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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain.

ABSTRACT: Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

SUBMITTER: Wu C

PROVIDER: S-EPMC7709165 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain.

Wu Chao C Qavi Abraham J AJ Hachim Asmaa A Kavian Niloufar N Cole Aidan R AR Moyle Austin B AB Wagner Nicole D ND Sweeney-Gibbons Joyce J Rohrs Henry W HW Gross Michael L ML Peiris J S Malik JSM Basler Christopher F CF Farnsworth Christopher W CW Valkenburg Sophie A SA Amarasinghe Gaya K GK Leung Daisy W DW

bioRxiv : the preprint server for biology 20201130

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. W ...[more]

PMID: 33269347

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Project description:AimSpike protein of coronavirus is responsible for virus binding, fusion and entry, and is a major inducer of neutralizing antibodies. This paper was to find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.MethodsThree fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.ResultsRBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST/RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST/RBD binding to Vero E6 cells were 77.08 and 352.73 respectively.ConclusionIn this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Project description:The clue behind the SARS-CoV-2 origin is still a matter of debate. Here, we report that SARS-CoV-2 has gained a novel spike protein S1-N-terminal domain (S1-NTD). In our CLuster ANalysis of Sequences (CLANS) analysis, SARS-CoV/SARS-CoV-2 S1-NTDs displayed a close relationship with OC43 and HKU1. However, in the complete and S1-NTD-free spike protein, SARS-CoV/SARS-CoV-2 revealed closeness with MERS-CoV. Further, we have divided the S1-NTD of SARS-CoV-2 related viruses into three distinct types (Type-I to III S1-NTD) and the S1-NTD of viruses associated with SARS-CoVs into another three classes (Type-A to C S1-NTD) using CLANS and phylogenetic analyses. In particular, the results of our study indicate that SARS-CoV-2, RaTG13, and BANAL-20-52 viruses carry Type-I-S1-NTD and other SARS-CoV-2-related-bat viruses have Type-II and III. In addition, it was revealed that the Pangolin-GX and Pangolin-Guangdong lineages inherited Type-I-like and Type-II-like S1-NTD, respectively. Then our CLANS study shows the potential for evolution of Type-I and Type-III S1-NTD from SARS-CoV-related viruses Type-A and Type-B S1-NTDs, respectively. Furthermore, our analysis clarifies the possibility that Type-II S1-NTDs may have evolved from Type-A-S1-NTD of SARS-CoV-related viruses through Type-I S1-NTDs. We also observed that BANAL-20-103, BANAL-20-236, and Pangolin-Guangdong-lineage viruses containing Type-II-like S1-NTD are very close to SARS-CoV-2 in spike genetic areas other than S1-NTD. Possibly, it suggests that the common ancestor spike gene of SARS-CoV-2/RaTG13/BANAL-20-52-like virus may have evolved by recombining the Pangolin-Guangdong/BANAL-20-103/BANAL-20-236-like spike gene to Pangolin-GX-like Type-I-like-S1-NTD in the unsampled bat or undiscovered intermediate host or possibly pangolin. These may then have evolved into SARS-CoV-2, RaTG13, and BANAL-20-52 virus spike genes by host jump mediated evolution. The potential function of the novel Type-I-S1-NTD and other types of S1-NTDs needs to be studied further to understand better its importance in the ongoing COVID-19 outbreak and for future pandemic preparedness.

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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain.

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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain.

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