key: cord-1042401-spaaz6u5
authors: Supekar, Nitin T; Shajahan, Asif; Gleinich, Anne S; Rouhani, Daniel S; Heiss, Christian; Chapla, Digantkumar Gopaldas; Moremen, Kelley W; Azadi, Parastoo
title: Variable post-translational modifications of SARS-CoV-2 nucleocapsid protein
date: 2021-05-13
journal: Glycobiology
DOI: 10.1093/glycob/cwab044
sha: 7841d492726c37317ac919cd0af7c1c82de6edb7
doc_id: 1042401
cord_uid: spaaz6u5

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and the most abundant protein in coronaviruses and thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having post-translational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells, one was in-house expressed and purified without a signal peptide sequence and the other was commercially produced with a signal peptide channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation, as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for coronavirus disease and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development point-of-care diagnostics.

coronavirus disease , spread rapidly and became a worldwide pandemic (Zhu, N., Zhang, D., et al. 2020) . According to the World Health Organization (WHO) report, as of mid-April 2021, there have been 137.9 million confirmed cases of COVID-19 globally, including 2. million deaths. In particular, to date, the USA alone has over 31.2 million confirmed cases and over 561,356 deaths due to COVID-19 (2020a) . There are currently more than 80 vaccine candidates in clinical trials, and 2 have been approved for full use in the United States (2020b).

However, the search for alternative vaccines to protect against SARS-CoV-2 still continues due to recent reports of mutations and new strains of SARS-CoV-2 (Wang, Z., Schmidt, F., et al. 2021) . Most of the current vaccine candidates targeting a specific protein are aimed at the virus's spike protein (Dong, Y., Dai, T., et al. 2020) . However, the nucleocapsid or N protein has also been suggested as vaccine or therapeutic target, as well as biomarker for the disease (Kumar, J., Qureshi, R., et al. 2020 , Kwarteng, A., Asiedu, E., et al. 2020 , Nikolaev, E.N., Indeykina, M.I., et al. 2020 . The N protein is an attractive target because it is highly conserved, strongly antigenic, and the most abundant protein in SARS-CoV-2 (Li, J.Y., Liao, C.H., et al. 2020 , Mu, J., Xu, J., et al. 2020 , Yoshimoto, F.K. 2020 . Since no detailed analysis of potential post-translational modifications (PTMs) of the SARS-CoV-2 N protein has been put forward to date, we decided to undertake this challenging analysis in our laboratory.

SARS-CoV-2 is a virus from the coronavirus family. The coronavirus virion consists of a nucleocapsid surrounded by a lipid envelope in which the membrane glycoprotein (M) and small transmembrane protein (E) are embedded. Protrusions composed of trimeric glycoproteins (Spike protein, S) are anchored in the lipid envelope and extend radially ( Figure 1A ). The S protein is the most studied protein in SARS-CoV-2 due to its exposed position on the virus

surface and its role in attaching to host cells, it has been chosen as a target for a number of vaccine candidates (Dong, Y., Dai, T., et al. 2020) . The S protein is highly glycosylated and binds to angiotensin converting enzyme 2 (hACE2) for entry into the host cell (Chen, Y., Guo, Y., et al. 2020 , Wang, Q., Zhang, Y., et al. 2020 , Zhao, P., Praissman, J.L., et al. 2020 . Through a combination of glycoproteomics and glycomics, we and others have recently deduced a detailed glycosylation profile of both S protein and its receptor hACE2 , Watanabe, Y., Allen, J.D., et al. 2020 , Zhao, P., Praissman, J.L., et al. 2020 . Moreover, a number of studies have highlighted the importance of spike protein glycosylation, which plays a critical role in the mechanism of viral attachment to the hACE2 receptor (Chen, Y., Guo, Y., et al. 2020 , Shajahan, A., Archer-Hartmann, S., et al. 2020 , Zhao, P., Praissman, J.L., et al. 2020 . However, there are reports suggesting that the S protein alone may be an insufficient target for vaccine and therapeutic development (Ferretti, A.P., Kula, T., et al. 2020 , Gouveia, D., Miotello, G., et al. 2020 , Ihling, C., Tänzler, D., et al. 2020 . A recent clinical study involving SARS-CoV-2 patients identified the majority of immunodominant CD8 + T cell epitopes (that activate CD8 + cells to kill virally infected cells) from virus proteins other than the S protein, leading to the conclusion that more protein targets will need to be included for new and less vulnerable vaccine designs (Ferretti, A.P., Kula, T., et al. 2020) .

In addition to N protein's role as a potential therapeutic or vaccine target, the N protein can also serve as an important diagnostic marker for coronavirus disease. Recent mass spectrometry (MS) -based studies of SARS-CoV-2 proteins from gargle-solution and nasal swab samples from COVID-19 patients detected the presence of N protein peptides, making them potential diagnostic biomarkers for COVID-19 (Gouveia, D., Miotello, G., et al. 2020 , Ihling, C., Tänzler, D., et al. 2020 . The presence of N protein peptides in the gargle and nasopharyngeal swabs could be used to develop a point-of-care high-throughput test for fast detection of SARS-

CoV-2. The authors demonstrated in these studies that SARS-CoV-2 peptidome detection through tandem mass spectrometry can be used as alternative methodologies to PCR and immunodiagnostics. The clinical study on gargle solution showed that peptide 41 RPQGLPNNTASWFTALTQHGK 61 from the N protein could be detected in the saliva of COVID-19 positive patients (Ihling, C., Tänzler, D., et al. 2020) . Another study demonstrated that N protein peptides 375 ADETQALPQR 385 and 170 GFYAQGSR 177 can be detected with intense signal within short retention time in nasopharyngeal samples from COVID-19 patients (Gouveia, D., Miotello, G., et al. 2020) . These recent findings on COVID-19 clinical samples show the presence of viral N protein in the host body fluids and its potential of the utilization as a diagnostic biomarker at the point-of-care.

The nucleocapsid (N) protein makes up the most abundant source of proteins in the coronavirus (Surjit, M. and Lal, S.K. 2008) . The N protein interacts with viral genome RNA to form long, flexible, helical ribonucleoprotein (RNP) complexes ( Figure 1A ). and contributes towards viral genome condensation and packaging (Chen, C.-Y., Chang, C.-K., et al. 2007 , Narayanan, K., Kim, K.H., et al. 2003 ). The C-terminal interactions between the N and M proteins result in specific genome encapsidation during the budding process of the viral particle (He, R., Leeson, A., et al. 2004) .

Mapping studies of SARS-CoV-2 and other coronaviruses, including the closely related SARS-CoV-1, have linked the RNA-binding function to a fragment of 55 amino acids located at the N-terminal half that resides in Domain 1, and the RNA-binding, as well as dimerization functions, to the C-terminal half from Domain 3 (Chen, C.-Y., Chang, C.-K., et al. 2007 ). The linker region (LKR) connects the N-and C-terminal domains of the protein and includes a Ser/Arg-rich (S/R-rich) region (Chang, C.-k., Hou, M.-H., et al. 2014) . Studies have shown that the S/R-rich LKR contains multiple putative sites of phosphorylation that may play a role in

regulating N protein function and N-M protein interactions (He, R., Leeson, A., et al. 2004 , Peng, T.-Y., Lee, K.-R., et al. 2008 , Surjit, M., Kumar, R., et al. 2005 , Wu, C.H., Yeh, S.H., et al. 2009 ).

Early studies on the N protein from coronaviruses suggested that the N protein is a phosphoprotein and does not bear glycans on its backbone (Fung, T.S. and Liu, D.X. 2018 , Laude, H. and Masters, P.S. 1995 , Parker, M.M. and Masters, P.S. 1990 ). To discover the PTMs of SARS-CoV-2 N protein, we explored several high-resolution MS-based approaches, including glycomics, glycoproteomics, and phosphoproteomics analyses, using N protein from two different, recombinant sources, a commercial source and a recombinant protein produced in our lab.

The N protein obtained from the commercial source was expressed in HEK293 with a signal peptide (SP), designed to cause the protein to enter the secretory pathway ( Figure 1B ).

For convenience, we will term this commercial N protein "N(SP+)". Evaluated by SDS-PAGE, N(SP+) had a molecular mass of 60 kDa, which differs from its theoretical protein mass of 47 kDa ( Figure 2 ). The added mass is not due to the signal peptide, which is removed during protein expression and no longer present in the final commercial product. Rather, the added mass is likely due to PTMs, including glycosylation and phosphorylation ( Figure 3 ) (Masters, P.S. 2006 ). In the following, we describe our characterization of the PTMs of N(SP+), focusing primarily on the glycoproteomic analysis. However, a detailed glycomic analysis of the detected glycans can be found in the supplementary material ( Figure S2 -S28 and Table SI-SII) .

We used trypsin and elastase proteases both separately and sequentially to produce three peptide samples. The LC-MS/MS data of these protease digests were analyzed by using

search algorithms in the Byonic software, employing all possible mammalian N-glycans, O-glycans, and phosphorylation as possible post-translational modifications.

There are five potential N-glycosylation sites (-NXS/T-with X≠P) on the N protein (N47, N77, N192, N196, N269) ( Figure 4) . The Byonic N-glycopeptide search suggested that only N47 and N269 were glycosylated in N(SP+). We manually validated this conclusion for accurate precursor mass (5 ppm) and precise glycan neutral loss in MS/MS experiments (<20 ppm). The data confirmed that the two N-glycosylation sites vary distinctly in their N-glycosylation profiles.

The site N47 was part of glycopeptide 41 RPQGLPNNTASWF 53 and had a glycan occupancy of about 53% ( Figure 5 and NeuAc1GalNAc1Gal1GlcNAc2Man3GlcNAc2Fuc1, which showed 6.56% glycan occupancy relative to other glycoforms at site N47. Moreover, all the glycans detected at this site featured core fucosylation. We also observed multiple doubly fucosylated glycans (~16%) and a minor amount of triply fucosylated structure (0.45%) ( Table SIII) .

The Byonic N-glycopeptide and manual search also resulted in detection of N-glycan on peptide 267 AYNVTQAFGR 276 at site N269. The glycan occupancy at site N269 was found to be higher (94%) than that of site N47 (53%). At site N269, we detected mostly high-mannose type glycosylation making up ~85% of total glycan ( Figure 5A ). The relative percentage of complex (4.5%) and hybrid type N-glycans was lower (~3.6%). Fucosylation and sialylated glycans were identified at trace levels (0.63% and 0.76%, respectively) ( Table SIII) . The tryptic peptide 69 GQGVPINTNSSPDDQIGYYR 88 was detected only in non-glycosylated form ( Figure S48 ), indicating that N77 is not occupied.

The N-glycosylation sites N192 and N196 occur in the linker region, which is rich in serine and arginine (SR-rich). Investigation of the N-glycosylation at these sites proved difficult because the two N-glycan sequons are located next to each other and are separated by only one arginine residue ( Figure 4 ). For site N192, the trypsin digest possibly generated the this stepwise approach, we could not identify any N-glycans at sites N192 and N196.

To further investigate the possibility of glycosylation at sites N192 and N196, we performed an 18 O labeling experiment where N-glycans are removed from glycoproteins with PNGase F in the presence of H 2 18 O, followed by enzymatic digestion with trypsin/elastase ( Figure S1 ). In the process of de-glycosylation, the N-glycan-bearing Asn is converted to 

T379; found on three glycopeptides) in total was very low (<10% for each site) (Figures S136-S138 (fragments c8, c9, z9 and c10) ( Figure S65 ). We observed that the glycan occupancy at S23 was only about 2% as shown in Figure S136 . HCD and CID MS 2 analysis of glycopeptide 144 DHIGTR 149 confirmed the O-glycosylation at T148 by the presence of glycan oxonium ions, peptide fragments (b and y ions) and glycan neutral losses ( Figures S70-S74 ). The overall glycan occupancy at site T148 was determined at ~81%, out of which mono-and di-sialyl Core 2 type glycans (~70%) were predominant ( Figure 5B , Table SIV ).

The Byonic glycopeptide search showed a strong indication of glycosylation on the peptide 159 LQLPQGTTLPK 169 , which contains two potential O-glycosylation sites ( Figures S75-S92 ). To further evaluate the glycosylation in this peptide, we manually validated full mass, HCD and CID MS 2 spectra that showed oxonium ions, b and y peptide fragments, along with peaks corresponding to the masses of the peptide and peptide+HexNAcHex ( Figure S76 ). The ETD experiment for site mapping of T165 or T166 in the peptide 159 LQLPQGTTLPK 169 detected two spectra that showed c and z ions indicating that some of these peptides are O-glycosylated on T165 and others on T166 ( Figures S91-S92 ). The ETD MS 2 spectra showed the fragment peaks z5 at m/z 908.57 and c7 at m/z 1120.57, diagnostic for the presence of glycans at site T165 ( Figure S91 ), as well as the fragment peaks z4 at m/z 807.36 and c8 at m/z 1221.65, diagnostic for the presence of glycans at site T166 ( Figure S92 ). The data demonstrated that

13 the peptide 159 LQLPQGTTLPK 169 is almost fully occupied with glycans. Moreover, Core 1 type structures were predominant, accounting for 77% of total glycans at these sites ( Figure 5B ).

However, we also detected Core 2 to Core 4 type glycans, including extended Core 2 glycans with fucose and sialic acid on the peptide 159 LQLPQGTTLPK 169 ( Figures S75-S92 ).

The peptide 204 GTSPAR 209 , located in the LKR region, showed O-glycans at both sites, T205 and S206. We were not able to deduce site specific information at these sites. Figure S132 ). At site T391, di-sialylated Core 1 to Core 2 type glycans were predominant over other glycans (Figures 5B and S114-S128, Table SIV) .

Interestingly, site S404 was found to contain only mono-and di-sialylated Core 2 type structures

14 as shown in Figures S129-S131. We confirmed the O-glycan structures and glycosylation sites through both Byonic software search and manual analysis.

In summary, as shown in Figure 5B , the sites T148, T165, T166, T205, S206, T391, and S404 comprised significant levels of O-glycosylation (47%-90%), and the sites S23, T245, T247 and T379 indicated a lower level (1%-9%) of O-glycosylation. We identified Core 1, Core 2, Core 3, and Core 4 type O-glycans at these sites, confirming the glycomics analysis findings (see supplementary material and Figure S2 , S5 and Table SII, SIV) .

We digested the N protein with elastase/trypsin and conducted a Byonic-based phosphate search in the elastase digest. In this experiment, we identified phosphorylation on glycopeptide 388 KQQTVTLLPA 397 , which contains two potential sites -T391 and T393 -for glycosylation or phosphorylation on N(SP+). As shown in the HCD MS 2 spectrum ( Figure   S133 ), the glycopeptide identity was confirmed by validating b and y ions and glycan neutral losses. The presence of a phosphate in the peptide was confirmed by CID MS 2 experiments using the high-resolution precursor mass and neutral losses of phosphate (loss of m/z 79.9663) from the glycopeptide ( Figure S134 ). As shown in the deconvoluted CID MS 2 spectrum Figure   6A , the peak at m/z 1584.77 represents the glycopeptide fragment 388 KQQTVTLLPA 397 with a phosphate group and a glycan, and the peak at m/z 1504.81 represents the same peptide with Figure S132 ).

Since the extensive glycosylation of the commercial N protein N(SP+) was likely an artefact resulting from the presence of the signal peptide during expression, we wanted to also determine the PTMs of properly expressed (i.e. without the signal peptide) N protein N(SP-) for comparison. We expressed N(SP-) in our laboratory in HEK293 cells and confirmed it by

Western blot using HRP anti-His Tag Antibody from Biolegend ( Figure 1C ). N(SP-) was purified from cell lysate using the MagneHis™ Protein Purification kit from Promega (V8500). The SDS-PAGE showed that, in contrast to N(SP+), whose mass was higher due to glycosylation, N(SP-) had a molecular mass of 47 kDa, as expected from the known protein sequence. N(SP-) was subjected to proteolysis and the enzymatic digests analyzed by LC-MS/MS for identification of PTM and peptide mass fingerprinting.

Protein databases suggest that SARS-CoV-2 N protein is a phosphoprotein. We For this site, the peptide without phosphate modification was also found, suggesting that the site Ser176 is not fully phosphorylated. Our analysis found that the N(SP-) at site S176 is 44.4% 16 experiments (<20 ppm) did not detect N-glycosylation on the sites N47, N77, and N269 in the tryptic digest of N(SP-). For these three sites, we were able to detect only peptides, 41 RPQGLPNNTASWFTALTQHGK 61 , 69 GQGVPINTNSSPDDQIGYYR 88 and 267 AYNVTQAF 274 , confirming that this region is covered by the tryptic digest. We did not obtain sequence coverage for N-glycosylation sites N192 and N196 that occur in the linker region. Surprisingly, we did not detect the peptides of the SR-rich linker region between amino acids G178 -R209, possibly due to presence of multiple PTMs, although no PTMs were detected in this region other than phosphorylation on S176. Further, we conducted Byonic-assisted and manual O-glycan search on the tryptic peptides of N(SP-). However, we did not identify peptides with O-glycans.

We observed cleavage of methionine at the N-terminal end and acetylation of the subsequent serine residue (Figures S139-S140). The cleavage of N-terminal methionine and acetylation of the N-terminus are the most common protein post-translational modification (Bonissone, S., Gupta, N., et al. 2013) . Two-thirds of the proteins in any proteome are suggested to be potential substrates for N-terminal methionine excision (NME), and methionine aminopeptidases (MetAPs) are expressed in all organisms from bacteria to eukaryotes (Giglione, C., Boularot, A., et al. 2004 ). For the methionine excision, the second or P2, amino acid in protein substrates is important as MetAP preferentially excises the N-terminal Met when the second residue is Gly, Ala, Ser, Thr, Cys, Pro, or Val. The substrates for N-acetyltransferase (NAT), which acetylates the N-terminus, are usually the four smallest residues (Gly, Ala, Ser, and Thr). In the case of the N protein the P2 amino acid is Ser, and thus our observation of post-translational modification at the N-terminus of the N-protein is following the conventional Nterminal processing of proteins (Bonissone, S., Gupta, N., et al. 2013) . Since NME and NTA activity is necessary for protein function and stability, such processing of the N protein could be critical for its activity in viral replication and infection.

The coronavirus N protein consists of three distinct but highly conserved domains, including an N-terminal RNA-binding domain (NTD), a C-terminal dimerization domain (CTD), and a central Ser/Arg (SR)-rich linker domain, which displays an intrinsically disordered structure and facilitates molecular movements to aid interactions (Figure 2 ). The NTD is reportedly responsible for RNA binding, CTD for oligomerization, and the SR-rich linker is generally known to be primarily involved in phosphorylation events ( A recent report used the same commercial glycosylated version of N protein and studied the binding antibodies isolated from the plasma of human Covid-19 patients (Rudberg, A.-S., Havervall, S., et al. 2020) . The authors compared the serological titer of antibodies with three antigens of SARS-CoV-2, including spike (S) protein and nucleocapsid (N) protein, and found that 235 out of 243 positive cases had antibodies that bound to this glycosylated version of the

That study shows that the glycosylated N protein can bind antibodies generated by Covid-19 patients, possibly by binding only to non-glycosylated regions of this N protein (Rudberg, A.-S., Havervall, S., et al. 2020) . However, additional epitopes might be revealed using a N protein without such post-translational modifications.

Recently, several enzyme-linked immunosorbent assay (ELISA)-based serological tests have been developed to detect serum immunoglobulins (Ig) against SARS-CoV-2. Serological assays are shown to be critical in assessing the population spectrum that has been exposed to the virus, as well as the heterogeneity of antibody response. A dual ELISA test against SARS-CoV-2 N protein showed the critical importance of epitope unmasking by de-glycosylation of protein produced in a mammalian system (Rump, A., Risti, R., et al. 2020) .

The phosphorylation sites in the SARS-CoV-2 N protein were investigated in a recent study and reported as occurring within the N-terminal portion of the protein, at or near the RNA binding region, but not at the C-terminal dimerization domain (Bouhaddou, M., Memon, D., et al. 2020) . There are clusters of phosphorylation sites within the arginine/serine (RS)-dipeptide rich region, which is C-terminal to the RNA binding region, and a conserved sequence across N proteins within the coronavirus family. In SARS-CoV-1 this region is phosphorylated by serinearginine (SR) protein kinases and modulates the role of SARS-CoV-1 N protein in host translation inhibition (Peng, T.-Y., Lee, K.-R., et al. 2008) . Phosphorylation of this same region can possibly play a similar role in SARS-CoV-2.

To our knowledge, the coronavirus N protein is the only phosphorylated structural protein on the virus, and this phosphorylation has been proposed to play a role in regulating its functions (Calvo, E., Escors, D., et al. 2005 , Chen, H., Gill, A., et al. 2005 , Lin, L., Shao, J., et al. 2007 ). Evidence of significant conformational changes in the N protein structure due to phosphorylation has been reported (Stohlman, S.A., Fleming, J.O., et al. 1983 ). N protein from SARS-CoV-1 has been shown to elicit a well-defined immunological response, which

underscores the importance of the N protein as a potent target for a vaccine against COVID-19

infection (Blicher, T., Kastrup, J.S., et al. 2005) . A recent study, aimed to investigate the effect of early SARS-CoV-2-specific humoral immune responses on disease outcome, found that deceased patients had stronger antibody responses towards N protein while survivors had much stronger antibody response to the S protein highlighting the importance of the N protein in disease outcome (Atyeo, C., Fischinger, S., et al. 2020) . Although several vaccine candidates have exhibited high efficacy, the majority are based on recognition of the S protein (2020b, Dong, Y., Dai, T., et al. 2020 , Mohan, P., Singhal, A., et al. 2020 , Polack, F.P., Thomas, S.J., et al. 2020 . There is a concern that the vaccine will no longer be effective if new SARS-CoV-2 strains with altered S protein sequence emerge (Koyama, T., Weeraratne, D., et al. 2020) . The N protein has been reported as less susceptible to mutations and thus merits further consideration as a target for future vaccine development, although another study has claimed that N protein is subject to increased mutations ( 

Sequencing-grade modified trypsin and elastase were purchased from Promega 

The The O-glycans were released from the glycopeptide peptide by reductive β-elimination reported elsewhere (Shajahan, A., Heiss, C., et al. 2017 by eluting with 5% acetic acid and dried by lyophilization. The borates were removed by the addition of a solution of methanol: acetic acid (9:1) and evaporation under a steam of nitrogen.

The released N-and O-linked glycans were then permethylated using NaOH/DMSO-methyl iodide method published previously (Shajahan, A., Heiss, C., et al. 2017) .

The permethylated N-and O-glycans were dissolved in 2 µL of methanol. 0.5 µL of sample was mixed with equal volume of DHB matrix solution (10 mg/mL in 1:1 methanol-water) and

spotted on to a MALDI plate. MALDI-MS spectra were acquired in positive ion and reflector mode using an AB Sciex 5800 MALDI-TOF-TOF mass spectrometer. 

The glycoprotein digests were analyzed on an Orbitrap Fusion Tribrid mass spectrometer equipped with a nanospray ion source and connected to a Dionex Ultimate 3000 

The raw data files and search results can be accessed from glycopost repository:

https://glycopost.glycosmos.org/preview/519892202600f0148a95a0 PIN CODE 7735 

dimerization domain -CTD, coronavirus disease -COVID-19, human angiotensinconverting enzyme 2 -hACE2, human embryonic kidney -HEK, dithiothreitol -DTT

Higher-energy Collisional Dissociation -HCD, product triggered -pd, Collision-Induced Dissociation -CID

Electron-transfer dissociation -ETD, ion trap-mass spectrometry -IT-MS

conceived of the paper; N.S. performed glycoproteomics and glycomics sample processing, A.S conducted glycomics data acquisition

M performed production of N protein at CCRC. P.A. and C.H. monitored the project. Competing interests The authors certify that they have do not have any competing interests. Supplemental material: Glycomics and Glycoproteomics workflow of N protein (S1), N-and Oglycan MALDI/ESI-MS data and annotated N-and O-glycan MS/MS

Supplemental Tables SI-SIV are also included as separate files for relative quantification via glycomics and glycoproteomics of N(SP+)

High-resolution structure of HLA-A*1101 in complex with SARS nucleocapsid peptide

N-terminal protein processing: a comparative proteogenomic analysis

The Global Phosphorylation Landscape of SARS-CoV-2 Infection

Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells

The SARS coronavirus nucleocapsid protein--forms and functions

Modular organization of SARS coronavirus nucleocapsid protein

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA

Mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using surface plasmon resonance

Structure analysis of the receptor binding of 2019-nCoV

A systematic review of SARS-CoV-2 vaccine candidates

The Nucleocapsid Protein of SARS-CoV-2: a Target for Vaccine Development

Unbiased Screens Show CD8 (+) T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein

Post-translational modifications of coronavirus proteins: roles and function

Protein N-terminal methionine excision. Cellular and molecular life sciences : CMLS

Proteotyping SARS-CoV-2 Virus from Nasopharyngeal Swabs: A Proof-of-Concept Focused on a 3 Min Mass Spectrometry Window

A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2

Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus

Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

Emergence of Drift Variants That May Affect COVID-19 Vaccine Development and Antibody Treatment

Designing of Nucleocapsid Protein Based Novel Multi-epitope Vaccine Against SARS-COV-2 Using Immunoinformatics Approach

Targeting the SARS-CoV2 nucleocapsid protein for potential therapeutics using immuno-informatics and structure-based drug discovery techniques

The Coronavirus Nucleocapsid Protein

2020. The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway

Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

The Molecular Biology of Coronaviruses

Novel coronavirus vaccine: An international holy grail

SARS-CoV-2-encoded nucleocapsid protein acts as a viral suppressor of RNA interference in cells

Characterization of N protein self-association in coronavirus ribonucleoprotein complexes

Mass-Spectrometric Detection of SARS-CoV-2 Virus in Scrapings of the Epithelium of the Nasopharynx of Infected Patients via Nucleocapsid N Protein

Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein

Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization

Mutational insights into the envelope protein of SARS-CoV-2

Evolutionary dynamics of SARS-CoV-2 nucleocapsid protein and its consequences

SARS-CoV-2 exposure, symptoms and seroprevalence in healthcare workers in Sweden

Dual ELISA using SARS-CoV-2 nucleocapsid protein produced in E. coli and CHO cells reveals epitope masking by N-glycosylation

Comprehensive characterization of N-and O-glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2

Glycomic and glycoproteomic analysis of glycoproteins-a tutorial

Deducing the N-and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation

The SARS-CoV nucleocapsid protein: a protein with multifarious activities

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

2021. mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

Site-specific glycan analysis of the SARS-CoV-2 spike

Phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication

The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor

A Novel Coronavirus from Patients with Pneumonia in China

Legends to figures

Financial support from the US National Institutes of Health (S10OD018530, P41GM103390, R24GM137782 and R01GM130915) is gratefully acknowledged.

Nucleocapsid protein -N protein, membrane -M, envelope -E, Spike -S, severe acute respiratory syndrome coronavirus 2 -SARS-CoV-2, N-terminal RNA-binding domain -NTD, C-