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The SARS-CoV-2 genome consists of an unsegmented sense RNA sequence and a forward-sense single-stranded RNA in the 5' untranslated region (UTR), a single open reading frame and a short 3' UTR, containing 14 The open reading frame (ORF) encodes 29 viral proteins, including 25 non-structural proteins and accessory proteins, and 4 structural proteins. Approximately two-thirds of the genome encodes two overlapping polyproteins pp1a and pp1ab, which are located at the 5' end. These two polyproteins are digested into 16 nonstructural proteins (NSPs) by two viral proteases. Polyprotein pp1a is proteolytically cleaved into 11 functional NSPs, while pp1ab is cleaved into 15 NSPs. Each NSP in the viral life cycle plays a unique role. Most non-structural proteins of SARS-CoV-2 have more than 85% amino acid sequence identity with SARS-CoV, which are used for virus replication and transcription and play an important role in viral RNA replication and immunity.
Figure 1. Proposed model for SARS-CoV-2 genome packaging. (Sources: Cubuk J, et al. 2021)
The number of nine auxiliary protein ORFs varies among different coronaviruses. ORF1a is the longest ORF in the genome, occupying almost two-thirds. ORF1b overlaps with ORF1a. The ORF at the 3' end of the viral genome encodes a set of typical structural proteins, including nucleocapsid (N) protein, spike (S) protein, membrane (M) protein and envelope (E) protein. The spike protein, membrane protein and envelope protein are located on the surface of the virus particle membrane and are packaged in combination with the N protein. The four structural proteins are responsible for the assembly of the virus to form mature viral particles. S protein plays a role in membrane fusion in host receptor binding and is an important element for the virus to enter the cell. The smallest structural protein, E protein, is mostly located at transport sites within cells and is involved in the assembly and budding of coronaviruses. M protein has the highest content and is an important protein involved in coronavirus assembly, which can determine the shape of the virus envelope. N protein is involved in the synthesis of viral genome and is closely related to the viral replication cycle and host cell response to viral infection. In addition, the ORF encodes eight auxiliary proteins scattered among these structural genes. Successful replication depends directly on efficient synthesis of viral RNA, and the replicase proteins responsible for this process are obvious antiviral drug targets. The structure of SARS-CoV-2 sheds light on the virus's life cycle and facilitates drug and vaccine development.
Like other coronaviruses, the nucleocapsid protein is one of the most critical structural components of SARS-CoV-2. This protein has 90% homology with the N protein of SARS-CoV and has important functional significance. The nucleocapsid protein N combines with the viral RNA genome to form the ribonucleoprotein core. It is a basic protein encoded by 419 amino acids and contains a short lysine-rich region. It is transcribed and translated to form a 1260 nucleotide length. The N gene is in a spiral shape. Nucleocapsid protein N can be divided into three putative domains, an N-terminal domain, an RNA-binding domain RBD, and a C-terminal domain. NTD and CTD are important structural and functional domains, which are separated by disordered linkers and flanked by disordered tails at both ends. The NTD domain is also known as the RNA-binding domain, which is responsible for the binding of viral RNA. It has a similar right-hand-shaped structural feature and can be divided into three regions, the protruding basic finger, the basic palm, and the acidic wrist. Most of the conserved residues are present in the alkaline palm region compared to the fingers and acidic wrist. The significant charge distribution on the NTD surface may be suitable for more efficient binding to its RNA genome. The CTD domain, also known as the homodimerization domain, is responsible for the dimerization of the N protein and contains a nuclear localization signal, a feature necessary for the creation of the viral nucleocapsid. It has also been shown to bind to viral RNA. These two domains are required for binding to the viral genomic RNA, helping to package it into approximately 100 nanoparticles. The SR domain is located in the LKR part, contains abundant phosphorylation sites, and is obviously more conserved than the linker domain. It is highly conserved among coronaviruses and exists in a highly phosphorylated state. It can be phosphorylated at multiple sites by serine/threonine protein kinase (SRPK1) in vitro. The most conserved amino acid residues are serine (Ser), threonine (hr) and arginine (Arg), which are closely related to the life cycle of SARS-CoV-2.
The basic function of the nucleocapsid protein N is to package the viral genomic RNA into a long helical ribonucleoprotein (RNP) complex, and participate in the assembly of viral particles by interacting with the viral genome and membrane protein M. Nucleocapsid protein N is also a phosphorylated protein. Glycogen synthase kinase-3 (GSK-3) and the SRPK family are involved in the phosphorylation of the SR-rich domain of the nucleocapsid protein. Phosphorylation of nucleocapsid protein N affects its protein-protein and protein-RNA interactions, and these functions may also affect the activation of nuclear factor kappa-B (NF-κB) and the life cycle of the virus. The functions of the protein include the replication and transcription of viral RNA, the formation and maintenance of ribonucleoprotein complexes. Nucleocapsid protein N plays an interactive role between the virus and the host, plays a role in the life cycle of the virus, participates in host cell cellular mechanisms such as interferon inhibition, RNA interference and apoptosis, and is also an immunodominant antigen in the host immune response. Nucleocapsid protein N is involved in encapsidation and packaging of viral genomic RNA. It binds to the 5' or 3' end of genomic RNA and may be involved in viral genome replication and subgenomic mRNA transcription. It can inhibit the activity of cyclin-dependent kinases, thereby interfering with the S phase progression of virus-infected cells. The gene fragment of nucleocapsid protein N is used as a serological ELISA detection antigen, providing a safe, cost-effective tool for detecting SARS-CoV-2 positive patients and a sensitive method for early detection of SARS-CoV-2 infection. According to the most common mutation types of SARS-CoV-2 in genomics, most of the mutation sites are located on the structural gene S. However, unlike the structural gene S, the mutation frequency of the N gene is low, indicating that the nucleocapsid protein N is relatively conservative and may become a marker protein for laboratory diagnosis.
References
| Target | Cat. No. | Product Name | Size | Species Reactivity | Application | Detection Sample | |
| SARS-CoV-2 NP | DEIASL017 | SARS-CoV-2 N ELISA Kit | 96T | Human | Quantitative | Serum, plasma | Inquiry |
| SARS-CoV-2 NP | DEIASL194 | SARS-CoV-2 Nucleocapsid Protein IgG Titer ELISA Kit | 96T | Quantitative | Serum, plasma | Inquiry | |
| SARS-CoV-2 NP | DEIA-NS2307-28 | COVID-19 Spike Protein Accquant ELISA Kit | 96T | Human | Quantitative | serum, plasma, tissue homogenates and other biological fluids. | Inquiry |
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