Structural characterization of the C-terminal domain of SARS-CoV-2 nucleocapsid protein

Zhou, Renjie; Zeng, Rui; Brunn, Albrecht von; Lei, Jian

doi:10.1186/s43556-020-00001-4

Cited by 92 publications

(113 citation statements)

References 51 publications

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“…3). Thus, our data align with the lowresolution structural model of SARS-CoV and SARS-CoV-2 N proteins, in which the CTD (residues 247-364), largely responsible for dimerization, and NTD, involved in RNA-binding, are only loosely associated [24,28,29].…”

Section: Sec-mals (supporting

confidence: 84%

“…Such organization allows for a vast conformational change, which in combination with positively charged surfaces [23], facilitates nucleic acid binding [24]. Indeed, the crystal structure of the N-terminal domain (NTD) reveals an RNA binding groove [25][26][27], while crystal structures of the Cterminal domain (CTD) show a highly interlaced dimer with additional nucleic acid binding capacity [28,29]. The N protein shows unusual properties in the presence of RNA, displaying concentration-dependent liquid-liquid phase separation [22,23,30,31] that is pertinent to the viral genome packaging mechanism [32,33].…”

Section: Introductionmentioning

confidence: 99%

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The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

Tugaeva

Hawkins

Smith

et al. 2020

Preprint

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The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.HighlightsSARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

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Section: Sec-mals (supporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

Tugaeva

Hawkins

Smith

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…The sequence identity for CTD of SARS-CoV-2 with SARS-CoV has been observed to be 96% except for notable changes in five residues; Q268, D291, H335, Q346 and N350 of SARS-CoV to A267, E290, T334, Q345 and N348 in case of SARS-CoV-2, respectively. In order to elucidate the structural similarities, one group overlaid the structure of the SARS-CoV-2 N-protein CTD domain determined by them with CTD structures available with PDB code 7C22 [ 111 ] (unpublished manuscript). The outcome revealed high structural similarity with r.m.s.d value of C α in the range 0.15–0.31 Å. Additionally; a positively charged surface area present in the CTD domain has been suggested to be responsible to facilitate RNA binding.…”

Section: Nucleocapsid Proteinmentioning

confidence: 99%

SARS-CoV-2: Insights into its structural intricacies and functional aspects for drug and vaccine development

Kaur

Sharma

Kumar

et al. 2021

International Journal of Biological Macromolecules

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Globally, SARS-CoV-2 has emerged as threat to life and economy. Researchers are trying to find a cure against this pathogen but without much success. Several attempts have been made to understand the atomic level details of SARS-CoV-2 in the past few months. However, one review with all structural details for drug and vaccine development has been missing. Hence, this review aims to summarize key functional roles played by various domains of SARS-CoV-2 genome during its entry into the host, replication, repression of host immune response and overall viral life cycle. Additionally, various proteins of SARS-CoV-2 for finding a potent inhibitor have also been highlighted. To mitigate this deadly virus, an understanding of atomic level information, pathogenicity mechanisms and functions of different proteins in causing the infection is imperative. Thus, these structural details would finally pave the way for development of a potential drug/vaccine against the disease caused by SARS-CoV-2.

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“…CoV N protein can bind to viral RNA forming ribonucleoprotein (RNP) complex. The N protein is involved in viral RNA transcription and replication, packaging of the viral genome, interferon inhibition, actin reorganization, host cell cycle progression and apoptosis 27 - 30 . As there is currently little information regarding the N protein of SARS-CoV-2, it is vital to determine if results obtained from other coronaviruses, such as MERS-CoV and SARS-CoV, can be applied to SARS-CoV-2.…”

Section: Introductionmentioning

confidence: 99%

MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein

Kim

Baek

et al. 2021

Theranostics

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Background: The molecular interactions between viral proteins form the basis of virus production and can be used to develop strategies against virus infection. The interactions of the envelope proteins and the viral RNA-binding nucleocapsid (N) protein are essential for the assembly of coronaviruses including the Middle East respiratory syndrome coronavirus (MERS-CoV). Methods: Using co-immunoprecipitation, immunostaining, and proteomics analysis, we identified a protein interacting with the spike (S) protein in the cells infected with MERS-CoV or SARS-CoV-2. To confirm the interaction, synthetic peptides corresponding to the C-terminal domain of the S protein (Spike CD) were produced and their effect on the interaction was investigated in vitro . In vivo effect of the Spike CD peptides after cell penetration was further investigated using viral plaque formation assay. Phylogeographic analyses were conducted to deduce homology of Spike CDs and N proteins. Results: We identified a direct interaction between the S protein and the N protein of MERS-CoV that takes place during virus assembly in infected cells. Spike CD peptides of MERS-CoV inhibited the interaction between the S and N proteins in vitro. Furthermore, cell penetration by the synthetic Spike CD peptides inhibited viral plaque formation in MERS-CoV-infected cells. Phylogeographic analyses of Spike CDs and N proteins showed high homology among betacoronavirus lineage C strains. To determine if Spike CD peptides can inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we used the same strategy and found that the SARS-CoV-2 Spike CD peptide inhibited virus replication in SARS-CoV-2-infected cells. Conclusions: We suggest that the interaction between the S protein and the N protein can be targeted to design new therapeutics against emerging coronaviruses, including SARS-CoV-2.

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Structural characterization of the C-terminal domain of SARS-CoV-2 nucleocapsid protein

Cited by 92 publications

References 51 publications

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

SARS-CoV-2: Insights into its structural intricacies and functional aspects for drug and vaccine development

MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein

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