Yiling Jiang1, Kuijie Tong1, Roubin Yao1, Yuanze Zhou2, Hanwen Lin1, Liubing Du1, Yunyun Jin1, Liu Cao1, Jingquan Tan2, Xing-Ding Zhang1, Deyin Guo1, Ji-An Pan3, Xiaoxue Peng4. 1. The Center for Infection and Immunity Study and Molecular Cancer Research Center, School of Medicine, Sun Yat-Sen University, Guangming Science City, Shenzhen, 518107, China. 2. Nanjing CRYCISION Biotechnology Co., Ltd, Nanjing, 211100, China. 3. The Center for Infection and Immunity Study and Molecular Cancer Research Center, School of Medicine, Sun Yat-Sen University, Guangming Science City, Shenzhen, 518107, China. panjan@mail.sysu.edu.cn. 4. The Center for Infection and Immunity Study and Molecular Cancer Research Center, School of Medicine, Sun Yat-Sen University, Guangming Science City, Shenzhen, 518107, China. pengxx9@mail.sysu.edu.cn.
Abstract
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: Analysis of viral protein-protein interactions is an esseene">ntial step to un class="Species">ncover 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. RESULTS: 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. CONCLUSIONS: 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.
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