As is mentioned above, the coronavirus N protein serves as a multifunctional protein with a complex structure and multiple functional domains, thus plays a critical role in the viral assembly and pathogenesis.

The N protein binds viral RNA via the RNA-binding domain (RBD) in its N-terminal domain (NTD) to form ribonucleoproteins (RNPs), a critical step for genome packaging and the accurate incorporation of the viral genome into new particles (Kuo et al., 2014; Korn et al., 2023). Beyond this structural role, the efficiency of subsequent assembly and budding constitutes a key innate immune evasion strategy. By ensuring rapid virion formation and egress, the N protein minimizes the cytosolic exposure of viral nucleic acids (Li et al., 2020). This process reduces their detection by intracellular pattern recognition receptors such as RIG-I and MDA5, thereby suppressing the initiation of the type I interferon response (Li et al., 2020; Kopecky-Bromberg et al., 2007). Also, the N protein is capable of forming dimers by oligomerization which enhances its RNA-binding capacity and facilitates the RNP complexes formation, thereby prompt the structural integrity of viral particles (Zhao et al., 2024).

Furthermore, as coronavirus M protein is essential for viral assembly (Zhang et al., 2022), N protein interacts with the M protein by the CTD to recruit the RNP complexes to the correct viral assembly sites and promote virion assembly and budding (Kuo et al., 2016; Siu et al., 2008; Boscarino et al., 2008). In the year of 2004, He et al. (2004a,b) investigated potential interaction between SARS-CoV N protein and the M protein by mammalian two-hybrid system, and found that the amino acids (168–208) within the N protein should be the key binding motif for the N protein-M protein interaction, which is necessary for N protein multimerization and correct conformation of N protein. Furthermore, coronavirus N protein interacts with the Ubl1 domain of its replicase-transcriptase subunit Nsp3 by its SR region shown in Figure 1B, which is critical to the RNA synthesis and initiation (Koetzner et al., 2022).

Coronavirus N protein also plays a regulatory role in the viral pathogenesis to evade host innate immune systems for viral replication by modulating the host innate immune responses, virus-host cells interaction and inflammatory responses. These functions make N protein a vital molecule in the coronavirus life cycle and pathogenic mechanisms.

The innate immune response is the host initial barrier in against pathogen invasion. Innate immune cells recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), thereby activating anti-infection signaling pathways and stimulating the production of cytokines and chemokines (Kasuga et al., 2021). PRRs that play a key role in the process of coronavirus infection mainly include retinoic acid-inducible gene I-like receptors (RLRs) and Toll-like receptors (TLRs), which effectively inhibit viral replication and transmission by recognizing viral RNA and activating downstream signaling. The feedback mechanism of interferon (IFN) as a key antiviral cytokine involves multilevel signaling (Yoneyama et al., 2015; Kawai and Akira, 2008). Followed that RLR family members RIG-I and MDA5 recognize the viral RNA, they form complexes with mitochondrial antiviral signaling protein (MAVS) and tumor necrosis factor receptor-associated factor 3 (TRAF3), which in turn activate IKKε/TBK1 kinase, promote phosphorylation of IFN regulators IRF3 and IRF7, and induce the production of type I IFNs. On the other hand, TLRs synergistically promote the expression of type I IFNs and inflammatory cytokines by activating NF-κB, MAPK signaling pathways, and IRF3 through MyD88 and TRIF adaptor proteins after recognizing viral RNA (Li S. et al., 2020). By binding to the target cell surface receptors, the secreted IFN activates the JAK-STAT signaling cascade and induces the expression of hundreds of interferon-stimulated genes (ISGs), forming a strong antiviral defense network, significantly inhibiting viral replication and blocking its diffusion process.

However, coronaviruses can antagonize the whole antiviral IFN signaling pathway in multiple ways by using their N proteins. In the early stage of IFN signaling activation, Hu et al. found that SARS-CoV and MERS-CoV N proteins interact with TRIM25 and inhibit RIG-I signaling, which in turn inhibit type I IFN production (Hu et al., 2017). Chang et al. also demonstrated that MERS-CoV N protein can act as an IFN antagonist, and its interaction with TRIM25 blocks the ubiquitination of RIG-I and the activation of the RIG-I pathway. During its infection cycle and transmission, N protein of SARS-CoV-2, a highly pathogenic human coronavirus, induces a complex network of host antiviral responses. Zheng et al. showed that the interaction of SARS-CoV-2 N protein with G3BP1 attenuates the formation of antiviral stress particles (avSG) and prevents cofactors G3BP1 and PACT from activating RIG-I, thereby inhibiting RIG-I/MDA5-mediated type I and III IFN responses. Furthermore, N protein also affects the RIG-I-mediated dsRNA recognition (Zheng et al., 2022). SARS-CoV N proteins exert IFN antagonism by targeting protein activators (PACT) of interferon-induced protein kinases and impairing PACT-RIG-I/MDA5 interactions (Ding et al., 2017). IBV N interferes with MDA5 recognition of double-stranded RNA (dsRNA) and targets LGP2, hindering the activation of MDA5 and LGP2-stimulated IFN-β promoters, inhibiting type I IFN production, and thereby inhibiting the host's innate immune response (Huang et al., 2023). Since RIG-I signaling triggers phosphorylation and translocation of key transcription factors, including NF-κB and IRF3, phosphorylation of IRF3 and NF-κB p65 subunits is attenuated in the presence of MERS-CoV N protein, resulting in inhibition of IFN production (Chang et al., 2020). Guo et al. found that SARS-CoV-2 N protein inhibits the activation of the NF-κB pathway and its downstream signaling by blocking the assembly of the TAK1-TAB2/3 complex through its CTD region, and therefore, SARS-CoV-2 N inhibits the occurrence of cellular inflammation and promote viral replication. In addition, Glu-290 and Gln-349 within the SARS-CoV-2 N protein CTD are key sites that inhibit the NF-κB pathway (Guo et al., 2025). SARS-CoV N protein interacts with the host transcription factor NF-κB to regulate interleukin-6 (IL-6) expression and induce innate inflammatory response (Zhang et al., 2007). TGEV N protein enhances neonatal Fc receptor (FcRn) promoter activity by activating the NF-κB signaling pathway to resist viral invasion in intestinal mucosal immunity; in addition, N-protein-mediated upregulation of FcRn promotes IgG transcytosis (Qian et al., 2019). However, to date, there is no evidence indicating that TGEV N protein affects IFN-β production. Although it has been reported that PEDV N protein can activate NF-κB through the TLR2, TLR3, and TLR9 pathways in porcine intestinal epithelial cells (Cao et al., 2015), Ding Z. et al. (2014) found PEDV N protein competitively inhibited the interaction between IRF-3 and TBK1 by targeting TANK-binding kinase 1 (TBK1), blocking the production of IFN-β by host cells. It also antagonizes IFN-λ production by preventing the transcription factor NF-κB nuclear translocation. Coronavirus N proteins can also block the downstream of IFN signaling by suppressing JAK-STAT signaling and the related ISGs expression. For example, as the only member of deltacoronavirus, PDCoV prevents the activation of the JAK-STAT signaling pathway by interacting with STAT1 through N protein interaction with STAT1 and inhibiting nuclear translocation of STAT1 by degrading KPNA2 (Hu et al., 2024). As histone deacetylase 1 (HDAC1) positively regulates the transcription of two antiviral IFN-stimulated genes (ISGs), ISG15 and OAS1, PEDV N protein impairs their expression by competitively binding to the transcription factor Sp1 and blocking the transcription of HDAC1 in the nucleus to disrupt STAT1 acetylation-phosphorylation balance, resulting in the inhibition of JAK-STAT1 signaling and facilitation of viral replication (Xu et al., 2021).

In summary, coronavirus N proteins employ a multifaceted strategy to evade the innate immune response by targeting two critical junctures: the initial detection of viral RNA and the subsequent signaling cascades. They disrupt upstream pathogen recognition by interfering with RLRs (RIG-I and MDA5) and their co-factors (TRIM25, PACT, G3BP1), thereby preventing the activation of key transcription factors like IRF3 and NF-κB. Simultaneously, they inhibit downstream signaling pathways, including the JAK-STAT pathway, the TBK1-IRF3 axis, and the NF-κB pathway, to block the production of interferons, inflammatory cytokines, and antiviral ISGs. This coordinated suppression of both the induction and effector phases of the interferon response is a central mechanism of coronavirus immune evasion (Figure 2). It is worth mentioning that, as the final key effectors in the IFN signaling transduction process, different ISGs target the coronavirus N proteins for their efficient antiviral functions (e.g., ISGylation) (Bang et al., 2024; Zhu et al., 2024).

However, there is few studies showing how N proteins exerts immune evasion by interacting these ISGs. Further study may be driven by this direction for a more comprehensive understanding of the pathogenic mechanism of coronaviruses. (Q3,Q5)

Coronavirus N protein also plays a key role in regulating the host cell cycle through multiple mechanisms to create favorable conditions for viral replication. As the host p53 is a key protein in cell cycle regulation and DNA damage response, researchers found that the N protein binds to p53, inhibiting its function and thereby interfering with the cell cycle progression (Xu et al., 2016). For example, PEDV N protein utilizes its nuclear localization signal (NLS) for the interaction with p53 in the nucleus, activating the p53-DREAM signaling pathway to induce the S-phase arrest and promote the viral replication (Su et al., 2021). Since p53 is reported to be an antiviral host factor, PEDV N protein could recruit the E3 ubiquitin ligase COP1 to inhibit COP1 self-ubiquitination and degradation, thus facilitating COP-1-mediated ubiquitination-dependent proteasomal degradation of p53 (Dong et al., 2024). Except for p53, Zhu et al. (2022) also screened the protein expression profiles of the PEDV N protein-induced S-phase arrested cells and found most of the differentially expressed proteins are associated with DNA replication, RNA transcription and protein synthesis, indicating that coronavirus may facilitate its own replication by utilizing ribosome proteins associated with protein synthesis involved in the cell cycle. For the SARS-CoV-2 infection, its N protein interacts with Smad3 to activate TGF-β-Smad3 pathway and the downstream Smad3-dependent G1 cell cycle arrest for acute kidney injury (Wang et al., 2022). Therefore, these clues illustrate that coronavirus N protein creates favorable conditions for the viral replication and infection in the host cells by disturbing the cell cycle. These mechanisms provide important references for understanding the role of the N protein in viral infection and pathogenicity.

Post-translational modifications (PTMs) refer to the covalent modification of a protein synthesized by a specific enzyme to add the corresponding functional group in the form of a covalent bond to one or more amino acid residues. These modifications play an important role in regulating protein activity, stability, subcellular localization, and mediating protein interactions. PTMs include phosphorylation, succinylation, methylation, acetylation, etc. During the interaction between N protein and host, PTMs of N protein promotes viral replication, assembly, release, and inhibition of IFN response, thereby promoting viral proliferation and immune escape (Wu et al., 2023; Cheng et al., 2022).

During PEDV infection, the PEDV N protein enhances STAT1 acetylation by downregulating deacetylase (HDAC1), subsequently blocking STAT1 phosphorylation and nuclear translocation, evading the host's innate immune response by inhibiting antiviral IFN signaling (Xu et al., 2023). Phosphorylation of coronaviruses N proteins is associated with glycogen synthase kinase 3 (GSK3). Studies have shown that GSK3α/β plays a major role in the SR motif of phosphorylated N protein. Phosphorylation of the SR motif of N protein promotes the proliferation of PEDV (Jia et al., 2025). Wu et al. showed that GSK-3 inhibition reduces SARS-CoV N protein Ser177 phosphorylation and viral replication, indicating this modification's importance for viral proliferation (Wu et al., 2009). The N protein of SARS-CoV-2 binds to the host GSK-3β via the GSK-3 interaction domain (GID), which plays a key role in N protein phosphorylation and viral replication (Yun et al., 2022). In terms of phosphorylation modification, it was found that the phosphorylation modification of SARS-CoV-2 N protein can enhance its affinity for RNA binding, promote viral replication and viral particle assembly, and interfere with the localization of stress granules in host cells (Carlson et al., 2020; Lu et al., 2021). Simultaneously, Carlson et al. confirmed that N protein phosphorylation sites (S176, S180, S184, S197, S206, T205) are the core targets for regulating N protein phase separation and functional switch. The low phosphorylation state of N protein promotes RNA packaging, while the high phosphorylation state shifts to virion assembly (Carlson et al., 2020). N protein can also block the phosphorylation of IRF3 and TBK1 in the JAK-STAT signaling pathway, thereby inhibiting the host immune response (Zhao et al., 2021). It is also reported that binding of phosphorylated SARS-CoV N^S177 and N^S180 to the host 14-3-3 protein in the cytoplasm was regulated nucleocytoplasmic N shuttling (Surjit et al., 2005). Tugaeva et al. reported that the binding of the N protein dimer of SARS-CoV-2 to the “bridge protein” 14-3-3 dimer requires serine phosphorylation at position 197 of the N protein, and that the interaction between 14-3-3 and the N protein can affect viral replication (Tugaeva et al., 2021). Tomer M et al. found that phosphorylation modification of SRAS-CoV-2 N protein requires the SR-specific protein kinase SRPK1/2 to act, thereby triggering further phosphorylation of GSK3α/β and CK1 to achieve broad phosphorylation of the SR-rich domain of N protein. Treated with the kinase inhibitor Alectinib, it is able to inhibit N phosphorylation of SRPK1/2 and restrict SARS-CoV-2 replication (Yaron et al., 2020). In 2022, Liu et al. reported that SARS-CoV-2 protein succinylation mainly occurs on M protein and N protein. K65 and K102 are situated within the RNA-binding domain of the N protein, while acetylation sites such as K266 are located in or near the dimerization region, potentially influencing N protein oligomerization. Succinylation of the N protein enhances viral replication by modulating host cell metabolism, including the accumulation of tricarboxylic acid (TCA) cycle intermediates (Liu et al., 2022). Cai et al. (2021) found that SARS-CoV-2 N proteins can also be methylated, with the methylation of the N proteins R95 and R177, the methylation of R95 inhibiting the formation of host cell stress granules and thus affecting the antiviral response, while the methylation of R177 promotes the binding of N protein to RNA and promotes viral replication. Acetylation of SARS-CoV-2 N protein at K61 impairs RNA-binding capacity and compromises viral assembly. CREB-binding protein (CBP)-mediated acetylation at K375 further demonstrates the regulatory role of this modification in the viral life cycle, highlighting potential antiviral strategies targeting N protein acetylation (Hatakeyama et al., 2021). Zhu et al. (2024) revealed that the N protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation mechanism, a modification that hinders the assembly of N functions into oligomers, ultimately inhibiting viral RNA synthesis and the ISGylation levels of N are controlled by direct deISGylation by NSP3 papain-like protease (PLpro) enzymatic activity. Chen et al. identified phosphorylation sites adjacent to the RNA-binding domain of IBV N protein using mass spectrometry. Surface plasmon resonance (SPR) kinetic analysis further demonstrated that phosphorylated N protein exhibits enhanced specificity for viral RNA recognition (Chen et al., 2005). The above studies show that PTMs play an important role in the interaction between viruses and hosts, and these PTMs of N protein may become potential targets for future antiviral drug development (Table 1).