Electronic Detection of SARS-CoV-2 N-Protein Before the Onset of Symptoms

AbstractExcessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

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Structure and Function of Major SARS-CoV-2 and SARS-CoV Proteins

Bioinformatics and Biology Insights ◽

10.1177/11779322211025876 ◽

2021 ◽

Vol 15 ◽

pp. 117793222110258

Author(s):

Ritesh Gorkhali ◽

Prashanna Koirala ◽

Sadikshya Rijal ◽

Ashmita Mainali ◽

Adesh Baral ◽

...

Keyword(s):

Genomic Organization ◽

Membrane Vesicles ◽

Structural Proteins ◽

Host Immune System ◽

Accessory Proteins ◽

Nonstructural Proteins ◽

N Protein ◽

Small Proteins ◽

Methylation Mark ◽

And Function

SARS-CoV-2 virus, the causative agent of COVID-19 pandemic, has a genomic organization consisting of 16 nonstructural proteins (nsps), 4 structural proteins, and 9 accessory proteins. Relative of SARS-CoV-2, SARS-CoV, has genomic organization, which is very similar. In this article, the function and structure of the proteins of SARS-CoV-2 and SARS-CoV are described in great detail. The nsps are expressed as a single or two polyproteins, which are then cleaved into individual proteins using two proteases of the virus, a chymotrypsin-like protease and a papain-like protease. The released proteins serve as centers of virus replication and transcription. Some of these nsps modulate the host’s translation and immune systems, while others help the virus evade the host immune system. Some of the nsps help form replication-transcription complex at double-membrane vesicles. Others, including one RNA-dependent RNA polymerase and one exonuclease, help in the polymerization of newly synthesized RNA of the virus and help minimize the mutation rate by proofreading. After synthesis of the viral RNA, it gets capped. The capping consists of adding GMP and a methylation mark, called cap 0 and additionally adding a methyl group to the terminal ribose called cap1. Capping is accomplished with the help of a helicase, which also helps remove a phosphate, two methyltransferases, and a scaffolding factor. Among the structural proteins, S protein forms the receptor of the virus, which latches on the angiotensin-converting enzyme 2 receptor of the host and N protein binds and protects the genomic RNA of the virus. The accessory proteins found in these viruses are small proteins with immune modulatory roles. Besides functions of these proteins, solved X-ray and cryogenic electron microscopy structures related to the function of the proteins along with comparisons to other coronavirus homologs have been described in the article. Finally, the rate of mutation of SARS-CoV-2 residues of the proteome during the 2020 pandemic has been described. Some proteins are mutated more often than other proteins, but the significance of these mutation rates is not fully understood.

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Persistent COVID-19 Symptoms Minimally Impact the Development of SARS-CoV-2-Specific T Cell Immunity

Viruses ◽

10.3390/v13050916 ◽

2021 ◽

Vol 13 (5) ◽

pp. 916

Author(s):

Hengsheng Fang ◽

Adam D. Wegman ◽

Kianna Ripich ◽

Heather Friberg ◽

Jeffrey R. Currier ◽

...

Keyword(s):

Cellular Immunity ◽

Viral Infections ◽

T Cell Immunity ◽

N Protein ◽

Cell Immunity ◽

Public Health Challenge ◽

Human Coronaviruses ◽

Cellular Immune ◽

Persistent Viral Infections ◽

Insight Into

SARS-CoV-2 represents an unprecedented public health challenge. While the majority of SARS-CoV-2-infected individuals with mild-to-moderate COVID-19 resolve their infection with few complications, some individuals experience prolonged symptoms lasting for weeks after initial diagnosis. Persistent viral infections are commonly accompanied by immunologic dysregulation, but it is unclear if persistent COVID-19 impacts the development of virus-specific cellular immunity. To this end, we analyzed SARS-CoV-2-specific cellular immunity in convalescent COVID-19 patients who experienced eight days or fewer of COVID-19 symptoms or symptoms persisting for 18 days or more. We observed that persistent COVID-19 symptoms were not associated with the development of an overtly dysregulated cellular immune response. Furthermore, we observed that reactivity against the N protein from SARS-CoV-2 correlates with the amount of reactivity against the seasonal human coronaviruses 229E and NL63. These results provide insight into the processes that regulate the development of cellular immunity against SARS-CoV-2 and related human coronaviruses.

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N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection

Nature Communications ◽

10.1038/s41467-021-22072-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dandan Shan ◽

Joseph M. Johnson ◽

Syrena C. Fernandes ◽

Hannah Suib ◽

Soyoon Hwang ◽

...

Keyword(s):

Single Molecule ◽

High Performance ◽

Point Of Care ◽

Capillary Blood ◽

Molecular Testing ◽

Sample Collection ◽

Protein Assay ◽

N Protein ◽

Protein Levels ◽

Percent Agreement

AbstractThe COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1–7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

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Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

iScience ◽

10.1016/j.isci.2021.102681 ◽

2021 ◽

pp. 102681

Author(s):

Chao Wu ◽

Abraham J. Qavi ◽

Asmaa Hachim ◽

Niloufar Kavian ◽

Aidan R. Cole ◽

...

Keyword(s):

N Protein ◽

Terminal Domain ◽

Functional Consequences

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The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic

Gene ◽

10.1016/0378-1119(79)90011-8 ◽

1979 ◽

Vol 8 (1) ◽

pp. 107-119 ◽

Cited By ~ 49

Author(s):

N.C. Franklin ◽

G.N. Bennett

Keyword(s):

Dna Sequence ◽

Bacteriophage Lambda ◽

N Protein

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The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

Nature Communications ◽

10.1038/s41467-020-20768-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shan Lu ◽

Qiaozhen Ye ◽

Digvijay Singh ◽

Yong Cao ◽

Jolene K. Diedrich ◽

...

Keyword(s):

Phase Separation ◽

Potential Role ◽

Rna Binding ◽

Stress Granule ◽

M Protein ◽

Rich Region ◽

Virion Assembly ◽

N Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

AbstractThe multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

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Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)61563-7 ◽

1987 ◽

Vol 262 (6) ◽

pp. 2696-2703 ◽

Cited By ~ 7

Author(s):

M.R. Maurizi

Keyword(s):

Escherichia Coli ◽

Bacteriophage Lambda ◽

Lon Protease ◽

N Protein

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SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽

10.3390/cells10030530 ◽

2021 ◽

Vol 10 (3) ◽

pp. 530

Author(s):

Soo Jin Oh ◽

Ok Sarah Shin

Keyword(s):

Nucleocapsid Protein ◽

Nuclear Translocation ◽

Nonstructural Protein ◽

Poly I:C ◽

Interferon Response ◽

Type I ◽

Antiviral Immune Response ◽

N Protein ◽

Nonstructural Protein 1 ◽

Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

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