Nucleocapsid Proteins Trigger Main Antibody-Mediated Immunity in COVID-19 Patients

Written by: Zohair Ahmed
Edited by: Esther Melamed

The genome of SARS-CoV-2, the virus that causes COVID-19, codes for structural and non-structural proteins (ex: proteins mediating the virus’ life cycle). The structural proteins include the envelope (E) protein, spike (S) protein, nucleocapsid (N) protein, and the membrane (M) protein. The  S protein plays a critical role in the virus’s ability to enter human cells by mediating recognition of the angiotensin 2 (ACE2) receptor on human cells and directing fusion with the human cell membrane. The S protein is trimeric (made of three protein components), and has differing conformations post and pre-fusion with the target human cell membrane. The E protein is a transmembrane protein that is involved in the viral life cycle, assembly, budding, and envelope formation. The M protein is embedded in the lipid membrane of SARS-CoV-2 and is the most abundant glycoprotein in the viral particle. Lastly, the N protein functions in viral replication, transcription, forms the virus’ capsid and protects the viral genome from harsh environments. 

While several studies have emphasized the role and importance of the S protein in infectivity, there is an emerging role for the N protein as a key player in the host immune response to SARS-CoV-2. For example, in a recent study published in Science Direct, by Smits et al., it was demonstrated that out of different SARS CoV-2 proteins, the N protein was the strongest predictor of antibody response in the host to the SARS-CoV-2 infection. In addition to promoting an antibody host response, the N protein appears to also be responsible for host cytokine generation and T cell immune responses. While cytokines typically represent a healthy immune response to an invading virus, in COVID-19, the generation of cytokines can sometimes become detrimental and lead to a cytokine overdrive, aka cytokine storm. In turn, cytokine storm can lead to destructive pro-inflammatory effects to the host and worsen clinical outcomes in patients with severe COVID-19 Thus, it appears that the N protein of SARS-CoV-2 is a critical viral protein that can incite dysregulated host immune responses and worsen disease severity in COVID-19. 

Going forward, it will be very important to continue to study the role of the N protein in immune response generation in order to design more specific therapies that target N-protein mediated immune responses in COVID-19 patients. 

The host immune response consists of an antibody response and T cell response, which together lead to the release of cytokines. While cytokines are intended to help the host’s battle of the virus, too much of a cytokine response can lead to an “immune overdrive”  and subsequent damage to the body itself. This overdrive of cytokines has been termed ‘Cytokine Storm’, and tends to be present in the most severe cases of COVID-19. An older study published in Nature Communications on Nov 27, 2020,  explained to us that the Nucleocapsid protein of SARS-CoV-2 has the ability to separate using a term called “liquid-liquid phase separation” (Savastano) which is able to create high-density protein/RNA condensates, effectively upregulating the recruitment of the RNA polymerase for SARS-CoV-2 and indirectly upregulating the transcription of viral RNA. This means that the Nucleocapsid of the virus is able to upregulate the viral process within the body of the host. Dr. Smits and others published recently on Jan 31, 2021, in Science Direct a study that compared the IgG antibody response against the three proteins found in SARS-CoV-2, the Nucleocapsid, Membrane, or Spike proteins (N, M, or S) using a dot blot analysis in order to see whether the antibodies and detection systems for each protein was effective within COVID-19 and control patients.

After protein expression, purification, cDNA subcloning, DNA sequencing, and introduction to E Coli, dot blot analysis revealed that the N protein is the most prevalent protein, and binds to viral RNA creating the ribonucleoprotein core. The N-Terminal Domain, or NTD of the nucleocapsid aids in RNA-binding activity. Quantification of dot blots revealed that there was a strong IgG response to the N protein, both complete and fragment pieces. Dot blot analysis also revealed that the N protein is the best candidate for antibody reactivity when against SARS-CoV-2. 

This finding that it is essentially the N protein creating the main humoral response is significant for several reasons. The humoral response found in the progression of the coronavirus has been found to be related to the severity of the disease progression. This is significant because if the N protein causes the strongest IgG response, there may be a link between the N protein and virus severity between patients. Another reason is that in some fatal cases of the coronavirus, aggressive humoral response induces cytokine storms which often fatally lead to ARDS. Although we are not sure of the cause of this storm, examining the mechanism of the nucleocapsid triggered humoral response may bring some light to revealing the exact biochemical pathways that activate the cytokines, and may provide a gateway in symptom eradication through specific medicines when treating the disease, as preventive medicine has formulated various vaccines until today, while the widespread contraction is still an imminent threat.