Molecular characterization of SARS-CoV-2 nucleocapsid protein

Abstract

Corona Virus Disease 2019 (COVID-19) is a highly prevalent and potent infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Until now, the world is still endeavoring to develop new ways to diagnose and treat COVID-19. At present, the clinical prevention and treatment of COVID-19 mainly targets the spike protein on the surface of SRAS-CoV-2. However, with the continuous emergence of SARS-CoV-2 Variants of concern (VOC), targeting the spike protein therapy shows a high degree of limitation. The Nucleocapsid Protein (N protein) of SARS-CoV-2 is highly conserved in virus evolution and is involved in the key process of viral infection and assembly. It is the most expressed viral structural protein after SARS-CoV-2 infection in humans and has high immunogenicity. Therefore, N protein as the key factor of virus infection and replication in basic research and clinical application has great potential research value. This article reviews the research progress on the structure and biological function of SARS-CoV-2 N protein, the diagnosis and drug research of targeting N protein, in order to promote researchers' further understanding of SARS-CoV-2 N protein, and lay a theoretical foundation for the possible outbreak of new and sudden coronavirus infectious diseases in the future.

Keywords: COVID-19; SARS-CoV-2; clinical application; diagnostics; nucleocapsid protein.

Figure 1

Schematic diagram illustrating the microstructural pattern and genome structure of human beta coronavirus (Chen et al., 2020b; Yang and Rao, 2021; Malone et al., 2022). (A) Schematic representation depicting the microstructure of beta coronavirus. The spike protein (S), membrane protein (M), and envelope protein (E) collectively assemble to form a structurally stable viral envelope. The N protein binds to the S, E, and M proteins on the viral envelope and associates with the viral RNA to form ribonucleoprotein (RNP) complexes, thereby constructing a stable viral particle. (B) Schematic representation illustrating the genome structures of five human beta coronaviruses. The blue boxes denote the non-structural protein-coding genes of the virus; the red boxes represent genes encoding viral structural proteins; the gray boxes indicate the genes encoding viral accessory proteins.

Figure 2

Schematic representation of SARS-CoV-2 N protein structure. (A) Schematic depiction of the N-protein domains. Within the NTD, basic structural motifs are present that facilitate binding to viral genomic RNA, resulting in RNP formation. CTD comprises complementary motifs that promote N-protein dimerization. (B) Schematic illustration of the N protein structure following dimer formation. (C) Schematic diagram illustrating NTD crystal structure analysis. The blue region on the left represents the resolved NTD crystal structure in the absence of RNA binding (PDB ID: 6M3M), while the area enclosed by the dashed line denotes the basic amino acid motif involved in RNA affinity. The purple segment on the right indicates the resolved NTD crystal structure post-RNA binding (PDB ID: 7ACT), whereas the yellow portion signifies the bound RNA. The black arrow denotes the three-dimensional structural alteration of the NTD basic amino acid motif following RNA binding. (D) Analysis of the crystal structure of CTD yielding dimer formation (PDB ID: 6WZO) (Matsuo, 2021).

Figure 3

Sequence alignment and crystal structure similarity analysis were conducted on conserved domains (NTD and CTD) of the N protein across SARS-CoV-1, SARS-CoV-2, and MERS-CoV. (A) Aligning amino acid sequences within the NTD of three human beta coronaviruses shows significant sequence resemblance. Using NTD^COV2 as the reference sequence, NTD^MERS shares 60.3% similarity, while NTD^cov1 exhibits 90.7% similarity. (B) Aligning amino acid sequences within the NTD of three human beta coronaviruses shows significant sequence resemblance. Using NTD^COV2 as the reference sequence, NTD^MERS shares 50.9% similarity, while NTD^cov1 exhibits 95.4% similarity. (C) Comparison of the crystal structures of the N protein NTD across three beta coronaviruses. On the left, resolved structures of SARS-CoV-1 (plum red, PDB ID: 2OFZ) and SARS-CoV-2 (blue, PDB ID: 6M3M) NTD crystals are juxtaposed. On the right, comparison of resolved structures of MERS-CoV (yellow, PDB ID: 4UD1) and SARS-CoV-2 (blue, PDB ID: 6M3M) NTD crystals is presented; Gray arrows indicate basic amino acid structural motifs capable of RNA binding. The findings indicate that the NTD crystal structures across the three viral N proteins are largely identical. (D) Comparison of the crystal structure of CTD across three beta coronaviruses. On the left, resolved structures of SARS-CoV-1 (plum red, PDB ID: 2CJR) and SARS-CoV-2 (blue, PDB ID: 7C22) CTD crystals are compared. On the right, comparison of resolved structures of MERS-CoV (yellow, PDB ID: 6G13) and SARS-CoV-2 (blue, PDB ID: 7C22) CTD crystals is presented. The results indicate that the crystal structures of the three viral N proteins are largely consistent (Matsuo, 2021).

Figure 4

Schematic diagram of the SARS-CoV-2 replication process (Yang and Rao, 2021; Malone et al., 2022). The SARS-CoV-2 spike protein attaches to the cell ACE2 receptor, facilitating virus entry into the cytoplasm via endocytosis, where viral genomic RNA is subsequently released. Translation of viral genomic RNA within the cytosol results in the production of 16 non-structural proteins, 9 accessory proteins, and 4 structural proteins (S, E, M, and N). The N protein binds to the progeny viral genomic RNA to form RNP, which subsequently assembles into the phase separation (PS) structure and interacts with S, E, and M proteins embedded in the Endoplasmic Reticulum Golgi Intermediate Compartment (ERGIC). Mature progeny virus assembly occurs, followed by release via cell exocytosis to complete the viral replication cycle.

Figure 5

The regulation and functional significance of SARS-CoV-2 N protein phase separation. The phase separation of the N protein is initiated by its binding with RNA molecules of various lengths and is influenced by electrostatic forces such as pH and NaCl concentration. (A) The phase separation of the N protein has several effects, including the formation of stress granules (SGs) and interaction with key factors involved in the host cell’s antiviral response, such as MAVS and RIG-I, ultimately leading to the inhibition of type I interferon (IFN) production. (B) During the assembly and budding of mature virions, the N protein interacts with viral genomic RNA (gRNA) to form condensates, which are then anchored at the ERGIC membrane through co-phase separation (co-PS) with the SARS-CoV-2 M protein. (C) The N protein phase separation also regulates host-cell innate immune pathways by interacting with NF-κB activators, resulting in the upregulation of cytokine production. This, in turn, elicits an inflammatory response and the secretion of various cytokines, further contributing to the host immune response against SARS-CoV-2 infection.