剂具有重要意义。邻近标记（PL）是一种研究蛋白质-蛋白质相互作用的新技术，但它尚未应用于病毒受体或共受体的鉴定。这里,我们试图通过使用TurboID催化的PL来鉴定SARS-CoV-2的共受体。膜蛋白血管紧张素转换酶2（ACE2）用作诱饵并与TurboID缀合，构建了稳定表达ACE2-TurboID的A549细胞系。在生物素和ATP存在下，SARS-CoV-2假病毒与ACE2-TurboID稳定表达的细胞系孵育，这可以启动TurboID的催化活性，并用生物素标记相邻的内源性蛋白。随后，收获生物素化的蛋白质并通过质谱鉴定。我们鉴定了一种膜蛋白,AXL,已在功能上显示可介导SARS-CoV-2进入宿主细胞。我们的数据表明PL可用于鉴定病毒进入的共受体。
