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1.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1968842

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
2.
Nucleic Acids Res ; 50(14): 8168-8192, 2022 08 12.
Article in English | MEDLINE | ID: covidwho-1961119

ABSTRACT

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.


Subject(s)
Coronavirus Nucleocapsid Proteins , RNA, Double-Stranded , SARS-CoV-2 , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Phosphoproteins/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/genetics , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , Temperature
3.
J Struct Biol ; 214(3): 107879, 2022 09.
Article in English | MEDLINE | ID: covidwho-1914725

ABSTRACT

14-3-3 proteins are important dimeric scaffolds that regulate the function of hundreds of proteins in a phosphorylation-dependent manner. The SARS-CoV-2 nucleocapsid (N) protein forms a complex with human 14-3-3 proteins upon phosphorylation, which has also been described for other coronaviruses. Here, we report a high-resolution crystal structure of 14-3-3 bound to an N phosphopeptide bearing the phosphoserine 197 in the middle. The structure revealed two copies of the N phosphopeptide bound, each in the central binding groove of each 14-3-3 monomer. A complex network of hydrogen bonds and water bridges between the peptide and 14-3-3 was observed explaining the high affinity of the N protein for 14-3-3 proteins.


Subject(s)
14-3-3 Proteins , Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , 14-3-3 Proteins/chemistry , COVID-19 , Coronavirus Nucleocapsid Proteins/chemistry , Humans , Phosphopeptides/chemistry , Phosphoproteins/chemistry , Protein Binding
4.
PLoS Comput Biol ; 18(5): e1010121, 2022 05.
Article in English | MEDLINE | ID: covidwho-1846916

ABSTRACT

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.


Subject(s)
Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , COVID-19 , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Microscopy, Electron , Molecular Dynamics Simulation , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
5.
Int J Mol Sci ; 23(6)2022 Mar 10.
Article in English | MEDLINE | ID: covidwho-1742485

ABSTRACT

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.


Subject(s)
Coronavirus Nucleocapsid Proteins/immunology , Coronavirus OC43, Human/immunology , Cross Reactions/immunology , Epitope Mapping/methods , Epitopes, B-Lymphocyte/immunology , SARS-CoV-2/immunology , Adult , Amino Acid Sequence , COVID-19/immunology , COVID-19/virology , Cohort Studies , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus OC43, Human/genetics , Coronavirus OC43, Human/physiology , Cross Reactions/genetics , Enzyme-Linked Immunosorbent Assay/methods , Epitopes, B-Lymphocyte/metabolism , HEK293 Cells , Health Personnel/statistics & numerical data , Humans , Protein Domains , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
6.
J Mol Biol ; 434(9): 167516, 2022 05 15.
Article in English | MEDLINE | ID: covidwho-1712817

ABSTRACT

Stress granule (SG) formation mediated by Ras GTPase-activating protein-binding protein 1 (G3BP1) constitutes a key obstacle for viral replication, which makes G3BP1 a frequent target for viruses. For instance, the SARS-CoV-2 nucleocapsid (N) protein interacts with G3BP1 directly to suppress SG assembly and promote viral production. However, the molecular basis for the SARS-CoV-2 N - G3BP1 interaction remains elusive. Here we report biochemical and structural analyses of the SARS-CoV-2 N - G3BP1 interaction, revealing differential contributions of various regions of SARS-CoV-2 N to G3BP1 binding. The crystal structure of the NTF2-like domain of G3BP1 (G3BP1NTF2) in complex with a peptide derived from SARS-CoV-2 N (residues 1-25, N1-25) reveals that SARS-CoV-2 N1-25 occupies a conserved surface groove of G3BP1NTF2 via surface complementarity. We show that a φ-x-F (φ, hydrophobic residue) motif constitutes the primary determinant for G3BP1NTF2-targeting proteins, while the flanking sequence underpins diverse secondary interactions. We demonstrate that mutation of key interaction residues of the SARS-CoV-2 N1-25 - G3BP1NTF2 complex leads to disruption of the SARS-CoV-2 N - G3BP1 interaction in vitro. Together, these results provide a molecular basis of the strain-specific interaction between SARS-CoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection.


Subject(s)
Coronavirus Nucleocapsid Proteins , DNA Helicases , Poly-ADP-Ribose Binding Proteins , Protein Interaction Domains and Motifs , RNA Helicases , SARS-CoV-2 , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Crystallography , DNA Helicases/chemistry , Humans , Mutation , Phosphoproteins/chemistry , Phosphoproteins/genetics , Poly-ADP-Ribose Binding Proteins/chemistry , RNA Helicases/chemistry , RNA Recognition Motif Proteins/chemistry
7.
PLoS One ; 17(1): e0262868, 2022.
Article in English | MEDLINE | ID: covidwho-1643287

ABSTRACT

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.


Subject(s)
Antibodies, Viral/chemistry , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/epidemiology , COVID-19 Serological Testing/standards , Coronavirus Nucleocapsid Proteins/chemistry , Epidemiological Monitoring , Female , Humans , Immunoglobulin A/chemistry , Immunoglobulin A/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin M/chemistry , Immunoglobulin M/immunology , Male , Middle Aged , New York/epidemiology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2/classification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry
8.
Virology ; 567: 1-14, 2022 02.
Article in English | MEDLINE | ID: covidwho-1628759

ABSTRACT

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Intracellular Membranes/metabolism , Viral Replicase Complex Proteins/metabolism , Amino Acid Motifs , Animals , Cell Line , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Mice , Murine hepatitis virus , Mutation , Protein Binding , Protein Domains , RNA, Viral/biosynthesis , Viral Replicase Complex Proteins/chemistry , Viral Replicase Complex Proteins/genetics , Viral Replication Compartments/metabolism
9.
Sci Rep ; 12(1): 1060, 2022 01 20.
Article in English | MEDLINE | ID: covidwho-1639278

ABSTRACT

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.


Subject(s)
Coronavirus Nucleocapsid Proteins , Gold/chemistry , Metal Nanoparticles/chemistry , SARS-CoV-2/chemistry , Surface Plasmon Resonance , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/chemistry , Phosphoproteins/analysis , Phosphoproteins/chemistry
10.
Int J Biol Macromol ; 200: 428-437, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1633983

ABSTRACT

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.


Subject(s)
COVID-19 Testing/methods , Coronavirus Nucleocapsid Proteins/chemistry , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Circular Dichroism , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Dimerization , Epitopes/chemistry , Escherichia coli/genetics , Humans , Immunoglobulin G/immunology , Models, Molecular , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sensitivity and Specificity
11.
Nature ; 602(7897): 487-495, 2022 02.
Article in English | MEDLINE | ID: covidwho-1585830

ABSTRACT

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.


Subject(s)
COVID-19/immunology , COVID-19/virology , Evolution, Molecular , Immune Evasion , Immunity, Innate/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , COVID-19/transmission , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Immunity, Innate/genetics , Interferons/immunology , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Proteomics , RNA, Viral/genetics , RNA-Seq , SARS-CoV-2/classification , SARS-CoV-2/growth & development
12.
ACS Appl Mater Interfaces ; 13(50): 60612-60624, 2021 Dec 22.
Article in English | MEDLINE | ID: covidwho-1569206

ABSTRACT

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Nasal Mucosa/virology , Polymers/chemistry , RNA, Viral/metabolism , SARS-CoV-2 , Biofouling , Biological Assay , Biosensing Techniques , Humans , Ions , Limit of Detection , Mass Spectrometry , Nasopharynx/virology , Phosphoproteins/chemistry , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Specimen Handling
13.
Infect Genet Evol ; 96: 105155, 2021 12.
Article in English | MEDLINE | ID: covidwho-1525880

ABSTRACT

The present study aimed to predict the binding potential of carbon nanotube and nano fullerene towards multiple targets of SARS-CoV-2. Based on the virulent functions, the spike glycoprotein, RNA-dependent RNA polymerase, main protease, papain-like protease, and RNA binding domain of the nucleocapsid proteins of SARS-CoV-2 were prioritized as the molecular targets and their three-dimensional (3D) structures were retrieved from the Protein Data Bank. The 3D structures of carbon nanotubes and nano-fullerene were computationally modeled, and the binding potential of these nanoparticles to the selected molecular targets was predicted by molecular docking and molecular dynamic (MD) simulations. The drug-likeness and pharmacokinetic features of the lead molecules were computationally predicted. The current study suggested that carbon fullerene and nanotube demonstrated significant binding towards the prioritized multi-targets of SARS-CoV-2. Interestingly, carbon nanotube showed better interaction with these targets when compared to carbon fullerene. MD simulation studies clearly showed that the interaction of nanoparticles and selected targets possessed stability and conformational changes. This study revealed that carbon nanotubes and fullerene are probably used as effectual binders to multiple targets of SARS-CoV-2, and the study offers insights into the experimental validation and highlights the relevance of utilizing carbon nanomaterials as a therapeutic remedy against COVID-19.


Subject(s)
Fullerenes/metabolism , Nanotubes, Carbon , SARS-CoV-2/metabolism , Viral Proteins/chemistry , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Fullerenes/chemistry , Fullerenes/pharmacokinetics , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Nanotubes, Carbon/chemistry , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Viral Proteins/metabolism
14.
FEBS Lett ; 595(23): 2872-2896, 2021 12.
Article in English | MEDLINE | ID: covidwho-1516705

ABSTRACT

The current work investigated SARS-CoV-2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC-based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress-stimulated NCAP to undergo liquid-liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , /metabolism , A549 Cells , Adaptor Proteins, Signal Transducing/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , DNA Helicases/metabolism , Humans , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Binding , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , RNA-Binding Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , eIF-2 Kinase/metabolism
15.
Cell Host Microbe ; 29(12): 1788-1801.e6, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1509671

ABSTRACT

Previous work found that the co-occurring mutations R203K/G204R on the SARS-CoV-2 nucleocapsid (N) protein are increasing in frequency among emerging variants of concern or interest. Through a combination of in silico analyses, this study demonstrates that R203K/G204R are adaptive, while large-scale phylogenetic analyses indicate that R203K/G204R associate with the emergence of the high-transmissibility SARS-CoV-2 lineage B.1.1.7. Competition experiments suggest that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly. Moreover, the 203K/204R virus shows increased infectivity in human lung cells and hamsters. Accordingly, we observe a positive association between increased COVID-19 severity and sample frequency of 203K/204R. Our work suggests that the 203K/204R mutations contribute to the increased transmission and virulence of select SARS-CoV-2 variants. In addition to mutations in the spike protein, mutations in the nucleocapsid protein are important for viral spreading during the pandemic.


Subject(s)
Amino Acid Substitution , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Genome, Viral , Mutation , SARS-CoV-2/genetics , Animals , COVID-19/epidemiology , COVID-19/virology , Cell Line , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Cricetulus , Epithelial Cells/pathology , Epithelial Cells/virology , Gene Expression , Genetic Fitness , Humans , Models, Molecular , Mutagenesis , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phylogeny , Protein Conformation , SARS-CoV-2/classification , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , Selection, Genetic , Severity of Illness Index , Virion/genetics , Virion/growth & development , Virion/pathogenicity , Virulence , Virus Replication
16.
J Virol ; 95(16): e0018721, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1486048

ABSTRACT

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Nucleocapsid Proteins/chemistry , Host-Pathogen Interactions/drug effects , Porcine epidemic diarrhea virus/drug effects , Quercetin/analogs & derivatives , Tumor Suppressor Protein p53/chemistry , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Binding Sites , Cell Line , Chlorocebus aethiops , Coronavirus Infections/drug therapy , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Gene Expression Regulation , High-Throughput Screening Assays , Host-Pathogen Interactions/genetics , Molecular Docking Simulation , Nuclear Localization Signals , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Quercetin/chemistry , Quercetin/pharmacology , S Phase Cell Cycle Checkpoints/drug effects , S Phase Cell Cycle Checkpoints/genetics , Signal Transduction , Swine , Swine Diseases/drug therapy , Swine Diseases/genetics , Swine Diseases/metabolism , Swine Diseases/virology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Vero Cells , Virus Replication/drug effects
17.
FEBS J ; 289(13): 3813-3825, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1476183

ABSTRACT

The ongoing outbreak of COVID-19 caused by SARS-CoV-2 has resulted in a serious public health threat globally. Nucleocapsid protein is a major structural protein of SARS-CoV-2 that plays important roles in the viral RNA packing, replication, assembly, and infection. Here, we report two crystal structures of nucleocapsid protein C-terminal domain (CTD) at resolutions of 2.0 Å and 3.1 Å, respectively. These two structures, crystallized under different conditions, contain 2 and 12 CTDs in asymmetric unit, respectively. Interestingly, despite different crystal packing, both structures show a similar dimeric form as the smallest unit, consistent with its solution form measured by the size-exclusion chromatography, suggesting an important role of CTD in the dimerization of nucleocapsid proteins. By analyzing the surface charge distribution, we identified a stretch of positively charged residues between Lys257 and Arg262 that are involved in RNA-binding. Through screening a single-domain antibodies (sdAbs) library, we identified four sdAbs targeting different regions of nucleocapsid protein with high affinities that have future potential to be used in viral detection and therapeutic purposes.


Subject(s)
Coronavirus Nucleocapsid Proteins , Single-Domain Antibodies , Amino Acid Sequence , Coronavirus Nucleocapsid Proteins/chemistry , Nucleocapsid/chemistry , Phosphoproteins/chemistry , SARS-CoV-2 , Single-Domain Antibodies/chemistry
18.
Glycobiology ; 31(9): 1080-1092, 2021 09 20.
Article in English | MEDLINE | ID: covidwho-1434394

ABSTRACT

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


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Protein Processing, Post-Translational/physiology , SARS-CoV-2/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Coronavirus Nucleocapsid Proteins/chemistry , Glycosylation , HEK293 Cells , Humans , Phosphorylation , SARS-CoV-2/chemistry
19.
Chem Commun (Camb) ; 57(79): 10222-10225, 2021 Oct 05.
Article in English | MEDLINE | ID: covidwho-1408635

ABSTRACT

We developed a one-minute, one-step SARS-CoV-2 antigen assay based on protein-induced fluorescence enhancement of a DNA aptamer. The system showed significant selectivity and sensitivity towards both nucleocapsid protein and SARS-CoV-2 virus lysate, but with marked improvements in speed and manufacturability. We hence propose this platform as a mix-and-read testing strategy for SARS-CoV-2 that can be applied to POC diagnostics in clinical settings, especially in low- and middle-income countries.


Subject(s)
Antigens, Viral/chemistry , Aptamers, Nucleotide/chemistry , COVID-19 Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2 , Biological Assay , Carbocyanines/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Phosphoproteins/chemistry
20.
Mol Syst Biol ; 17(9): e10079, 2021 09.
Article in English | MEDLINE | ID: covidwho-1406892

ABSTRACT

We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further ˜29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Host-Pathogen Interactions/genetics , Protein Processing, Post-Translational , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Transport Systems, Neutral/chemistry , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Binding Sites , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , Computational Biology/methods , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Models, Molecular , Molecular Mimicry , Neuropilin-1/chemistry , Neuropilin-1/genetics , Neuropilin-1/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Multimerization , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viroporin Proteins/chemistry , Viroporin Proteins/genetics , Viroporin Proteins/metabolism , Virus Replication
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