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Nucleocapsid Protein
Nucleocapsid Protein of Coronavirus
The coronavirus nucleocapsid protein (N protein) is a structural protein that forms complexes with genomic RNA, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly.
Amino acid sequence comparisons have shown that CoV N proteins have three distinct and highly conserved domains: two structural and independently folded structural regions, namely the N terminal domain (NTD/domain 1) and C-terminal domain (CTD/domain 3), which are separated by a intrinsically disordered central region (RNA-binding domain/domain 2); all three domains have been shown in different CoVs to bind with viral RNA[1].
There are several common characteristics of CoV N protein NTDs, including predicted secondary structures such as a central β-sheet platform flanked by α-helices[3], with a basic RNA binding groove along the β-platform and an extended β-hairpin. The NTD is enriched in aromatic and basic residues and the folded shape resembles a hand with basic fingers that extend far beyond the protein core, a hydrophobic palm, and an acidic―wrist. It has been proposed that the flexible, positively charged finger-like β-hairpin extension in the NTD of both IBV and SARS-CoV N protein is able to grasp RNA by neutralizing its phosphate groups, while the base moieties can make contact with exposed aromatic residues from the hydrophobic palm[4].
The NTD is separated from the CTD by an intrinsically disordered middle region referred to as the linker region (LKR). The charged LKR is also known as the SR-domain because it is rich in serine and arginine residues[5], and it is involved in cell signaling[6]. The flexible LKR is capable of direct interaction with RNA under in vitro conditions[7].Potential phosphorylation sites have been mapped to the Ser/Arg-rich portion of the LKR of SARS-CoV N[8]. These LKR phosphorylation sites are thought to function in binding M protein, heteronuclear ribonucleoprotein (hnRNP-A1) and RNA to the N protein with high binding affinity[9].
Domain organization of the Severe Acute Respiratory Syndrome human coronavirus (SARS-CoV) nucleocapsid protein[2]. IDR (a.a. 1–44; 182–247; 366–422)—intrinsically disordered regions; NTD (a.a. 45–181)—N terminal domain; LKR (182–247)—linker region; CTD (248–365)—C-terminal domain. The charged SR rich (striated box) and the nuclear localization signal (NLS, solid box) motifs are shown.
The CTD, which is a hydrophobic, helix-rich terminal, has been mapped for SARS-N to aa 248–365. This domain is also referred to as the dimerization domain because it contains residues responsible for self-association to form homodimers, as well as homo-oligomers through a domain-swapping mechanism[10]. Oligomerization of N protein is necessary to produce a stable conformation because in its monomeric form, the CTD folds into an extended conformation with a large cavity in its center, making it unstable. Sequence comparison shows that the dimerization domain of the N protein is conserved at least among the alpha, beta and gamma groups of CoVs, suggesting a common structural and functional role for this domain[11].
The coronavirus N protein is abundantly produced within infected cells. N protein has multiple functions, including binding to viral RNA to form the ribonucleocapsid and has also been proposed to have roles in virus replication, transcription and translation. In host cells, N proteins have been shown to cause deregulation of the cell-cycle, inhibit the production of interferon, up-regulate the production of COX2, up-regulate the activity of AP1,and induce apoptosis in serum deprived cells—of all which may have possible pathological consequences.
| 1. Virus Life Cycle | Function |
|---|---|
| 1.1. Viral Core Formation |
Primary role of CoV N is packaging the viral genome into long, flexible, helical RNP complexes. |
| 1.2. Viral Assembly |
CoV N protein dimerization and association with viral genomic RNA is critical for viral assembly. Interaction amongst the viral structural proteins (N, E, S and M), as well as a host membrane envelope obtained from the site of budding is required for viral assembly. |
| 1.3. Virus Budding/envelope formation |
Association of CoV N with the ER-Golgi complex plays a role in virus budding. Presence of N results in increased yields of VLPs and complete virion formation. |
| 1.4. Genomic mRNA replication/genomic RNA synthesis |
Intracellular co-localization of N with replicase components is required for RNA synthesis. Translation of N protein is implicated in stimulation of gRNA infection during RNA synthesis. |
| 2. Cellular Response | |
| 2.1. Chaperone activity |
All CoV N proteins involved in proper folding of nucleic acids by RNA chaperone proteins. |
| 2.2. Cell cycle regulation |
SARS-CoV N modulates the host cell cycle by regulating cyclin-CDKactivity. Leads to the arrest in progression of S phase. |
| 2.3. Cell stress responses—host translational shutoff |
SARS-N and MHV-N interact with cellular hnRNP-A1, which could act as a switch that redirects viral activity from RNA synthesis to nucleocapsid formation. Interaction of N protein CTD with elongation factor 1α (EF1α), a major translational factor in mammalian cells, can suppress translation. |
| 2.4. Viral pathogenesis—Immune system interference |
N protein plays an important role in viral pathogenesis. Mice infected with JMHV protected by anti-N monoclonal antibodies. Synthesis of type-1 interferon (1FN) inhibited by SARS-CoV N. The CTD of N has been shown to be a critical antagonist of 1FN induction. |
| 2.5. Signal transduction |
Activation of host cell signal transduction pathways and kinases leads to phosphorylation of N. |
Proteins
| Cat.No. | Product Name | Sequence | Tag | Source | Activity | More | |
|---|---|---|---|---|---|---|---|
| PKSR030486 | Recombinant 2019-nCoV NP NTD domain (His Tag) | / | N-His | E.coli | / | ||
| PKSR030487 | Recombinant 2019-nCoV NP CTD domain Protein | / | / | E.coli | / | ||
| PKSR030485 | Recombinant 2019-nCoV Nucleocapsid Protein (His Tag) | Met1-Ala419 | N-His | E.coli | / | ||
| PKSR030497 | Recombinant 2019-nCoV Nucleocapsid Protein (His Tag) | Met1-Ala419 | C-His | Baculovirus-Insect Cells | / | ||
| PKSR030512 | Recombinant 2019-nCoV Nucleocapsid Protein, Biotinylated (His Tag) | (Met1-Ala419(335Gly/Ala)) | C-His | Baculovirus-Insect Cells | / | ||
Antibodies
| Cat.No. | Product Name | Source | Reactivity | Applications | ||
|---|---|---|---|---|---|---|
| E-AB-V1011 | SARS-COV-2 NP Monoclonal Antibody(2019-nCoV) | Mouse / Human | IgG1 | ELISA | ||
| E-AB-V1012 | SARS-COV-2 NP ScFv Monoclonal Antibody(2019-nCoV) | Mouse / Human | IgG1 | ELISA | ||
| E-AB-V1013 | SARS-COV-2 NP Monoclonal Antibody(2019-nCoV) | Mouse | IgG1 | WB,ELISA | ||
| E-AB-V1014 | SARS-COV-2 NP Monoclonal Antibody(2019-nCoV) | Rabbit | IgG | WB,ELISA | ||
| E-AB-V1015 | SARS-COV-2 NP Polyclonal Antibody(2019-nCoV) | Rabbit | IgG | WB,ELISA | ||
| E-AB-V1016 | SARS-COV-2 NP Polyclonal Antibody(2019-nCoV) | Rabbit | IgG | ELISA | ||
Reference
- 1.Laude, H.; Masters, P. The coronavirus nucleocapsid protein. In Coronaviruses and Arteriviruses; Plenum Press: New York, NY, USA,1995; pp. 141–163.
- 2.McBride R, Van Zyl M, Fielding B C. The coronavirus nucleocapsid is a multifunctional protein[J]. Viruses, 2014, 6(8): 2991-3018.3. Jayaram, H.; Fan, H.; Bowman, B.R.; et al. X-ray structures of the N-and C-terminal domains of a coronavirus nucleocapsid protein: Implications for nucleocapsid formation. J. Virol.2006,80, 6612–6620.
- 3.Jayaram, H.; Fan, H.; Bowman, B.R.; Ooi, A.; Jayaram, J.; Collisson, E.W.; Lescar, J.; Prasad, B.V.X-ray structures of the N-and C-terminal domains of a coronavirus nucleocapsid protein: Implications for nucleocapsid formation. J. Virol.2006,80, 6612–6620.
- 4.Fan, H.; Ooi, A.; Tan, Y.W.; et al. The nucleocapsid protein of coronavirus infectious bronchitis virus: Crystal structure of its N-terminal domain and multimerization properties. Structure 2005,13, 1859–1868.
- 5.Hurst, K.R.; Koetzner, C.A.; Masters, P.S. Identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein. J. Virol.2009,83, 7221–7234.
- 6.Parker, M.M.; Masters, P.S. Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein.Virology 1990, 179, 463–468.
- 7.Chang, C.K.; Hsu, Y.L.; Chang, Y.H.; et al. Multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: Implications for ribonucleocapsid protein packaging. J. Virol.2009, 83, 2255–2264.
- 8.Peng, T.Y.; Lee, K.R.; Tarn, W.Y. Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization. FEBS J. 2008, 275,4152–4163.
- 9.Masters, P. Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus. Arch. Virol. 1992, 125, 141–160.
- 10.Chang, C.K.; Sue, S.C.; Yu, T.H.; et al. The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure. FEBS Lett. 2005,579, 5663–5668.
- 11.Yu, I.M.; Oldham, M.L.; Zhang, J.; Chen, J. Crystal structure of the severe acute respiratorysyndrome (SARS) coronavirus nucleocapsid protein dimerization domain reveals evolutionarylinkage between corona- and arteriviridae. J. Biol. Chem. 2006, 281, 17134–17139.
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