scholarly journals SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans

2020 ◽  
Author(s):  
Nitin T. Supekar ◽  
Asif Shajahan ◽  
Anne S. Gleinich ◽  
Daniel Rouhani ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Point Of Care ◽  
Variable Mass ◽  
Phosphorylation Site ◽  
Post Translational Modifications ◽  
N Protein ◽  
Point Of Care Diagnostics ◽  
Glycosylation Sites

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

ATR-Spin: An open-source 3D printed device for direct cytocentrifugation onto Attenuated Total Reflectance crystals.

Lab on a Chip ◽  
2021 ◽  
Author(s):  
David Perez-Guaita ◽  
Zack Richardson ◽  
G. Quintas ◽  
Julia Kuligowski ◽  
Diana Eva Bedolla ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Molecular Level ◽  
Point Of Care ◽  
Attenuated Total Reflectance ◽  
Cell Preparation ◽  
Total Reflectance ◽  
Point Of Care Diagnostics ◽  
3D Printed ◽  
Rapid Characterization

Infrared Spectroscopy (IR) enables the direct and rapid characterization of cells at the molecular level. Achieving a rapid and consistent cell preparation is critical for the development of Point-of-Care diagnostics...

scholarly journals Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

2020 ◽  
Author(s):  
Corinne A. Lutomski ◽  
Tarick J. El-Baba ◽  
Jani R. Bolla ◽  
Carol V. Robinson
Keyword(s):  
Antibody Response ◽  
Nucleocapsid Protein ◽  
Electron Transfer Dissociation ◽  
Rna Binding ◽  
Vaccine Development ◽  
Cyclophilin A ◽  
Proteolytic Processing ◽  
Full Length ◽  
Oligomeric State ◽  
N Protein

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

scholarly journals Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses

2020 ◽  
Author(s):  
Noah Avery Schuster
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Close Association ◽  
Phylogenetic Analyses ◽  
N Gene ◽  
Nucleocapsid Proteins ◽  
Nucleocapsid Gene ◽  
N Protein ◽  
Global Pandemic ◽  
The Relationship

An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 16,500,000 cases and 650,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the β-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (S37 and A267) were found to exist only in its N protein, along with an extended β-hairpin that bends towards the nucleotide binding site. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, these data will enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral and vaccine development.

scholarly journals Swab Sample Transfer for Point-Of-Care Diagnostics: Characterization of Swab Types and Manual Agitation Methods

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e105786 ◽  
Author(s):  
Nuttada Panpradist ◽  
Bhushan J. Toley ◽  
Xiaohong Zhang ◽  
Samantha Byrnes ◽  
Joshua R. Buser ◽  
...  
Keyword(s):  
Point Of Care ◽  
Swab Sample ◽  
Point Of Care Diagnostics ◽  
Sample Transfer

scholarly journals The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

2020 ◽  
Author(s):  
Kristina V. Tugaeva ◽  
Dorothy E. D. P. Hawkins ◽  
Jake L. R. Smith ◽  
Oliver W. Bayfield ◽  
De-Sheng Ker ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Phosphorylation Site ◽  
Dependent Manner ◽  
Nucleocytoplasmic Shuttling ◽  
Genome Packaging ◽  
Rich Region ◽  
N Protein ◽  
Proteomic Data ◽  
Cellular Pathways ◽  
Micromolar Range

AbstractThe coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Phosphorylated SARS-CoV N was shown to bind to the host 14-3-3 protein in the cytoplasm. Proteomic data indicate that seven human 14-3-3 proteins are highly abundant in human tissues vulnerable to SARS-CoV-2 infection, collectively reaching ~1.8% of all proteins in the lungs, ~1.4% in the gastrointestinal system, ~2.3% in the nervous system. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its mechanism and the specific critical phosphosites were unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein hub, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and determine the apparent KD to selected isoforms in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, located within the SR-rich region of N. The tight 14-3-3/pN association suggests it could regulate nucleocytoplasmic shuttling of N, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

scholarly journals A Chemoenzymatic Method for Glycoproteomic N-glycan Type Quantitation

2019 ◽  
Author(s):  
Henghui Li ◽  
Leyuan Li ◽  
Kai Cheng ◽  
Zhibin Ning ◽  
Janice Mayne ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Cancer Cell Line ◽  
The Novel ◽  
Post Translational Modifications ◽  
Glycosylation Sites ◽  
Type Composition ◽  
Dimethyl Labeling ◽  
High Mannose ◽  
Biomarker Research ◽  
Glycan Type

ABSTRACTGlycosylation is one of the most important post-translational modifications in biological systems. Current glycoproteome methods mainly focus on qualitative identification of glycosylation sites or intact glycopeptides. However, the systematic quantitation of glycoproteins has remained largely unexplored. Here, we developed a chemoenzymatic method to quantitatively investigate N-glycoproteome based on the N-glycan types. Taking advantage of the specificity of different endoglycosidases and isotope dimethyl labeling, six N-glycan types of structures linked on each glycopeptide, including high-mannose/hybrid, bi-antennary and tri-antennary with/without core fucose, were quantified. As a proof of principle, the glycoproteomic N-glycan type quantitative (glyco-TQ) method was first used to determine the N-glycan type composition of immunoglobulin G1 (IgG1) Fc fragment. Then we applied the method to analyze the glycan type profile of proteins in the breast cancer cell line MCF7, and quantitatively revealed the N-glycan type micro-heterogeneity at both the glycopeptide and glycoprotein levels. The novel quantitative strategy to evaluate the relative intensity of the six states of N-glycan type glycosylation on each site provides a new avenue to investigate function of glycoproteins in broad areas, such as cancer biomarker research, pharmaceuticals characterization and anti-glycan vaccine development.

scholarly journals Adjuvant Potential of CD24 on Immunogenicity and Lethal Challenge Protection of a DNA vector Expressing Nucleocapsid Protein of Crimean Congo Hemorrhagic Fever Virus

2018 ◽  
Author(s):  
Touraj Aligholipour Farzani ◽  
Alireza Hanifehnezhad ◽  
Katalin Foldes ◽  
Koray Ergunay ◽  
Erkan Yilmaz ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Foreign Gene ◽  
Lethal Challenge ◽  
Crimean Congo Hemorrhagic Fever ◽  
N Protein ◽  
Hemorrhagic Fever Virus ◽  
Dna Vector

Crimean Congo hemorrhagic fever virus (CCHFV) is the causative agent of a globally-spread tick-borne zoonotic infection with an eminent risk of fatal human disease. Imminent public health threat posed by disseminated virus activity and lack of an approved therapeutic make CCHFV an urgent target for vaccine development. We described the construction of a DNA vector expressing nucleocapsid protein (N) of CCHFV (pV-N13) and investigated its potential to stimulate cytokine and total/specific antibody responses in BALB/c and challenge experiment in IFNAR-/- mice. Due to lack of sufficient antibody stimulation towards N protein, we have selected CD24 protein as a potential adjuvant which has proliferative effect on B and T cells. Overall, our N expressing construct when administered solely or in combination with pCD24 vector elicited significant cellular and humoral responses in BALB/c, despite variations in particular cytokines and total antibodies. However, the stimulated antibodies produced due to expression of N protein have shown no neutralizing ability in VNA. Furthermore, challenge experiments were revealed protection potential of N expressing construct in IFNAR -/- mice model. In conclusion, we have shown that CD24 has prominent effect as a genetic adjuvant when co-delivers with a synergic foreign gene expressing vector. Besides, targeting of S segment of CCHFV can be considered as a practical way in developing vaccine against this virus due to its ability to induce immune response which leads to protection in challenge assays in IFN-gamma defective mice models.

scholarly journals Lethal Encephalitis of Unknown Origin—Elucidation by Metagenomics

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 59
Author(s):  
Martin Beer
Keyword(s):  
Point Of Care ◽  
Animal Health ◽  
Unknown Origin ◽  
Emerging Viruses ◽  
Viral Pathogens ◽  
Point Of Care Diagnostics ◽  
Close Relationship ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Novel and (re)emerging viruses cause frequent threats to both human and animal health. Diagnostic metagenomics using unbiased next-generation sequencing (NGS) is the key method for the identification of new pathogens. With today’s available state-of-the-art platforms, NGS can be broadly used to also identify novel and unknown pathogens in different sample materials (even in point-of-care diagnostics) or to characterize the complete genomes of all types of pathogens. Nevertheless, the extreme numbers of sequence fragments resulting from NGS analyses requires not only novel diagnostic pipelines including powerful software tools for big data analysis but also a new dimension of knowledge and resources. We therefore developed and validated a universal workflow for metagenome diagnostics for the analysis of disease syndromes in both animals and humans. The metagenomics pipeline will be presented, and several examples with the detection and characterization of novel viruses will be shown. The power of diagnostic metagenomics will be presented with different examples focusing on lethal encephalitis cases in both animals and humans where we were able to identify a series of novel or unexpected viral pathogens. Furthermore, the detection of zoonotic pathogens was only possible by a “one-health” approach and the close relationship between veterinary and human medicine. The major aim of the presentation is to give an idea about the capabilities of modern NGS-based metagenomics and to learn more about the newly detected viral pathogens. Since a large proportion of severe encephalitis cases still remain unexplained, a main conclusion is the recommendation that those cases should be analyzed by using a modern and powerful metagenomics workflow.

scholarly journals Generation and Characterization of DNA Vaccines Targeting the Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus

2004 ◽  
Vol 78 (9) ◽  
pp. 4638-4645 ◽  
Author(s):  
Tae Woo Kim ◽  
Jin Hyup Lee ◽  
Chien-Fu Hung ◽  
Shiwen Peng ◽  
Richard Roden ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Immune Responses ◽  
Vaccine Development ◽  
Dna Vaccines ◽  
Global Scale ◽  
Target Antigen ◽  
Humoral And Cellular Immunity ◽  
N Protein ◽  
Histocompatibility Complex

ABSTRACT Severe acute respiratory syndrome (SARS) is a serious threat to public health and the economy on a global scale. The SARS coronavirus (SARS-CoV) has been identified as the etiological agent for SARS. Thus, vaccination against SARS-CoV may represent an effective approach to controlling SARS. DNA vaccines are an attractive approach for SARS vaccine development, as they offer many advantages over conventional vaccines, including stability, simplicity, and safety. Our investigators have previously shown that DNA vaccination with antigen linked to calreticulin (CRT) dramatically enhances major histocompatibility complex class I presentation of linked antigen to CD8+ T cells. In this study, we have employed this CRT-based enhancement strategy to create effective DNA vaccines using SARS-CoV nucleocapsid (N) protein as a target antigen. Vaccination with naked CRT/N DNA generated the most potent N-specific humoral and T-cell-mediated immune responses in vaccinated C57BL/6 mice among all of the DNA constructs tested. Furthermore, mice vaccinated with CRT/N DNA were capable of significantly reducing the titer of challenging vaccinia virus expressing the N protein of the SARS virus. These results show that a DNA vaccine encoding CRT linked to a SARS-CoV antigen is capable of generating strong N-specific humoral and cellular immunity and may potentially be useful for control of infection with SARS-CoV.

scholarly journals Contribution of Nanotechnology in the Fight Against COVID-19

2020 ◽  
Vol 11 (1) ◽  
pp. 8233-8241 ◽  
Keyword(s):  
Rapid Detection ◽  
Vaccine Development ◽  
Point Of Care ◽  
Advanced Technology ◽  
Development Research ◽  
New Materials ◽  
The Earth ◽  
Point Of Care Diagnostics ◽  
Funding Agencies ◽  
New Research

Coronavirus disease (COVID-19) is a respiratory infectious disease caused by a newly discovered virus strain, severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2). This pandemic spread quickly across nations with a high mortality rate in immunocompromised patients. This contagious disease posed a serious threat to health systems. It impacted the continents of the earth in a way that could not have been predicted. Therefore, many leading funding agencies announced the call for proposal to diagnosis and treatment of COVID-19 pandemic using advanced technology-based methods, including nanotechnology. The researchers coming from the nanotechnology community can contribute their efforts to cope with COVID-19. As a community member of nanotechnology, we suggest some new research targets that can be designed and improved, optimized, and developed the existing/new materials in the sub-field of diagnostics and healthcare of nanotechnology. The potential research targets to fight against COVID-19 includes Point-of-care diagnostics (POCD), surveillance and monitoring, novel therapeutics, vaccine development, research, and development, repurposing existing drugs with potential therapeutic applications, development of antiviral nanocoating/antimicrobial spray-based coating for PPE, magnetic nanoparticles and viral RNA and rapid detection kits.

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