Analysis of viral protein–protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein–protein interactions. Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

中文翻译：

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蛋白质-蛋白质相互作用以及病毒蛋白质参与 SARS-CoV-2 复制的全基因组分析

病毒蛋白-蛋白相互作用的分析是揭示病毒蛋白功能和病毒蛋白复合物组装分子机制的重要步骤。我们采用哺乳动物双杂交系统来筛选 SARS-CoV-2 的所有病毒蛋白的蛋白质-蛋白质相互作用。我们的研究检测到 48 种相互作用，其中 14 种是本文首次报道的。与 SARS-CoV 的 Nsp1 不同，SARS-CoV-2 的 Nsp1 在所有病毒蛋白中具有最多的相互作用伙伴，并且可能充当病毒蛋白的枢纽。确认了 5 种自身相互作用，其中 Nsp1/Nsp3.1、Nsp3.1/N、Nsp3.2/Nsp12、Nsp10/Nsp14 和 Nsp10/Nsp16 5 种相互作用被确定为双向正向相互作用。利用SARS-CoV-2的复制子报告系统，我们筛选了所有病毒Nsp对病毒复制的影响，发现Nsp3的N末端Nsp3.1显着抑制复制子报告基因的表达。我们发现Nsp3通过其N末端的酸性区域与N相互作用，而N通过其富含碱性氨基酸的NTD与Nsp3相互作用。此外，使用纯化的截短的 N 和 Nsp3 蛋白，我们确定了 Nsp3 和 N 蛋白之间的直接相互作用。我们的研究结果为了解冠状病毒蛋白的功能提供了基础，并支持相互作用作为抗病毒药物开发目标的潜力。