*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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.
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 CellTraceTM 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.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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