Human coronavirus 2019-nCov Nucleocapsid

SARS-CoV-2 Nucleocapsid

The Nucleocapsid (N) is a structural protein playing a key role in the SARS-CoV-2 (2019-nCoV) life cycle, including replication, transcription, and genome packaging [1]. N is located inside the viral particle, where it associates with the viral RNA to form the ribonucleoprotein core [1]. The identification of two functional domains has made SARS-CoV-2 N an attractive therapeutic drug-target [2, 3].  Similar to the S protein, N is a major immunogen of SARS-CoV-2. Elevated Anti-SARS-CoV-2 N IgG and IgM antibody titers have been reported in patients’ sera approximately ten days after the onset of COVID-19 symptoms [4-6]. Thus, SARS-CoV-2 N is also an attractive target for early diagnosis [5-7]. 

Nucleocapsid protein overview

SARS-CoV-2 Nucleocapsid domain organization

Most of our current knowledge about the SARS-CoV-2 N protein comes from previous studies on SARS-CoV. The N proteins from these two β-coronaviruses exhibit ~92% homology in their amino acid (a.a.) sequences [1]. SARS-CoV-2 N is a flexible 419 a.a. multidomain protein.  It features two important functional domains [1-3, 8]:

• NTD (N-terminal domain) interacts with both the RNA genome and Membrane/Matrix (M) proteins to form virions.  The NTD contains the RNA-binding domain (RBD). The N protein interaction with the RNA forms the ribonucleoprotein core, which is packed into a helical “beads-on-a-string” conformation.
• CTD (C-terminal domain) allows RNA synthesis through the binding of replication-transcription complexes (RTCs), oligomerization of multiple N proteins through its dimerization domain, and genome incorporation into the new virion.

The NTD and CTD functional domains are connected by a central linker (LINK or LKR_IDR) and flanked by an N-terminal (N_IDR) and C-terminal region (C_IDR) [8]. These 3 domains are unfolded (i.e. they lack secondary structures) and are thus called "intrinsically disordered regions (IDR)". LK_IDR ensures flexibility of the N protein, with the NTD and CTD domains behaving independently. Previous work on  SARS-CoV N predicts that the N_IDR, C_IDR, and LK_IDR contain protein-protein and protein-RNA interaction sites [8]. Structural studies are ongoing to better understand how the various folded and IDR domains interact with one another and support SARS-CoV-2 N functions.

Nucleocapsid-mediated RNA packaging model

Model for Nucleocapsid-mediated RNA packaging

The nucleocapsid protein may have evolved to drive compaction of the viral genomic RNA (gRNA) via protein-RNA liquid-liquid phase separation (LLPS), a cellular process by which biological material is organized into compartments [1, 8].  One structural study has led to an RNA packaging model whereby a single-genome condensate forms through the interaction between the N protein and viral gRNA . This interaction is driven by specific gRNA sequences (packaging signals) at the 5’ and 3’ end of the genome (step 1). The N protein preferentially binds to these packaging signals in the genome and self-dimerizes to form a local cluster (step 2). A high local concentration of the N protein then drives the condensation of distal regions of the genome to form a stable single-genome condensate (step 3). Ultimately, the single-genome condensate undergoes maturation as an amorphous ribonuclear particle, which then recruits E, S, and M for virion assembly [8].

SARS-CoV-2 Nucleocapsid in vaccination strategies

While SARS-CoV-2 S is the leading target antigen in vaccine development, the N protein is an interesting candidate. This idea is supported by the specific immunoglobulin and T cell responses detected in COVID-19 patients [4-6]. Contrary to the S protein which is exposed at the virus surface, the N protein is inside the particle. Therefore, anti-SARS-CoV-2 N antibodies are most likely to be raised upon the release of the N protein from infected cells and/or disassembled virions and are not expected to neutralize the virus.  Further research is needed to understand whether the N-specific immunoglobulin and CD4+ and CD8+ T cell responses observed in COVID-19 patients plays a role in viral clearance.

References

1. Mu, J. et al. 2020. SARS-CoV-2-encoded nucleocapsid protein acts as a viral suppressor of RNA interference in cells. Sci China Life Sci 63, 1-4.
2. Krokhin O. et al. 2003. Mass spectrometric characterization of proteins from the SARS virus. Mol. & Cell. Prot. 2:346-356.
3. Kang, S. et al. 2020. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm Sin B. doi:10.1016/j.apsb.2020.04.009.
4. Grifoni A. et al. 2020. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell. 181:1489-1501.
5. Liu, W. et al. 2020. Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for Detecting Antibodies against SARS-CoV-2. J Clin Microbiol 58.
6. Guo L. et al. 2020. Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19). Clinical Infectious Diseases. 71(15) :778-785.
7. To K. K-W. et al. 2020. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. The Lancet Infectious Diseases. 20(5):565-574.
8. Cubuk, J. et al. 2020. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. bioRxiv. doi:10.1101/2020.06.17.158121.