The role of SARS-CoV-2 N protein in diagnosis and vaccination in the context of emerging variants: present status and prospects

Abstract

Despite many countries rapidly revising their strategies to prevent contagions, the number of people infected with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to surge. The emergent variants that can evade the immune response significantly affect the effectiveness of mainstream vaccines and diagnostic products based on the original spike protein. Therefore, it is essential to focus on the highly conserved nature of the nucleocapsid protein as a potential target in the field of vaccines and diagnostics. In this regard, our review initially discusses the structure, function, and mechanism of action of N protein. Based on this discussion, we summarize the relevant research on the in-depth development and application of diagnostic methods and vaccines based on N protein, such as serology and nucleic acid detection. Such valuable information can aid in designing more efficient diagnostic and vaccine tools that could help end the SARS-CoV-2 pandemic.

Keywords: SARS-CoV-2; diagnostic methods; immune escape; nucleocapsid protein; vaccine development; variants of concern.

FIGURE 1

Structural overview of the SARS-CoV-2 N protein. (A) Schematic diagram of the modular structure of the N protein of SARS-CoV-2. The N-arm, the Ser/Arg (SR)-rich central LKR, and Ctail, and the N-terminal domain (NTD) and C-terminal domain (CTD)–the three intrinsically disordered regions–are depicted. (B) SARS-CoV-2 N-NTD (PDB ID: 7CDZ) and N-CTD (PDB ID: 7DE1) electrostatic surfaces (PDB ID 7CEO). Blue represents a positively charge potential, and red represents a negative one. PyMOL was utilized to prepare all the structural figures.

FIGURE 2

Role of the N protein in the innate immune processes of host cells. (A) The N protein is a crucial factor that inhibits RNA interference (RNAi) in host cells infected with RNA viruses. It exerts this inhibition through two mechanisms: Firstly, the N protein binds with double-strand RNA (dsRNA) to prevent its recognition and cleavage by Dicer, thus preventing RNAi at the initial stage. Secondly, the N protein can also inhibit RNA degradation caused by small interfering RNA (siRNA) during the effector stage of RNAi. These mechanisms contribute to the stable preservation of viral RNA and enable the replication and proliferation of viruses in host cells, ultimately causing severe damage to them. (B) Viruses use their N proteins to interact with RIG-I and suppress the innate immune response by inhibiting IFN-β production in host cells. This interaction is mediated through the helicase domain of DExD/H-box helicases. SARS-CoV-2’s N proteins inhibit IFN-β by targeting its initial activation step. (C) SARS-CoV-2’s N proteins can impede the host’s innate immune response by functioning as type I interferon (IFN) signaling antagonists, ultimately compromising the immune system’s ability to fight against the virus. Specifically, they inhibit the phosphorylation and nuclear translocation of the transcription factors STAT1 and STAT2. This results in the subsequent attenuation of ISGF3, the IFN-stimulated gene factor 3 transcription complex, and a reduction in the expression of genes that are activated by IFN. Consequently, the presence of N proteins in SARS-CoV-2 infected cells reduces the immune system’s activity, which may increase the severity of the disease in some people.

FIGURE 3

Antigen-antibody-based serological detection of SARS-COV-2. (A) Traditional ELISA method based on a double antibody sandwich assay. (B) Magnetic beads adsorption of antigens and antibodies. (C) Using a lateral flow assay or double antibody sandwich, the quick quantum dot and colloidal gold immunodiagnostic approach for SARS-CoV-2 antibody is based on high specificity for the recombinant protein and quantum dot/colloidal gold immunofluorescence probes.

FIGURE 4

Nucleic acid-based detection of SARS-CoV-2. CRISPR/Cas system: Purified RNA can be amplified in an isothermal instrument using either reverse transcription recombinant polymerase amplification (RT-RPA) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). The amplified product can be reported using either the chromogenic substances in the amplification system or the CRISPR/Cas system for additional specific cleavage of nucleic acids and determination of virus infection. After the guide RNA binds to the CRISPR-associated Cas protein, the resulting complex can specifically cleave the viral nucleic acid sequence. The result can be reported by fluorescence quenching molecules in the reaction, by reporting the fluorescence signal, or by the side stream chromatography color development strip of the cleaved nucleic acid fragment.

FIGURE 5

Mutation prevalence across lineages. The Outbreak platform provides N protein mutation prevalence across the major subtypes of Omicron strains across lineages. (Mutations with > 75% prevalence in at least one lineage) (https://outbreak.info). The figure describes in detail the mutation prevalence in the Omicron lineage. Blank indicates that the mutation has not been detected. White to purple indicates the prevalence of the mutation in all sequences.