Introduction
The ongoing devastating coronavirus disease 2019 (COVID-19) pandemic is caused by the newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ), which can cause lower respiratory tract infections and atypical pneumonia.1–3 SARS-CoV-2 is more contagious.4–6 SARS-CoV-2 virion is composed of the trimeric Spike protein (S), the membrane glycoprotein (M), and the envelope protein (E) on its surface, and nucleocapsid phosphoprotein (N, or NCP) in its core.7 1,8–11 SARS-CoV-2 have not been investigated thoroughly.
Being the most abundant structural protein, NCP constitutes the coronavirus (CoV) core that encapsulates and stabilizes the viral genome.12 12–16 17–19 20 21 SARS-CoV-2 is a more sensitive indicator of early infections than IgG or viral RNA.22,23 SARS-CoV-2 NCP shares high homology with its SARS-CoV counterpart,24 23,25–28 29 SARS-CoV-2 NCP has also been proposed as a candidate target for the development of vaccines30–32 33–36 SARS-CoV-2 protein S, which mutates rapidly worldwide.37–39 SARS-CoV-2 patients are more likely to develop T cell responses against NCP than against protein S, and are able to generate durable B cell memory for both NCP and protein S.40–42
The SARS-CoV NCP was previously found to contain several important post-translational modifications (PTMs), primarily in the form of phosphorylation.7,43–45 SARS-CoV-2 NCP was also recently proposed as a key factor in modulating nucleocapsid assembly.45,46 SARS-CoV-2 NCP PTM profile using high-resolution mass spectrometry, based on which a protein structural model was proposed to highlight possible biological or immunological implications. For the first time, we show that SARS-CoV-2 NCP is decorated with N -glycans and ubiquitin in addition to phosphoryl groups.
Results
Landscape of PTMs on SARS-CoV-2 NCP
To portray the PTM landscape, liquid chromatography with tandem mass spectrometry (LC-MSMS) data of peptides, derived by digestion of SARS-CoV-2 NCP with multiple proteases, were subjected to iterative spectra-database matching for common PTMs (Figure 1 ). Detailed PTM identification can be found in Supplementary Table 1, https://links.lww.com/IMD/A9 SARS-CoV-2 NCP is decorated with a plethora of PTMs (Figure 2 A), including acetylation (K342, K375), succinylation (K387, K388), di-Gly (K169, K374, K388), phosphorylation (S2, S26, S176, S180, S184, T265, T379, T391, T393, S410, S416) and methylation (E136) as summarized in Table 1 . All domains of NCP were decorated with PTMs, with the majority of PTMs clustered in the N terminal domain (NTD) and C tail of NCP. As NCP is expected to be heavily modified by phosphorylation, this study identified five novel NCP phosphorylation sites in addition to previous archived sites,47,48 49 SARS-CoV-2 NCP (Figure 2 B, Supplementary Table 2, https://links.lww.com/IMD/A10 https://links.lww.com/IMD/A12
Figure 1: Overall workflow for comprehensive PTM survey of SARS-CoV-2 NCP using multi-enzyme digestion and enrichment techniques . DTT: dithiothreitol; FASP: filter aided sample preparation; HILIC: hydrophilic interaction liquid chromatography; IAA: iodacetamide; NCP: nucleocapsid phosphoprotein; PTM: post-translational modification; SARS-CoV-2 : severe acute respiratory syndrome coronavirus 2; SIMAC: sequential elution from IMAC (immobilized metal ion affinity based chromatography).
Figure 2: Summary of PTMs in SARS-CoV-2 NCP . A: Distribution of PTMs identified in this study related to functional subunits and domains of SARS-CoV-2 NCP. PTM notation can be found in right lower legend. Predominant N -glycan forms were directly marked on both glycosylation sites. B: Venn diagram summary of SARS-CoV-2 NCP phosphorylation sites identified in our and previous studies. C: Lysates from SARS-CoV-2 NCP expressed 293T cells were subjected to N and IgG pull-down followed by immunoblotting analyses. CTD: C terminal domain; NCP: nucleocapsid phosphoprotein; NTD: N terminal domain; PTM: post-translational modification; SARS-CoV-2 : severe acute respiratory syndrome coronavirus 2.
Table 1 -
Summary of PTMs identified from
SARS-CoV-2 NCP
Domain
Sites
PTM
Sequence identified
N-arm
2
Phosphorylation
S∗ DNGPQNQRNAPRITF
26
Phosphorylation
ITFGGPSDSTGS∗ NQNGER
NTD
77
N -glycosylation
GQGVPINTN∗ SSPDDQIGYYR
136
Methylation
DGIIWVATE∗ GALNTPK
169
di-Gly
NPANNAAIVLQLPQGTTLPK∗
176
Phosphorylation
GFYAEGS∗ RGGSQASSRGFYAEGS∗ R
180
Phosphorylation
GFYAEGSRGGS∗ QASSR
184
Phosphorylation
GFYAEGSRGGSQASS∗ R
CTD
265
Phosphorylation
QKRTAT∗ KAYNVTQAFGR
269
N -glycosylation
AYN∗ VTQAFGR
342
Acetylation
LDDK∗ DPNFK
C-tail
374
di-Gly
K∗ KADETQALPQR
375
Acetylation
K∗ ADETQALPQR
379
Phosphorylation
KADET∗ QALPQRADET∗ QALPQR
387
Succinylation
QK∗ KQQTVTLLPAADLDDFSK
388
di-Gly
K∗ QQTVTLLPAADLDDFSK
388
Succinylation
QKK∗ QQTVTLLPAADLDDFSK
391
Phosphorylation
KQQT∗ VTLLPAADLDDFSKQT∗ VT∗ LLPAADLDDFSK
393
Phosphorylation
QT∗ VT∗ LLPAADLDDFSK
410
Phosphorylation
KQQTVTLLPAADLDDFSKQLQQS∗ MSSADS∗ TQA
416
Phosphorylation
CTD: C terminal domain; NCP, nucleocapsid phosphoprotein; NTD: N terminal domain.
∗ Modified sites. Tryptic digestion resulted in di-Gly residue on lysine modified by ubiquitin.
Confirmation of ubiquitination of SARS-CoV-2 NCP
The di-Gly residue on lysine can be the result of tryptic digestion either by ubiquitin or ISG15 modifier. To resolve this ambiguity, SARS-CoV-2 NCP expressed in 293T cells was immunoprecipitated and then immunoblotted with either anti-ubiquitin or anti-ISG15 antibodies. As compared to the negative control sample (IgG immunoprecipitated), SARS-CoV-2 NCP was clearly modified by ubiquitin rather than by ISG15 (Figure 2 C).
Determination of N-glycosylation on SARS-CoV-2 NCP
To characterize glycosylation, glycopeptides from SARS-CoV-2 NCP digested with multiple proteases were enriched by hydrophilic interaction liquid chromatography-solid phase extraction (HILIC SPE). To determine glycosylated sites, peptides were deglycosylated by PNGase F in H2 O18 creating a +2.98 Da mass shift to mark glycosylated sites. Out of five possible N-X-S/T glycosylation sites, two sites (N77, N269, Table 1 ) in NCP were confirmed by intact glycopeptide characterization, while no evidence of N -glycosylation was found for the other three potential sites (N47, N192, and N196). Example mass spectra showing evidence of glycosylation were shown in Supplementary Figure 2A and 2B, https://links.lww.com/IMD/A12
Intact glycopeptides were also analyzed directly by LC-MSMS to resolve the highly heterogeneous glycan components. Our analysis identified 55 unique N -glycopeptides, deemed as a unique amino acid sequence with a unique N -glycan composition (Supplementary Table 3, https://links.lww.com/IMD/A11 Figure 3 A) suggested that N77 was preferentially occupied by complex type N -glycans, with 13 possible configurations comprising nearly 95% of total LC-MS intensity of all glycopeptides involving site N77, while the remaining 5% intensity was contributed by a hybrid N -glycan. According to LC-MS intensity, the most dominant glycan on N77 was Fuc2Hex3HexNAc6, which is predicted to be a fucosylated bi-antennary complex. In comparison, nearly 79% of LC-MS intensity of all N269-related glycopeptides can be attributed to six high-mannose oligosaccharides, while the remaining 21% was contributed by complex and hybrid N -glycans. The most dominant glycan on N269 was Hex5HexNAc2. So far, no evidence of sialic acid conjugation to NCP glycans was found at either site. Example N -glycopeptides spectra can be found in Supplementary Figure 2C and 2D, https://links.lww.com/IMD/A12
Figure 3: SARS-CoV-2 NCP decorated with glycansN -glycan categories on both sites. B: The most likely glycan structure on site N77 was added to the NTD RNA-binding domain SARS-CoV-2 NCP based on NTD binding model with dsRNA (Protein Data Bank ID: 7ACS, sequence 44-180). C: The most likely glycan structure on site N269 was added to the CTD dimerization domain SARS-CoV-2 NCP based on model of four CTD subunits polymerized (Protein Data Bank ID: 6WZQ, sequence 247-364), with each subunit colored differently. All NCP sequences were represented in cartoon mode while glycans were represented in sphere mode. CTD: C terminal domain; dsRNA: double-stranded RNA; LC-MS: liquid chromatography with mass spectrometry; NCP: nucleocapsid phosphoprotein; NTD: N terminal domain; PTM: post-translational modification; SARS-CoV-2 : severe acute respiratory syndrome coronavirus 2.
Based on the structures of the most likely N -glycans derived in this study and the SARS-CoV-2 NCP structure models, we generated atomic models that represented the most likely spatial distribution of the N -glycans on the SARS-CoV-2 NCP (Figure 3 B and 3C). Since the full structure of NCP has not been determined yet, currently available partial structure models of the N-terminal RNA-binding domain (7ACS) and C-terminal dimerization domain (6WZQ) were used to resolve glycans on site N77 and N296, respectively.
Alignment of PTMs with immunogenic epitopes on SARS-CoV-2 NCP
As PTMs, particularly in the form of glycans, can drastically change immunogenicity of a protein, we checked whether PTMs are located in potential epitope areas of SARS-CoV-2 NCP. However, because currently there is a lack of experimental data on SARS-CoV-2 NCP immune recognition, we resorted to bioinformatic prediction tools to generate potential epitope sequences. Out of 11 B cell epitopes of NCP predicted by Bepipred, six involved phosphorylation, ubiquitination, succinylation, acetylation, and/or glycosylation (Table 2 ). In particular, multiple phosphorylation sites in the SR-rich domain (S176, S180, S184) and ubiquitination (K169) were found in epitope 165-216. Another C-tail epitope (358-402) was decorated by a variety of PTMs including phosphorylation (T379, T391, T393), ubiquitination (K374, K388), acetylation (K375), and succinylation (K387, K388). To highlight, glycosylation (N77) was also found in a B cell epitope (59-105). In addition, as a viral core protein, the NCP epitope is likely to be endogenously processed by CD8+ T cells and presented as major histocompatibility complex class I (MHC-I)–associated peptides. Out of 11 T-cell epitope peptides predicted by NetMHCpan, only two contain PTMs, including ubiquitination (K169) in epitope 165–173 and acetylation (K342) in epitope 338-346.
Table 2 -
Predicted immuno-epitopes in
SARS-CoV-2 NCP aligned with PTMs
Start
End
Epitope sequence
Length
Score
PTM
B cell epitope sequences predicted by Bepipred algorithm
4
15
NGPQNQRNAPRI
12
0.60
–
17
48
FGGPSDSTGSNQNGERSGARSKQRRPQGLPNN
32
0.68
Phosphorylation: S26
59
105
HGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLS
47
0.56
Glycosylation: N77
119
127
AGLPYGANK
9
0.54
–
137
163
GALNTPKDHIGTRNPANNAAIVLQLPQ
27
0.58
–
165
216
TTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGD
52
0.66
Ubiquitination: K169; Phosphorylation: S176, S180, S184
226
267
RLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKA
42
0.60
Phosphorylation: S265
276
299
RRGPEQTQGNFGDQELIRQGTDYK
24
0.56
–
343
348
DPNFKD
6
0.53
–
358
402
DAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDD
45
0.62
Ubiquitination: K374, K388; Acetylation K375; Phosphorylation: T379, T391, T393; Succinylation: K387, K388
404
416
SKQLQQSMSSADS
13
0.58
Phosphorylation: S410, S416
MHC-I epitope sequences predicted by Net MHC pan EL algorithm
112
121
YLGTGPEAGL
10
0.229
–
158
167
VLQLPQGTTL
10
0.197
–
165
173
TTLPKGFYA
9
0.095
Ubiquitination: K169
220
230
ALLLLDRLNQL
11
0.956
–
226
234
RLNQLESKM
9
0.126
–
305
313
AQFAPSASA
9
0.176
–
316
324
GMSRIGMEV
9
0.422
–
330
339
WLTYTGAIKL
10
0.066
–
338
346
KLDDKDPNF
9
0.552
Acetylation: K342
351
359
ILLNKHIDA
9
0.125
–
397
407
AADLDDFSKQL
11
0.191
–
Bepipred score were calculated for each amino acid, and score for a peptide (selection threshold >0.5) were calculated as average of all amino acids. For MHC peptides prediction, the panEL prediction score was provided, and peptides of top 2% ranking score (corresponding to >0.095 in our analysis) were selected.MHC-I: major histocompatibility complex class I; NCP: nucleocapsid phosphoprotein; PTM: post-translational modification; SARS-CoV-2 : severe acute respiratory syndrome coronavirus 2.
Discussion
In coronavirus-infected cells, NCP is mainly located in the cytoplasm. It is able to self-associate into an oligomer and bind to viral RNA via its NTD, thus forming the ribonucleoprotein core complex that protects viral RNA and aids virus replication.12–16 13,14,17,20,50,51 SARS-CoV-2 NCP.
CoV NCP is widely known as a phosphoprotein.7,43,44 44,52,53 47,48 SARS-CoV-2 NCP is heavily decorated by phosphoryl groups across all domains. Compared with previous studies,47,48 SARS-CoV-2 NCP, and the technical bias of the peptide-centric LC-MS approach against hydrophilic phosphorylated sequences, more phosphorylation sites are likely to be mapped in future studies with complementary techniques or different expression systems. Future studies are also warranted to explore virological or immunological implications of the complex NCP phosphorylation profile, particularly for those sites outside of the SR-rich region, such as the C-tail region, which has been demonstrated to have high antigenicity on SARS-CoV NCP.29 SARS-CoV-2 NCP. Acylation can reduce the charge state and increase the hydrophobicity of modified sites. Further studies are needed to investigate possible biological roles of these PTMs.
To the best of our knowledge, this is the first time ubiquitination has ever been identified in a CoV protein. Other than causing protein degradation events, ubiquitination also triggers signaling or protein translocation. The locations of the newly identified ubiquitination sites on SARS-CoV-2 NCP are interesting: site K169 resides immediately before the SR-rich domain, while sites K374 and K388 both sit within the predicted bipartite nuclear localization signal domain (NLS-BP, KKKKADETQALPQRQKKQ), which is responsible for NCP translocation from the cytoplasm to the nucleus. Notably, alignment analysis (Supplementary Figure 3A, https://links.lww.com/IMD/A12 SARS-CoV-2 (K169, K374, K388), SARS-CoV (K170, K375, K389) and bat CoV HKU3 (K169, K374, K388). In fact, ubiquitination, rather than ISGylation, can also be detected by immunoblotting of NCP from SARS-CoV (Supplementary Figure 3B, https://links.lww.com/IMD/A12 54,55 17 SARS-CoV-2 NCP can be ubiquitinated by TRIM25.
Glycosylation is typically found on the viral surface or envelope proteins, and it has been shown to be a critical determinant for viral pathogenesis and immunogenicity,56–58 59,60 SARS-CoV-2 , are heavily masked by glycan camouflage,61–64 58,65–73 74,75 SARS-CoV-2 NCP was evidenced indirectly by PNGase F treatment in H2 O18 , which leaves an isotopic mark to the glycosylation locus. Alignment analysis of CoV sequences (Supplementary Figure 4, https://links.lww.com/IMD/A12 SARS-CoV-2 NCP. In contrast, potential glycosylation sites that are homologous to N269 of SARS-CoV-2 NCP can be found across multiple CoV species: SARS-CoV (N270), H-CoV-229E (N260), Bat-CoV-HKU3 (N269). To further confirm NCP glycosylation, the glycan component in these sites was determined directly by LC-MSMS as well. Typically, due to incomplete glycan maturation, viral protein glycans are mainly composed of short oligomannose branches, as in the case of site N269 in the C terminal domain domain.76 N -glycan patterns greatly extend conformational flexibility and epitope diversity of NCP and may profoundly contribute to the unique biological and clinical characteristics of SARS-CoV-2 as compared to other CoVs.
The ongoing global efforts to generate vaccines or antibodies for SARS-CoV-2 primarily focus on protein S as the target antigen.37,38,77 24 SARS-CoV-2 patients.40–42 78,79 SARS-CoV-2 patients developed stronger T cell responses against NCP than against protein S.40 36 SARS-CoV-2 NCP proposed in this study, can provide guidance to produce new vaccines or therapeutics to join the current vaccination and treatment paradigm in our arduous battle against the COVID-19 pandemic.
Materials and methods
Expression and purification of SARS-CoV-2 NCP
HEK293T (Human embryonic kidney, ATCC® CRL-3216TM ) cells were grown in Dulbecco modified Eagle medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco BRL). The full-length SARS-CoV-2 NCP gene (GenBank number QHD43419.1) was cloned into the pcDNA3.1 vector with a C-terminal 2 X Strep tag. HEK293T cells or Vero cells were transfected with the plasmid using Lipofectamine2000 Transfection Reagent (Invitrogen, CA, USA, 11668019). After 40–60 hours, the cells were lysed in 1 mL cold immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5% Nonidet P 40 Substitute (NP-40, Solarbio) and 1× complete EDTA-free protease inhibitor cocktail (Roche, Switzerland, 11873580001) for 30 minutes at 4°C. The lysates were then centrifuged for 30 minutes at 15000×g. The remaining lysate was incubated with 30 μL of Strep-Tactin Sepharose beads (IBA Lifesciences, Germany) in 0.6 mL IP Buffer for 2 hours with constant rotation at 4°C. The beads were washed three times with 1 mL of IP buffer containing 0.05% NP-40 followed by one final wash in detergent-free IP buffer in a fresh detergent-free tube. Proteins were eluted by agitating beads in 40 μL IP buffer supplemented with 2.5 mM D-Desthiobiotin (IBA Lifesciences) on a vortex mixer at room temperature for 30 minutes.
Sample preparation for PTM survey
NCP was digested into peptides according to a modified filter aided sample preparation protocol.80 Figure 1 .
Sample preparation for protein glycosylation characterization
Peptides derived from nucleocapsid protein by multiple protease digestion were used to enrich intact N -glycopeptides by house-made zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) stage-tip. Briefly, stage-tip packed with Exsil Pure ZIK HILIC 5 μm beads (Dr. Maisch GmbH, Germany, 6136918) was pre-conditioned with 0.1% trifluoroacetic acid (TFA) in 80% acetonitrile (ACN). Peptides in 0.1% TFA in 80% ACN were loaded to the beads and unbound flow-through fraction was collected. The column was washed twice with 0.1% TFA in 80% ACN. Glycopeptides were eluted first by 0.1% TFA and then by 50 mM ABC. Other than direct analyses by LC-MSMS, glycopeptides were also dried by Speed Vac and deglycosylated by PNGase F (NEB, 1:100) overnight at 37°C in 50 mM ABC in pure H2 O18 .
LC-MSMS experiments
Peptides (500 ng) were separated by C18 nano-column using an Ultimate 3000 nanoflow liquid chromatography system (Thermo Scientific, MA, USA) and analyzed by the Q-Exactive HFX mass spectrometry (Thermo Scientific) operated under the data-dependent mode. The MS spectra were recorded by Xcalibur software 2.3 (Thermo Scientific). All operation parameters for nanoLC and MS systems are detailed in the Supplementary Methods based on previously published methods, https://links.lww.com/IMD/A12 64
Bioinformatics
All MS raw data files of directed peptides analyses were searched by MaxQuant (version 1.6.10.43) against the Uniprot SARS-CoV-2 sequences. Mass tolerance of 4.5 ppm was set for the search. Trypsin/chymotrypsin/LysC with up to two missed cleavages was set. Carbamidomethylation on Cys was set as fixed modification, while oxidation on Met and O18 deamidation on Asn were set as variable modification. Identification was further screened by N -glycosylation possible sequons (N-X-S/T, X≠P). To explore additional PTM forms, phosphorylation (S/T), acetylation (K), methylation (K/R/E), succinylation (K), crotonylation (K), di-Gly (K), farnesylation (K/Nterm), myristoylation (K/Nterm), palmitoylation or prenylation, and oxidation on proline were separately specified in individual searches. Peptide level 1% FDR was set to filter the results. Confident identification of PTM was based on localization probability of 95%.
To reveal N -glycosylation forms, MS raw files from ZIC-HILIC fractioned peptides were analyzed by pGlyco (version 2.2.2)81 81
Immunogenic epitopes prediction
Identified PTM positions were aligned with possible immunogenic epitopes on SARS-CoV-2 NCP predicted by bioinformatic tools. Briefly, predictions of B cell epitopes were performed by Bepipred (v2.0) available via IEDB database (http://tools.iedb.org/main/bcell 82 83
Immunoblotting analysis
To confirm protein ubiquitination or ISGylation status, SARS-CoV-2 NCP expressed in 293T cells was immunoprecipitated by Strep-Tactin Sepharose beads (IBA Lifesciences, 2-1502-001), separated by 4%–15% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis before transferred to PVDF membranes. Immunoblotting was performed by Strep-Tactin-HRP conjugate antibody (IBA Lifesciences, 2-1502-001, 1:1000), anti-Ubiquitin (CST 3933, 1:1000), anti-ISG15 (CST 2758, 1:1000), and anti-GAPDH (CST 5174, 1:1000) antibody and visualized by chemiluminescence detection kit.
Acknowledgments
The authors thank the proteomics and metabolomics platform in the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases at Zhejiang University for mass spectrometry analyses.
References
[1]. Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China.
Nature 2020;579(7798):265–269.
[2]. Dong E, Du H, Gardner L. An interactive web-based dashboard to track COVID-19 in real time.
Lancet Infect Dis 2020;20(5):533–534.
[3]. Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin.
Nature 2020;579(7798):270–273.
[4]. Guan WJ, Ni ZY, Hu Y, et al. Clinical characteristics of coronavirus disease 2019 in China.
N Engl J Med 2020;382(18):1708–1720.
[5]. Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study.
Lancet 2020;395(10229):1054–1062.
[6]. Wang D, Hu B, Hu C, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China.
JAMA 2020;323(11):1061–11061.
[7]. Satarker S, Nampoothiri M. Structural proteins in severe acute respiratory syndrome coronavirus-2.
Arch Med Res 2020;51(6):482–491.
[8]. Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus.
J Virol 2020;94(7):e00127–e00220.
[9]. Hoffmann M, Kleine-Weber H, Schroeder S, et al.
SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
Cell 2020;181(2):271–280.e8.
[10]. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the
SARS-CoV-2 spike glycoprotein.
Cell 2020;181(2):281–292.e6.
[11]. Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
Science 2020;367(6483):1260–1263.
[12]. Hurst KR, Ye R, Goebel SJ, Jayaraman P, Masters PS. An interaction between the nucleocapsid protein and a component of the replicase-transcriptase complex is crucial for the infectivity of coronavirus genomic RNA.
J Virol 2010;84(19):10276–10288.
[13]. Chang CK, Hou MH, Chang CF, Hsiao CD, Huang TH. The SARS coronavirus nucleocapsid protein--forms and functions.
Antiviral Res 2014;103:39–50.
[14]. McBride R, van Zyl M, Fielding BC. The coronavirus nucleocapsid is a multifunctional protein.
Viruses 2014;6(8):2991–3018.
[15]. Dinesh DC, Chalupska D, Silhan J, et al. Structural basis of RNA recognition by the
SARS-CoV-2 nucleocapsid phosphoprotein.
PLoS Pathog 2020;16(12):e1009100.
[16]. Savastano A, Ibanez de Opakua A, Rankovic M, Zweckstetter M. Nucleocapsid protein of
SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.
Nat Commun 2020;11(1):6041.
[17]. Hu Y, Li W, Gao T, et al. The severe acute respiratory syndrome coronavirus nucleocapsid inhibits type I interferon production by interfering with TRIM25-mediated RIG-I ubiquitination.
J Virol 2017;91(8):e02143–e2216.
[18]. Li JY, Liao CH, Wang Q, et al. The ORF6, ORF8 and nucleocapsid proteins of
SARS-CoV-2 inhibit type I interferon signaling pathway.
Virus Res 2020;286:198074.
[19]. Chen K, Xiao F, Hu D, et al.
SARS-CoV-2 nucleocapsid protein interacts with RIG-I and represses RIG-mediated IFN-beta production.
Viruses 2020;13(1):47.
[20]. Tsai TL, Lin CH, Lin CN, Lo CY, Wu HY. Interplay between the poly(A) tail, poly(A)-binding protein, and coronavirus nucleocapsid protein regulates gene expression of coronavirus and the host cell.
J Virol 2018;92(23):e01162–e01218.
[21]. Che XY, Qiu LW, Pan YX, et al. Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome.
J Clin Microbiol 2004;42(6):2629–2635.
[22]. Liu L, Liu W, Zheng Y, et al. A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (
SARS-CoV-2 ) in 238 admitted hospital patients.
Microbes Infect 2020;22(4–5):206–211.
[23]. Guo L, Ren L, Yang S, et al. Profiling early humoral response to diagnose novel coronavirus disease (COVID-19).
Clin Infect Dis 2020;71(15):778–785.
[24]. Basu BV, Brown OR. Comparative analysis of Coronaviridae nucleocapsid and surface glycoprotein sequences.
Front Biosci 2020;25:1894–1900.
[25]. McAndrews KM, Dowlatshahi DP, Dai J, et al. Heterogeneous antibodies against
SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications on COVID-19 immunity.
JCI Insight 2020;5(18):e142386.
[26]. Li T, Wang L, Wang H, et al. Serum
SARS-CoV-2 nucleocapsid protein: a sensitivity and specificity early diagnostic marker for
SARS-CoV-2 infection.
Front Cell Infect Microbiol 2020;10:470.
[27]. Chia WN, Tan CW, Foo R, et al. Serological differentiation between COVID-19 and SARS infections.
Emerg Microbes Infect 2020;9(1):1497–1505.
[28]. Liu D, Wu F, Cen Y, et al. Comparative research on nucleocapsid and spike glycoprotein as the rapid immunodetection targets of COVID-19 and establishment of immunoassay strips.
Mol Immunol 2021;131:6–12.
[29]. Liu G, Hu S, Hu Y, et al. The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity.
Genomics Proteomics Bioinformatics 2003;1(3):193–197.
[30]. Dutta NK, Mazumdar K, Gordy JT. The nucleocapsid protein of
SARS-CoV-2 : a target for vaccine development.
J Virol 2020;94(13):e00647–e00720.
[31]. Kumar A, Kumar P, Saumya KU, Kapuganti SK, Bhardwaj T, Giri R. Exploring the
SARS-CoV-2 structural proteins for multi-epitope vaccine development: an in-silico approach.
Expert Rev Vaccines 2020;19(9):887–898.
[32]. Ong E, Wong MU, Huffman A, He Y. COVID-19 coronavirus vaccine design using reverse vaccinology and machine learning.
Front Immunol 2020;11:1581.
[33]. Kwarteng A, Asiedu E, Sakyi SA, Asiedu SO. Targeting the SARS-CoV2 nucleocapsid protein for potential therapeutics using immuno-informatics and structure-based drug discovery techniques.
Biomed Pharmacother 2020;132:110914.
[34]. Ray M, Sarkar S, Rath SN. Druggability for COVID-19: in silico discovery of potential drug compounds against nucleocapsid (N) protein of
SARS-CoV-2 .
Genomics Inform 2020;18(4):e43.
[35]. Tatar G, Ozyurt E, Turhan K. Computational drug repurposing study of the RNA binding domain of
SARS-CoV-2 nucleocapsid protein with antiviral agents.
Biotechnol Prog 2021;37(2):e3110.
[36]. Lang Y, Chen K, Li Z, Li H. The nucleocapsid protein of zoonotic betacoronaviruses is an attractive target for antiviral drug discovery.
Life Sci 2020;282:118754.
[37]. McCarthy KR, Rennick LJ, Nambulli S, et al. Recurrent deletions in the
SARS-CoV-2 spike glycoprotein drive antibody escape.
Science 2021;371(6534):1139–1142.
[38]. Starr TN, Greaney AJ, Addetia A, et al. Prospective mapping of viral mutations that escape antibodies used to treat COVID-19.
Science 2021;371(6531):850–854.
[39]. Dos Santos WG. Impact of virus genetic variability and host immunity for the success of COVID-19 vaccines.
Biomed Pharmacother 2021;136:111272.
[40]. Reynolds CJ, Swadling L, Gibbons JM, et al. Discordant neutralizing antibody and T cell responses in asymptomatic and mild
SARS-CoV-2 infection.
Sci Immunol 2020;5(54):eabf3698.
[41]. Hartley GE, Edwards ESJ, Aui PM, et al. Rapid generation of durable B cell memory to
SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence.
Sci Immunol 2020;5(54):eabf8891.
[42]. Dan JM, Mateus J, Kato Y, et al. Immunological memory to
SARS-CoV-2 assessed for up to 8 months after infection.
Science 2021;371(6529):eabf4063.
[43]. Wang J, Ji J, Ye J, et al. The structure analysis and antigenicity study of the N protein of SARS-CoV.
Genomics Proteomics Bioinformatics 2003;1(2):145–154.
[44]. Peng TY, Lee KR, Tarn WY. 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.
FEBS J 2008;275(16):4152–4163.
[45]. Carlson CR, Asfaha JB, Ghent CM, et al. Phosphoregulation of phase separation by the
SARS-CoV-2 N protein suggests a biophysical basis for its dual functions.
Mol Cell 2020;80(6):1092–1103.e4.
[46]. Carlson CR, Asfaha JB, Ghent CM, et al. Phosphoregulation of phase separation by the
SARS-CoV-2 N protein auggests a biophysical basis for its dual functions.
Mol Cell 2020;80(6):1092–1103. e4.
[47]. Bouhaddou M, Memon D, Meyer B, et al. The global phosphorylation landscape of
SARS-CoV-2 infection.
Cell 2020;182(3):685–712.e19.
[48]. Davidson AD, Williamson MK, Lewis S, et al. Characterisation of the transcriptome and proteome of
SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
Genome Med 2020;12(1):68.
[49]. Wang C, Xu H, Lin S, et al. GPS 5.0: an update on the prediction of kinase-specific phosphorylation sites in proteins.
Genomics Proteomics Bioinformatics 2020;18(1):72–80.
[50]. Kopecky-Bromberg SA, Martinez-Sobrido L, Frieman M, Baric RA, Palese P. Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists.
J Virol 2007;81(2):548–557.
[51]. Surjit M, Liu B, Chow VT, Lal SK. The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells.
J Biol Chem 2006;281(16):10669–10681.
[52]. Surjit M, Kumar R, Mishra RN, Reddy MK, Chow VT, Lal SK. The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation.
J Virol 2005;79(17):11476–11486.
[53]. Luo H, Chen Q, Chen J, Chen K, Shen X, Jiang H. The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1.
FEBS Lett 2005;579(12):2623–2628.
[54]. Choudhury NR, Heikel G, Michlewski G. TRIM25 and its emerging RNA-binding roles in antiviral defense.
Wiley Interdiscip Rev RNA 2020;11(4):e1588.
[55]. El-Asmi F, McManus FP, Brantis-de-Carvalho CE, Valle-Casuso JC, Thibault P, Chelbi-Alix MK. Cross-talk between SUMOylation and ISGylation in response to interferon.
Cytokine 2020;129:155025.
[56]. Yang TJ, Chang YC, Ko TP, et al. Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans.
Proc Natl Acad Sci U S A 2020;117(3):1438–1446.
[57]. Vigerust DJ, Shepherd VL. Virus glycosylation: role in virulence and immune interactions.
Trends Microbiol 2007;15(5):211–218.
[58]. Raman R, Tharakaraman K, Sasisekharan V, Sasisekharan R. Glycan-protein interactions in viral pathogenesis.
Curr Opin Struct Biol 2016;40:153–162.
[59]. Chen WH, Du L, Chag SM, et al. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.
Hum Vaccin Immunother 2014;10(3):648–658.
[60]. Kumar S, Maurya VK, Prasad AK, Bhatt MLB, Saxena SK. Structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-nCoV) and SARS coronavirus (SARS-CoV).
Virusdisease 2020;31(1):13–21.
[61]. Shajahan A, Supekar NT, Gleinich AS, Azadi P. Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus
SARS-CoV-2 .
Glycobiology 2020;30(12):981–988.
[62]. Walls AC, Tortorici MA, Frenz B, et al. Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy.
Nat Struct Mol Biol 2016;23(10):899–905.
[63]. Watanabe Y, Allen JD, Wrapp D, McLellan JS, Crispin M. Site-specific glycan analysis of the
SARS-CoV-2 spike.
Science 2020;369(6501):330–333.
[64]. Sun Z, Ren K, Zhang X, et al. Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
Engineering (Beijing) 2020;doi: 10.1016/j.eng.2020.07.014. [Published ahead of print August 30].
[65]. Fukushi M, Yoshinaka Y, Matsuoka Y, et al. Monitoring of S protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus.
J Virol 2012;86(21):11745–11753.
[66]. de Groot RJ. Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses.
Glycoconj J 2006;23(1–2):59–72.
[67]. Chang D, Zaia J. Why glycosylation matters in building a better flu vaccine.
Mol Cell Proteomics 2019;18(12):2348–2358.
[68]. Li W, Hulswit RJG, Widjaja I, et al. Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.
Proc Natl Acad Sci U S A 2017;114(40):E8508–E8517.
[69]. Parsons LM, Bouwman KM, Azurmendi H, de Vries RP, Cipollo JF, Verheije MH. Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding.
J Biol Chem 2019;294(19):7797–7809.
[70]. Shih YP, Chen CY, Liu SJ, et al. Identifying epitopes responsible for neutralizing antibody and DC-SIGN binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus.
J Virol 2006;80(21):10315–10324.
[71]. York IA, Stevens J, Alymova IV. Influenza virus N-linked glycosylation and innate immunity.
Biosci Rep 2019;39(1):BSR20171505.
[72]. Zheng J, Yamada Y, Fung TS, Huang M, Chia R, Liu DX. Identification of N-linked glycosylation sites in the spike protein and their functional impact on the replication and infectivity of coronavirus infectious bronchitis virus in cell culture.
Virology 2018;513:65–74.
[73]. Zhou Y, Lu K, Pfefferle S, et al. A single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms.
J Virol 2010;84(17):8753–8764.
[74]. Liang JQ, Fang S, Yuan Q, et al. N-Linked glycosylation of the membrane protein ectodomain regulates infectious bronchitis virus-induced ER stress response, apoptosis and pathogenesis.
Virology 2019;531:48–56.
[75]. Ma HC, Fang CP, Hsieh YC, Chen SC, Li HC, Lo SY. Expression and membrane integration of SARS-CoV M protein.
J Biomed Sci 2008;15(3):301–310.
[76]. Watanabe Y, Bowden TA, Wilson IA, Crispin M. Exploitation of glycosylation in enveloped virus pathobiology.
Biochim Biophys Acta Gen Subj 2019;1863(10):1480–1497.
[77]. Rappazzo CG, Tse LV, Kaku CI, et al. Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.
Science 2021;371(6531):823–829.
[78]. Zhao P, Cao J, Zhao LJ, et al. Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine.
Virology 2005;331(1):128–135.
[79]. Zhu MS, Pan Y, Chen HQ, et al. Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine.
Immunol Lett 2004;92(3):237–243.
[80]. Sun Z, Liu X, Jiang J, et al. Toward biomarker development in large clinical cohorts: an integrated high-throughput 96-well-plate-based sample preparation workflow for versatile downstream proteomic analyses.
Anal Chem 2016;88(17):8518–8525.
[81]. Liu MQ, Zeng WF, Fang P, et al. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.
Nat Commun 2017;8(1):438.
[82]. Jespersen MC, Peters B, Nielsen M, Marcatili P. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes.
Nucleic Acids Res 2017;45(W1):W24–W29.
[83]. Reynisson B, Alvarez B, Paul S, Peters B, Nielsen M. NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data.
Nucleic Acids Res 2020;48(W1):W449–W454.
