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Research
Ji-An Pan
Sun Yat-Sen University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2842-4126
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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 proteins 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. In summary, 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.
Keywords
SARS-CoV-2, interaction map, N, Nsp3, replication
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This is a list of supplementary files associated with this preprint. Click to download.
- FigS1.png
The conserved domains of Nsp3 of SARS-CoV-2. The diagram of Nsp3 with indicated conserved domains was produced according to the description of YP_009725299.1 of CDD of NCBI. The separation sites aa 749 for Nsp3.1 and aa 1462 for Nsp3.2 were labeled in the diagram.
- FigS2.png
Acid-base analysis for the viral proteins encoded by SARS-CoV-2. Viral proteins encoded by SARS-CoV-2 were listed in the order of PI values. Note that N is the most basic protein and Nsp3.1 is among the most acidic proteins of SARS-CoV-2.
- FigS3.png
The plasmid map of the replicon of SARS-CoV-2. The cDNA of SARS-CoV-2 genome was inserted in the BAC vector. Its expression was driven by the human Cytomegalovirus (CMV) promoter upstream of its sequence. The bovine growth hormone polyadenylation (BGH) was employed to terminate the transcription and was removed by a hepatitis delta virus ribozyme (RZ) to generate the authentic 3’ terminal sequence of SARS-CoV-2. The S gene was replaced with firefly luciferase to eliminate the generation of live virus and to quantify the replication of replicon.
- Table1.png
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On 21 Mar, 2021
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On 16 Mar, 2021
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On 16 Mar, 2021
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On 16 Mar, 2021
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This preprint is under consideration at Cell & Bioscience. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Ji-An Pan
Sun Yat-Sen University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-2842-4126
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 19 Mar, 2021
No community comments so far
Editorial decision: Major revision
On 11 Apr, 2021
Review #4 received
Received 11 Apr, 2021
Review #3 received
Received 11 Apr, 2021
Review #1 received
Received 25 Mar, 2021
Review #2 received
Received 25 Mar, 2021
Reviewer #4 agreed
On 21 Mar, 2021
Reviewer #3 agreed
On 20 Mar, 2021
Reviewer #2 agreed
On 16 Mar, 2021
First submitted
On 16 Mar, 2021
Editor assigned
On 16 Mar, 2021
Reviewers invited
Invitations sent on 16 Mar, 2021
Reviewer #1 agreed
On 16 Mar, 2021
Submission checks complete
On 16 Mar, 2021
Editor invited
On 16 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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 proteins 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. In summary, 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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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