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Abstract：
The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 is a critical viral protein that suppresses host gene expression by blocking the assembly of the ribosome on host mRNAs. To understand the mechanism of inhibition of host gene expression, we sought to identify cellular proteins that interact with nsp1. Using proximity-dependent biotinylation followed by proteomic analyses of biotinylated proteins, here we captured multiple dynamic interactions of nsp1 with host cell proteins. In addition to ribosomal proteins, we identified several pre-mRNA processing proteins that interact with nsp1, including splicing factors and transcription termination proteins, as well as exosome, and stress granule (SG)-associated proteins. We found that the interactions with transcription termination factors are primarily governed by the C-terminal region of nsp1 and are disrupted by the mutation of amino acids K164 and H165 that are essential for its host shutoff function. We further show that nsp1 interacts with Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and colocalizes with G3BP1 in SGs under sodium arsenite-induced stress. Finally, we observe that the presence of nsp1 disrupts the maturation of SGs over a long period. Isolation of SG core at different times shows a gradual loss of G3BP1 in the presence of nsp1.
摘要：
严重急性呼吸综合征冠状病毒和严重急性呼吸综合征冠状病毒2的非结构蛋白1（nsp1）是一种关键的病毒蛋白，通过阻断核糖体在宿主mRNA上的组装来抑制宿主基因表达。了解抑制宿主基因表达的机制,我们试图鉴定与nsp1相互作用的细胞蛋白。使用邻近依赖的生物素化，然后对生物素化的蛋白质进行蛋白质组学分析，在这里，我们捕获了nsp1与宿主细胞蛋白的多种动态相互作用。除了核糖体蛋白,我们确定了几种与nsp1相互作用的前mRNA加工蛋白，包括剪接因子和转录终止蛋白，以及外来体，和应激颗粒（SG）相关蛋白。我们发现与转录终止因子的相互作用主要受nsp1的C末端区域控制，并被其宿主关闭功能所必需的氨基酸K164和H165的突变所破坏。我们进一步显示，nsp1与RasGTP酶激活蛋白SH3结构域结合蛋白1（G3BP1）相互作用，并在亚砷酸钠诱导的应激下与SGs中的G3BP1共定位。最后,我们观察到nsp1的存在会长期破坏SGs的成熟。在不同时间的SG核心的分离显示了在nsp1存在下G3BP1的逐渐损失。
