Cell surface expression of SARS-CoV-2 nucleocapsid protein and its role in immunity

By Neha MathurReviewed by Danielle Ellis, B.Sc.Dec 15 2021

In a recent pre-print study posted to the bioRxiv* server, a team of researchers demonstrated the unexpected roles of nucleocapsid (N) protein in innate and adaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Study: Cell Surface SARS-CoV-2 Nucleocapsid Protein Modulates Innate and Adaptive Immunity. Image Credit: MedMoMedia/Shutterstock

The N protein, one of the four major structural proteins of SARS-CoV-2, is localized in the viral surface envelope that binds to viral RNA and induces antibody (Ab) and T-cell immune responses by manipulating innate and adaptive immunity. It is crucial to gather extensive knowledge of innate and adaptive immunity to SARS-CoV-2 for reducing SARS-CoV-2-related acute and chronic diseases and discovering vaccine candidates that induce cross-reactive B and T cell immunity against SARS-CoV-2 variants.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

About the study

In the present study, the researchers first detected cell surface expression of SARS-CoV-2 N in seven different types of cells - Vero, BHK-21_humanACE2(hACE2), Caco-2, Calu-3, CHOK1_hACE2, and HEK293-FT_hACE2 cells. Twenty-four hours post-infection (hpi) with wild-type (wt) or a recombinant SARS-CoV-2 expressing eGFP (SARS-CoV-2_eGFP), these cells were incubated with primary and fluorophore-conjugated secondary antibodies at 4°C before fixation and mounting for confocal imaging.

Next, they examined the binding of exogenous N to the cell surface to understand that surface expression of N requires its synthesis in the cell. For this, the researchers incubated BHK-21, CHO-K1, or HEK293-FT cells with exogenous purified recombinant N (rN) for 15 min at 37°C. Further, they performed strong flow cytometry staining with anti-N monoclonal antibodies (mAbs) relative to control cells.

The researchers also assessed the contribution of glycosaminoglycans (GAGs), negatively charged molecule on the plasma membrane, in N cell surface binding, which was demonstrated using a panel of GAG-deficient CHO cells. N binding to heparin was also directly demonstrated using biolayer interferometry (BLI).

Finally, the researchers determined whether N could be transferred from infected to non-infected cells for which they co-cultured infectable (hACE2 expressing) and non-infectable (non-hACE2 expressing) CHO-K1 cells. The non-infectable cells were pre-stained with CellTrace^TM Violet to enable unambiguous flow identification after co-culture.

Results

Confocal imaging showed N on the surface of Vero, BHK-21_hACE2, Caco-2, Calu-3, CHOK1_hACE2, and HEK293-FT_hACE2 cells infected with wt or SARS-CoV-2_eGFP at 24 hpi.  Flow cytometry analyses helped researchers gather quantitative measures of N surface expression and understand its kinetics. Remarkably, surface N was detected on a sub-population of the spike(S) or eGFP expressing cells for each of the seven cell types examined. While in live Vero and BHK-21_hACE2 infected cells, N signals appeared at 8 hpi, they appeared at 12 hpi for A549_hACE2 cells. In addition, levels of N on these cells equaled or exceeded cell surface S on all but one of the seven cell types indicating the abundance of N on the cell surface and the retention of S in the early secretory pathway.

Depending on the cell type and marker (S vs. eGFP), cells expressing N but not S or eGFP ranged from less than 1% to 43%. N-surface expression increased between 24 and 72 hpi, further demonstrating that N binding to the cell surface is due to a specific association with heparan sulfate and heparin. The results of experiments with GAG-deficient cell lines showed that each of them failed to bind rN over levels observed with recombinant GFP. The results of BLI showed N-specific affinity binding to heparan sulfate and heparin but not to other sulfated GAGs. Together, these findings indicated that N binds to the cell surface by interacting with heparan sulfate and heparin through electrostatic forces.

The flow cytometry results showed the interaction of N with negatively charged viral RNA through its positively charged RNA-binding domains. On treating rN-coated cells with polybrene, which neutralizes surface electrostatic charges, rN bound to live BHK-21, CHO-K1, and HEK293-FT cells decreased at the same magnitude as it would have decreased with bound N. The results of immunofluorescence and flow cytometry experiments showed that uninfected CHO-K1 cells showed a higher cell surface N signal than infected cells indicating that N synthesized inside a cell is released and vigorously transferred to non-synthesizing cells, where it is retained by binding heparin/heparan sulfate.

Conclusions

The remarkable ability of surface N to bind chemokines and block immune effector cells justifies its export and binding to infected and neighboring uninfected cells from an evolutionary point of view.

The N-specific binding to heparin suggests the possible role for secreted N in the chronic low-level inflammation that causes “long coronavirus disease 2019 (COVID-19)” symptoms.

To conclude, the study findings demonstrate unforeseen roles for N in innate and adaptive immunity to SARS-CoV-2. Interestingly, N may be an Ab and T cell target for future “universal” vaccines offering broader protection against future SARS-CoV-2 strains as well as other human coronaviruses.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources