Respiratory Syndrome Coronavirus Nucleocapsid Protein Research Articles

Electrochemiluminescence Lateral Flow Immunoassay Using Ruthenium(II) Complex‐Loaded Dendritic Mesoporous Silica Nanospheres for Highly Sensitive and Quantitative Detection of SARS‐CoV‐2 Nucleocapsid Protein

AbstractLateral flow immunoassays (LFIA) are widely used for the cost‐effective and rapid detection of diverse analytes. However, traditional LFIA suffers from difficulties in providing quantitative results and has low sensitivity. Herein, LFIA is combined with electrochemiluminescence (ECL), a leading transduction technique with high sensitivity and wide dynamic range, to achieve highly sensitive and quantitative detection of severe acute respiratory syndrome coronavirus nucleocapsid protein (SARS‐CoV‐2 N protein). Ruthenium(II)‐based complexes are synthesized and loaded into dendritic mesoporous silica nanospheres (PEI‐Ru/dSiO2), which possessed central‐radial pore channels and served as tags for ECL‐LFIA. The electrodes are fabricated on a nitrocellulose (NC) membrane, which simplifies the structure of the ECL‐LFIA. PEI‐Ru/dSiO2 is captured on the electrode surface via a sandwich immunoreaction, which enhances the ECL signal by decreasing the distance between PEI‐Ru/dSiO2 and the electrode surface. Using 2,2‐bis(hydroxymethyl)‐2,2′,2′'‐nitrilotriethanol (BIS‐TRIS) as coreactant, the ECL‐LFIA is used for detecting SARS‐CoV‐2 N protein, with a linear range of 1–104 ng mL−1 and a limit of detection (LOD) of 0.52 ng mL−1. ECL‐LFIA can also be used to detect analytes in complex matrices. These results demonstrate that the prepared ECL‐LFIA has great potential as a point‐of‐care testing platform for the rapid and quantitative detection of disease biomarkers.

Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Suppresses Type I and Type III Interferon Induction by Targeting RIG-I Signaling.

Type I and type III interferons (IFNs) are the frontline of antiviral defense mechanisms that trigger hundreds of downstream antiviral genes. In this study, we observed that MERS-CoV nucleocapsid (N) protein suppresses type I and type III IFN gene expression. The N protein suppresses Sendai virus-induced IFN-β and IFN-λ1 by reducing their promoter activity and mRNA levels, as well as downstream IFN-stimulated genes (ISGs). Retinoic acid-inducible gene I (RIG-I) is known to recognize viral RNA and induce IFN expression through tripartite motif-containing protein 25 (TRIM25)-mediated ubiquitination of RIG-I caspase activation and recruitment domains (CARDs). We discovered that MERS-CoV N protein suppresses RIG-I-CARD-induced, but not MDA5-CARD-induced, IFN-β and IFN-λ1 promoter activity. By interacting with TRIM25, N protein impedes RIG-I ubiquitination and activation and inhibits the phosphorylation of transcription factors IFN-regulatory factor 3 (IRF3) and NF-κB that are known to be important for IFN gene activation. By employing a recombinant Sindbis virus-EGFP replication system, we showed that viral N protein downregulated the production of not only IFN mRNA but also bioactive IFN proteins. Taken together, MERS-CoV N protein functions as an IFN antagonist. It suppresses RIG-I-induced type I and type III IFN production by interfering with TRIM25-mediated RIG-I ubiquitination. Our study sheds light on the pathogenic mechanism of how MERS-CoV causes disease.IMPORTANCE MERS-CoV causes death of about 35% of patients. Published studies showed that some coronaviruses are capable of suppressing interferon (IFN) expression in the early phase of infection and MERS-CoV proteins can modulate host immune response. In this study, we demonstrated that MERS-CoV nucleocapsid (N) protein suppresses the production of both type I and type III IFNs via sequestering TRIM25, an E3 ubiquitin ligase that is essential for activating the RIG-I signaling pathway. Ectopic expression of TRIM25 rescues the suppressive effect of the N protein. In addition, the C-terminal domain of the viral N protein plays a pivotal role in the suppression of IFN-β promoter activity. Our findings reveal how MERS-CoV evades innate immunity and provide insights into the interplay between host immune response and viral pathogenicity.

CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and in mediating vaccine-induced protective immunity. In contrast, relatively little is known about the role of T cell responses and the antigenic targets of MERS-CoV that are recognized by CD8+ T cells. In this study, the highly conserved MERS-CoV nucleocapsid (N) protein served as a target immunogen to elicit MERS-CoV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for generating MVA-MERS-N expressing recombinant N protein. Overlapping peptides spanning the whole MERS-CoV N polypeptide were used to identify major histocompatibility complex class I/II-restricted T cell responses in BALB/c mice immunized with MVA-MERS-N. We have identified a H2-d restricted decamer peptide epitope in the MERS-N protein with CD8+ T cell antigenicity. The identification of this epitope, and the availability of the MVA-MERS-N candidate vaccine, will help to evaluate MERS-N-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of MERS-CoV infection.

Development, Characterization, and Application of Monoclonal Antibodies against Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Five monoclonal antibodies (MAbs) against recombinant nucleocapsid protein (NP) of severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) were developed by hybridoma technology. Epitope mapping by Western blotting showed that these anti-SARS-CoV NP MAbs bind to distinct domains of NP. These anti-SARS-CoV NP MAbs, with their high specificity, are potentially ideal candidates for developing early and sensitive diagnostic assays for SARS-CoV.

Novel Immunodominant Peptide Presentation Strategy: a Featured HLA-A*2402-Restricted Cytotoxic T-Lymphocyte Epitope Stabilized by Intrachain Hydrogen Bonds from Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β(2)-microglobulin (β(2)m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.

MRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with Kd = 1.7 nm. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication.

Severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of the human FGL2 gene

Among the structural and nonstructural proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), the nucleocapsid (N) protein plays pivotal roles in the biology and pathogenesis of viral infection. N protein is thought to dysregulate cell signalling and the transcription of cellular genes, including FGL2, which encodes a prothrombinase implicated in vascular thrombosis, fibrin deposition and pneumocyte necrosis. Here, we showed that N protein expressed in cultured human cells was predominantly found in the cytoplasm and was competent in repressing the transcriptional activity driven by interferon-stimulated response elements. However, the expression of N protein did not influence the transcription from the FGL2 promoter. More importantly, N protein did not modulate the expression of FGL2 mRNA or protein in transfected or SARS-CoV-infected cells. Taken together, our findings did not support the model in which SARS-CoV N protein specifically modulates transcription of the FGL2 gene to cause fibrosis and vascular thrombosis.

MRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus 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

Coronavirus nucleocapsid protein is abundant in infected cells and participates in viral RNA replication and transcription. The central domain of the nucleocapsid protein contains several arginine/serine (RS) dipeptides, the biological significance of which has not been well investigated. In the present study, we demonstrate that the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated primarily within the RS‐rich region in cells and by SR protein kinase 1 in vitro. The nucleocapsid protein could suppress translation and its RS motif is essential for such an activity. Moreover, phosphorylation of the RS motif could modulate the translation inhibitory activity of the nucleocapsid protein. We further found that RS motif phosphorylation did not significantly affect RNA binding of the nucleocapsid protein but impaired its multimerization ability. We observed that the nucleocapsid protein could translocate to cytoplasmic stress granules in response to cellular stress. Deletion or mutations of the RS motif enhanced stress granule localization of the nucleocapsid protein, whereas overexpression of SR protein kinase 1 inhibited nucleocapsid protein localization to stress granules. The nucleocapsid protein lacking the RS motif formed high‐order RNP complexes, which may also account for its enhanced stress granule localization. Taken together, phosphorylation of the severe acute respiratory syndrome‐CoV nucleocapsid protein modulates its activity in translation control and also interferes with its oligomerization and aggregation in stress granules.

Severe acute respiratory syndrome coronavirus nucleocapsid protein confers ability to efficiently produce virus-like particles when substituted for the human immunodeficiency virus nucleocapsid domain

We replaced the HIV-1 nucleocapsid (NC) domain with different N-coding sequences to test SARS-CoV nucleocapsid (N) self-interaction capacity, and determined the capabilities of each chimera to direct virus-like particle (VLP) assembly. Analysis results indicate that the replacement of NC with the carboxyl-terminal half of the SARS-CoV N resulted in the production of wild type (wt)-level virus-like particles (VLPs) with the density of a wt HIV-1 particle. When co-expressed with SARS-CoV N, chimeras containing the N carboxyl-terminal half sequence efficiently packaged N. However, the same was not true for the chimera bearing the N amino-terminal half sequence, despite its production of substantial amounts of VLPs. According to further analysis, HIV-1 NC replacement with N residues 2–213, 215–421, or 234–421 resulted in efficient VLP production at levels comparable to that of wt HIV-1, but replacement with residues 215–359, 302–421, 2–168, or 2–86 failed to restore VLP production to wild-type levels. The results suggest that the domain conferring the ability to direct VLP assembly and release in SARS-CoV N is largely contained between residues 168 and 421.

Induction of T-cell response by a DNA vaccine encoding a novel HLA-A*0201 severe acute respiratory syndrome coronavirus epitope.

The severe acute respiratory syndrome coronavirus nucleocapsid protein (SARS-CoV N) is one of the major targets for SARS vaccine due to its high potency in triggering immune responses. In this study, we have identified a novel HLA-A*0201 restricted epitope, N220 (LALLLLDRL), of the SARS-CoV N-protein through bioinformatics analysis. The N-protein peptide N220 shows a high binding affinity towards human MHC class I in T2-cells, and is capable of activating cytotoxic T-cells in human peripheral blood mononuclear cells (PBMCs). The application of using the N220 peptide sequence with a single-chain-trimer (SCT) approach to produce a potential DNA vaccine candidate was investigated in HLA-A2.1Kb transgenic mice. Cytotoxicity assay clearly showed that the T-cells obtained from the vaccinated animals were able to kill the N-protein expressing cells with a cytotoxicity level of 86% in an effector cells/target cells ratio of 81:1 one week after the last vaccination, which is significantly higher than other N-protein peptides previously described. The novel immunogenic N-protein peptide revealed in the present study provides valuable information for therapeutic SARS vaccine design.

Antigenic and cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies

A member of the family of coronaviruses has previously been identified as the cause of the severe acute respiratory syndrome (SARS). In this study, several monoclonal antibodies against the nucleocapsid protein have been generated to examine distribution of the nucleocapsid in virus-infected cells and to study antigenic regions of the protein. Confocal microscopic analysis identified nucleocapsids packaged in vesicles in the perinuclear area indicating viral synthesis at the endoplasmic reticulum and Golgi apparatus. The monoclonal antibodies bound to the central and carboxyterminal half of the nucleocapsid protein indicating prominent exposure and immunogenicity of this part of the protein. Antibodies recognised both linear and conformational epitopes. Predictions of antigenicity using mathematical modelling based on hydrophobicity analysis of SARS nucleoprotein could not be confirmed fully. Antibody binding to discontinuous peptides provides evidence that amino acids 274–283 and 373–382 assemble to a structural unit particularly rich in basic amino acids. In addition, amino acids 286–295, 316–325 and 361–367 that represent the epitope recognised by monoclonal antibody 6D11C1 converge indicating a well-structured C-terminal region of the SARS virus nucleocapsid protein and functional relationship of the peptide regions involved. Alternatively, dimerisation of the nucleocapsid protein may result in juxtaposition of the amino acid sequences 316–325 and 361–367 on one nucleoprotein molecule to amino acid 286–295 on the second peptide. The monoclonal antibodies will be available to assess antigenicity and immunological variabilities between different SARS CoV strains.

Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein Is Phosphorylated and Localizes in the Cytoplasm by 14-3-3-Mediated Translocation

The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.

Intracellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Absence of Nucleolar Accumulation during Infection and after Expression as a Recombinant Protein in Vero Cells

The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS) and nucleolar localization signal motifs, failed to restore nuclear localization to an NLS-minus mutant Rev protein. These data indicate that nuclear localization is not a conserved property among all nidoviruses.

Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice

Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.

Antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein.

Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein. Methods: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli. After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments. Results: The full-length protein and two other fragments reacted with all 52 sera tested. Four important regions with possible epitopes were identified and named as EP1 (amino acids 51–71), EP2 (134–208), EP3 (249–273), and EP4 (349–422), respectively. EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera. EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested. Conclusion: The N protein is a highly immunogenic protein of the SARS-CoV. Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH2 terminus. The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV.