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1.
Biophys J ; 120(14): 2890-2901, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1604873

ABSTRACT

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.


Subject(s)
COVID-19 , SARS-CoV-2 , Coronavirus Nucleocapsid Proteins , Humans , Nucleocapsid/metabolism , Phosphoproteins , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism
2.
Nat Commun ; 12(1): 502, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1387327

ABSTRACT

The 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.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Matrix Proteins/metabolism , COVID-19/genetics , COVID-19/metabolism , Cell Membrane/virology , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , Humans , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Binding , Protein Domains , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Recognition Motif Proteins/genetics , RNA Recognition Motif Proteins/metabolism , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics
3.
BMC Res Notes ; 14(1): 10, 2021 Jan 06.
Article in English | MEDLINE | ID: covidwho-1388820

ABSTRACT

OBJECTIVE: This study describes the occurrence of a silent mutation in the RNA binding domain of nucleocapsid phosphoprotein (N protein) coding gene from SARS-CoV-2 that may consequence to a missense mutation by onset of another single nucleotide mutation. RESULTS: In the DNA sequence isolated from severe acute respiratory syndrome (SARS-CoV-2) in Iran, a coding sequence for the RNA binding domain of N protein was detected. The comparison of Chinese and Iranian DNA sequences displayed that a thymine (T) was mutated to cytosine (C), so "TTG" from China was changed to "CTG" in Iran. Both DNA sequences from Iran and China have been encoded for leucine. In addition, the second T in "CTG" in the DNA or uracil (U) in "CUG" in the RNA sequences from Iran can be mutated to another C by a missense mutation resulting from thymine DNA glycosylase (TDG) of human and base excision repair mechanism to produce "CCG" encoding for proline, which consequently may increase the affinity of the RNA binding domain of N protein to viral RNA and improve the transcription rate, pathogenicity, evasion from human immunity system, spreading in the human body, and risk of human-to-human transmission rate of SARS-CoV-2.


Subject(s)
COVID-19/genetics , Coronavirus Nucleocapsid Proteins/genetics , RNA, Viral/genetics , RNA-Binding Motifs/genetics , SARS-CoV-2/genetics , China , Databases, Genetic , Humans , Iran , Mutation, Missense , Phosphoproteins/genetics , Sequence Analysis, DNA , Silent Mutation
4.
J Biomol Struct Dyn ; 39(12): 4433-4448, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1317842

ABSTRACT

The emergence of the coronavirus disease-2019 pandemic has led to an outbreak in the world. The SARS-CoV-2 is seventh and latest in coronavirus family with unique exonucleases for repairing any mismatches in newly transcribed genetic material. Therefore, drugs with novel additional mechanisms are required to simultaneously target and eliminate the virus. Thus, a newly deciphered N protein is taken as a target that belongs to SARS-CoV-2. They play a vital role in RNA transcription, viral replication and new virion formation. This study used virtual screening, molecular modeling and docking of the 8987 ligands from Asinex and PubChem databases against this novel target protein. Three hotspot sites having DScore ≥1 (Site 1, Site 2 and Site 3) for ligand binding were selected. Subsequently, high throughput screening, standard precision and extra precision docking process and molecular dynamics concluded three best drugs from two libraries. Two antiviral moieties from Asinex databases (5817 and 6799) have docking scores of -10.29 and -10.156; along with their respective free binding energies (ΔG bind) of -51.96 and -64.36 on Site 3. The third drug, Zidovudine, is from PubChem database with docking scores of -9.75 with its binding free energies (ΔG bind) of -59.43 on Site 3. The RMSD and RMSF were calculated for all the three drugs through molecular dynamics simulation studies for 50 ns. Zidovudine shows a very stable interaction with fluctuation starting at 2.4 Å on 2 ns and remained stable at 3 Å from 13 to 50 ns. Thus, paving the way for further biological validation as a potential treatment.Communicated by Ramaswamy H. Sarma.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Molecular Docking Simulation , Nucleocapsid , Phosphoproteins , RNA-Binding Motifs , Virion
5.
J Biomol Struct Dyn ; 39(12): 4243-4255, 2021 08.
Article in English | MEDLINE | ID: covidwho-1317834

ABSTRACT

Recent outbreak of novel Coronavirus disease () pandemic around the world is associated with severe acute respiratory syndrome. The death toll associated with the pandemic is increasing day by day. SARS-CoV-2 is an enveloped virus and its N terminal domain (NTD) of Nucleocapsid protein (N protein) binds to the viral (+) sense RNA and results in virus ribonucleoprotien complex, essential for the virus replication. The N protein is composed of a serine-rich linker region sandwiched between NTD and C terminal (CTD). These terminals play a role in viral entry and its processing post entry. The NTD of SARS-CoV-2 N protein forms orthorhombic crystals and binds to the viral genome. Therefore, there is always a quest to target RNA binding domain of nucleocapsid phosphoprotein (NTD-N-protein which in turn may help in controlling diseases caused by SARS-CoV-2 in humans. The role of Chloroquine and Hydroxychloroquine as potential treatments for is still under debate globally because of some side effects associated with it. This study involves the In silico interactions of Chloroquine and Hydroxychloroquine with the NTD-N-protein of SARS-CoV-2. With the help of various computational methods, we have explored the potential role of both of these antiviral drugs for the treatment of patients by comparing the efficacy of both of the drugs to bind to NTD-N-protein. In our research Hydroxychloroquine exhibited potential inhibitory effects of NTD-N-protein with binding energy -7.28 kcal/mol than Chloroquine (-6.30 kcal/mol) at SARS-CoV-2 receptor recognition of susceptible cells. The outcomes of this research strongly appeal for in vivo trials of Hydroxychloroquine for the patients infected with . Furthermore, the recommended doses of Hydroxychloroquine may reduce the chances of catching to the healthcare workers and staff who are in contact with or delivering direct care to coronavirus patients as long as they have not been diagnosed with . We further hypothesize that the comparative NTD-N-protein -drug docking interactions may help to understand the comparative efficacy of other candidate repurposing drugs until discovery of a proper vaccine.Communicated by Ramaswamy H. Sarma.


Subject(s)
COVID-19 , Hydroxychloroquine , Antiviral Agents/pharmacology , COVID-19/drug therapy , Chloroquine/pharmacology , Computer Simulation , Drug Repositioning , Humans , Hydroxychloroquine/pharmacology , Nucleocapsid , Nucleocapsid Proteins , RNA-Binding Motifs , SARS-CoV-2
6.
Netw Model Anal Health Inform Bioinform ; 10(1): 44, 2021.
Article in English | MEDLINE | ID: covidwho-1265590

ABSTRACT

Coronavirus disease 2019 (COVID-19) is caused by a Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2), which is a positive-strand RNA virus. The SARS-CoV-2 genome and its association to SAR-CoV-1 vary from ca. 66 to 96% depending on the type of betacoronavirideae family members. With several drugs, viz. chloroquine, hydroxychloroquine, ivermectin, artemisinin, remdesivir, azithromycin considered for clinical trials, there has been an inherent need to find distinctive antiviral mechanisms of these drugs. Curcumin, a natural bioactive molecule has been shown to have therapeutic potential for various diseases, and its effect on COVID-19 is also currently being explored. In this study, we show the binding potential of curcumin targeted to a variety of SARS-CoV-2 proteins, viz. spike glycoproteins (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), spike protein-ACE2 (PDB ID: 6M17) along with nsp10 (PDB ID: 6W4H) and RNA dependent RNA polymerase (PDB ID: 6M71) structures. Furthermore, representative docking complexes were validated using molecular dynamics simulations and mechanistic studies at 100 ns was carried on nucleocapsid and nsp10 proteins with curcumin complexes which resulted in stable and efficient binding energies and correlated with that of docked binding energies of the complexes. Both the docking and simulation studies indicate that curcumin has the potential as an antiviral against COVID-19.

7.
BMC Genomics ; 22(1): 371, 2021 May 20.
Article in English | MEDLINE | ID: covidwho-1238703

ABSTRACT

BACKGROUND: Brazil is the third country most affected by Coronavirus disease-2019 (COVID-19), but viral evolution in municipality resolution is still poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We aimed to track molecular evolution and spread of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Esteio (Southern Brazil) using phylogenetics and phylodynamics inferences from 21 new genomes in global and regional context. Importantly, the case fatality rate (CFR) in Esteio (3.26%) is slightly higher compared to the Rio Grande do Sul (RS) state (2.56%) and the entire Brazil (2.74%). RESULTS: We provided a comprehensive view of mutations from a representative sampling from May to October 2020, highlighting two frequent mutations in spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in spike Receptor Binding Domain (RBD) characteristic of the B.1.351 and P.1 lineages, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). E484K was found in two genomes from mid-October, which is the earliest description of this mutation in Southern Brazil. Lineages containing this substitution must be subject of intense surveillance due to its association with immune evasion. We also found two epidemiologically-related clusters, including one from patients of the same neighborhood. Phylogenetics and phylodynamics analysis demonstrates multiple introductions of the Brazilian most prevalent lineages (B.1.1.33 and B.1.1.248) and the establishment of Brazilian lineages ignited from the Southeast to other Brazilian regions. CONCLUSIONS: Our data show the value of correlating clinical, epidemiological and genomic information for the understanding of viral evolution and its spatial distribution over time. This is of paramount importance to better inform policy making strategies to fight COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Brazil/epidemiology , Genome, Viral , Genomics , Humans
8.
J Med Virol ; 93(4): 2406-2419, 2021 04.
Article in English | MEDLINE | ID: covidwho-1227754

ABSTRACT

The analyses of 2325 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes revealed 107, 162, and 65 nucleotide substitutions in the coding region of SARS-CoV-2 from the three continents America, Europe, and Asia, respectively. Of these nucleotide substitutions 58, 94, and 37 were nonsynonymous types mostly present in the Nsp2, Nsp3, Spike, and ORF9. A continent-specific phylogram analyses clustered the SARS-CoV-2 in the different group based on the frequency of nucleotide substitutions. Detailed analyses about the continent-specific amino acid changes and their effectiveness by SNAP2 software was investigated. We found 11 common nonsynonymous mutations; among them, two novel effective mutations were identified in ORF9 (S194L and S202N). Intriguingly, ORF9 encodes nucleocapsid phosphoprotein possessing many effective mutations across continents and could be a potential candidate after the spike protein for studying the role of mutation in viral assembly and pathogenesis. Among the two forms of certain frequent mutation, one form is more prevalent in Europe continents (Nsp12:L314, Nsp13:P504, Nsp13:Y541, Spike:G614, and ORF8:L84) while other forms are more prevalent in American (Nsp12:P314, Nsp13:L504, Nsp13:C541, Spike:D614, and ORF8:L84) and Asian continents (Spike:D614), indicating the spatial and temporal dynamics of SARS-CoV-2. We identified highly conserved 38 regions and among these regions, 11 siRNAs were predicted on stringent criteria that can be used to suppress the expression of viral genes and the corresponding reduction of human viral infections. The present investigation provides information on different mutations and will pave the way for differentiating strains based on virulence and their use in the development of better antiviral therapy.


Subject(s)
COVID-19/virology , Mutation , SARS-CoV-2/genetics , Antiviral Agents/pharmacology , Asia/epidemiology , COVID-19/drug therapy , COVID-19/epidemiology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Papain-Like Proteases/genetics , Europe/epidemiology , Gene Silencing , Genes, Viral , Genome, Viral , Humans , Open Reading Frames , Phosphoproteins/genetics , Phylogeny , RNA, Small Interfering/genetics , SARS-CoV-2/classification , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics
9.
Clin Proteomics ; 18(1): 15, 2021 May 10.
Article in English | MEDLINE | ID: covidwho-1223761

ABSTRACT

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) global pandemic has had a profound, lasting impact on the world's population. A key aspect to providing care for those with COVID-19 and checking its further spread is early and accurate diagnosis of infection, which has been generally done via methods for amplifying and detecting viral RNA molecules. Detection and quantitation of peptides using targeted mass spectrometry-based strategies has been proposed as an alternative diagnostic tool due to direct detection of molecular indicators from non-invasively collected samples as well as the potential for high-throughput analysis in a clinical setting; many studies have revealed the presence of viral peptides within easily accessed patient samples. However, evidence suggests that some viral peptides could serve as better indicators of COVID-19 infection status than others, due to potential misidentification of peptides derived from human host proteins, poor spectral quality, high limits of detection etc. METHODS: In this study we have compiled a list of 636 peptides identified from Sudden Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) samples, including from in vitro and clinical sources. These datasets were rigorously analyzed using automated, Galaxy-based workflows containing tools such as PepQuery, BLAST-P, and the Multi-omic Visualization Platform as well as the open-source tools MetaTryp and Proteomics Data Viewer (PDV). RESULTS: Using PepQuery for confirming peptide spectrum matches, we were able to narrow down the 639-peptide possibilities to 87 peptides that were most robustly detected and specific to the SARS-CoV-2 virus. The specificity of these sequences to coronavirus taxa was confirmed using Unipept and BLAST-P. Through stringent p-value cutoff combined with manual verification of peptide spectrum match quality, 4 peptides derived from the nucleocapsid phosphoprotein and membrane protein were found to be most robustly detected across all cell culture and clinical samples, including those collected non-invasively. CONCLUSION: We propose that these peptides would be of the most value for clinical proteomics applications seeking to detect COVID-19 from patient samples. We also contend that samples harvested from the upper respiratory tract and oral cavity have the highest potential for diagnosis of SARS-CoV-2 infection from easily collected patient samples using mass spectrometry-based proteomics assays.

10.
Anal Chem ; 93(14): 5963-5971, 2021 04 13.
Article in English | MEDLINE | ID: covidwho-1164779

ABSTRACT

Biofouling caused by the accumulation of biomolecules on sensing surfaces is one of the major problems and challenges to realize the practical application of electrochemical biosensors, and an effective way to counter this problem is the construction of antifouling biosensors. Herein, an antifouling electrochemical biosensor was constructed based on electropolymerized polyaniline (PANI) nanowires and newly designed peptides for the detection of the COVID-19 N-gene. The inverted Y-shaped peptides were designed with excellent antifouling properties and two anchoring branches, and their antifouling performances against proteins and complex biological media were investigated using different approaches. Based on the biotin-streptavidin affinity system, biotin-labeled probes specific to the N-gene (nucleocapsid phosphoprotein) of COVID-19 were immobilized onto the peptide-coated PANI nanowires, forming a highly sensitive and antifouling electrochemical sensing interface for the detection of COVID-19 nucleic acid. The antifouling genosensor demonstrated a wide linear range (10-14 to 10-9 M) and an exceptional low detection limit (3.5 fM). The remarkable performance of the genosensor derives from the high peak current of PANI, which is chosen as the sensing signal, and the extraordinary antifouling properties of designed peptides, which guarantee accurate detection in complex systems. These crucial features represent essential elements for future rapid and decentralized clinical testing.


Subject(s)
Biosensing Techniques , Electrochemical Techniques , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , Humans , Molecular Probes , Peptides
11.
Pathogens ; 10(3)2021 Mar 10.
Article in English | MEDLINE | ID: covidwho-1154465

ABSTRACT

Here we describe the first molecular test developed in the early stage of the pandemic to diagnose the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Sardinian patients in February-March 2020, when diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The "Caterina assay" is a SYBR®Green real-time reverse-transcription polymerase chain reaction (rRT-PCR), designed to detect the nucleocapsid phosphoprotein (N) gene that exhibits high discriminative variation RNA sequence among bat and human coronaviruses. The molecular method was applied to detect SARS-CoV-2 in nasal swabs collected from 2110 suspected cases. The study article describes the first molecular test developed in the early stage of the declared pandemic to identify the coronavirus disease 2019 (COVID-19) in Sardinian patients in February-March 2020, when a diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The assay presented high specificity and sensitivity (with a detection limit ≥50 viral genomes/µL). No false-positives were detected, as confirmed by the comparison with two certified commercial kits. Although other validated molecular methods are currently in use, the Caterina assay still represents a valid and low-cost detection procedure that could be applied in countries with limited economic resources.

12.
Biomolecules ; 11(3)2021 03 19.
Article in English | MEDLINE | ID: covidwho-1148287

ABSTRACT

The huge global expansion of the COVID-19 pandemic caused by the novel SARS-corona virus-2 is an extraordinary public health emergency. The unavailability of specific treatment against SARS-CoV-2 infection necessitates the focus of all scientists in this direction. The reported antiviral activities of guanidine alkaloids encouraged us to run a comprehensive in silico binding affinity of fifteen guanidine alkaloids against five different proteins of SARS-CoV-2, which we investigated. The investigated proteins are COVID-19 main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and a non-structural protein (nsp10) (PDB ID: 6W4H). The binding energies for all tested compounds indicated promising binding affinities. A noticeable superiority for the pentacyclic alkaloids particularly, crambescidin 786 (5) and crambescidin 826 (13) has been observed. Compound 5 exhibited very good binding affinities against Mpro (ΔG = -8.05 kcal/mol), nucleocapsid phosphoprotein (ΔG = -6.49 kcal/mol), and nsp10 (ΔG = -9.06 kcal/mol). Compound 13 showed promising binding affinities against Mpro (ΔG = -7.99 kcal/mol), spike glycoproteins (ΔG = -6.95 kcal/mol), and nucleocapsid phosphoprotein (ΔG = -8.01 kcal/mol). Such promising activities might be attributed to the long ω-fatty acid chain, which may play a vital role in binding within the active sites. The correlation of c Log P with free binding energies has been calculated. Furthermore, the SAR of the active compounds has been clarified. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) studies were carried out in silico for the 15 compounds; most examined compounds showed optimal to good range levels of ADMET aqueous solubility, intestinal absorption and being unable to pass blood brain barrier (BBB), non-inhibitors of CYP2D6, non-hepatotoxic, and bind plasma protein with a percentage less than 90%. The toxicity of the tested compounds was screened in silico against five models (FDA rodent carcinogenicity, carcinogenic potency TD50, rat maximum tolerated dose, rat oral LD50, and rat chronic lowest observed adverse effect level (LOAEL)). All compounds showed expected low toxicity against the tested models. Molecular dynamic (MD) simulations were also carried out to confirm the stable binding interactions of the most promising compounds, 5 and 13, with their targets. In conclusion, the examined 15 alkaloids specially 5 and 13 showed promising docking, ADMET, toxicity and MD results which open the door for further investigations for them against SARS-CoV-2.


Subject(s)
Alkaloids/chemistry , Antiviral Agents/chemistry , Coronavirus Nucleocapsid Proteins/chemistry , Porifera/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Animals , Antiviral Agents/pharmacology , Antiviral Agents/toxicity , Blood-Brain Barrier , Crystallography, X-Ray , Ligands , Membrane Glycoproteins/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Phosphoproteins/chemistry , Protease Inhibitors/chemistry , Rats , Software , Viral Proteases/chemistry
13.
Sci Rep ; 11(1): 6614, 2021 03 23.
Article in English | MEDLINE | ID: covidwho-1147848

ABSTRACT

There is a plethora of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) serological tests based either on nucleocapsid phosphoprotein (N), S1-subunit of spike glycoprotein (S1) or receptor binding domain (RBD). Although these single-antigen based tests demonstrate high clinical performance, there is growing evidence regarding their limitations in epidemiological serosurveys. To address this, we developed a Luminex-based multiplex immunoassay that detects total antibodies (IgG/IgM/IgA) against the N, S1 and RBD antigens and used it to compare antibody responses in 1225 blood donors across Greece. Seroprevalence based on single-antigen readouts was strongly influenced by both the antigen type and cut-off value and ranged widely [0.8% (95% CI 0.4-1.5%)-7.5% (95% CI 6.0-8.9%)]. A multi-antigen approach requiring partial agreement between RBD and N or S1 readouts (RBD&N|S1 rule) was less affected by cut-off selection, resulting in robust seroprevalence estimation [0.6% (95% CI 0.3-1.1%)-1.2% (95% CI 0.7-2.0%)] and accurate identification of seroconverted individuals.


Subject(s)
Antigens/immunology , COVID-19/diagnosis , Serologic Tests/methods , Adolescent , Adult , Aged , Antibodies, Viral/blood , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoassay , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Middle Aged , Phosphoproteins/immunology , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology , Young Adult
14.
Biochem Biophys Res Commun ; 538: 54-62, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1125913

ABSTRACT

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/chemistry , RNA-Binding Proteins/chemistry , SARS-CoV-2/genetics , Coronavirus Nucleocapsid Proteins/genetics , Cryoelectron Microscopy , Genome, Viral , Humans , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Binding , Protein Domains , Protein Multimerization , RNA-Binding Proteins/genetics
15.
Pathogens ; 10(3)2021 Mar 10.
Article in English | MEDLINE | ID: covidwho-1125099

ABSTRACT

Here we describe the first molecular test developed in the early stage of the pandemic to diagnose the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Sardinian patients in February-March 2020, when diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The "Caterina assay" is a SYBR®Green real-time reverse-transcription polymerase chain reaction (rRT-PCR), designed to detect the nucleocapsid phosphoprotein (N) gene that exhibits high discriminative variation RNA sequence among bat and human coronaviruses. The molecular method was applied to detect SARS-CoV-2 in nasal swabs collected from 2110 suspected cases. The study article describes the first molecular test developed in the early stage of the declared pandemic to identify the coronavirus disease 2019 (COVID-19) in Sardinian patients in February-March 2020, when a diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The assay presented high specificity and sensitivity (with a detection limit ≥50 viral genomes/µL). No false-positives were detected, as confirmed by the comparison with two certified commercial kits. Although other validated molecular methods are currently in use, the Caterina assay still represents a valid and low-cost detection procedure that could be applied in countries with limited economic resources.

16.
J Biomol Struct Dyn ; : 1-26, 2021 Feb 15.
Article in English | MEDLINE | ID: covidwho-1081511

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the most cryptic pandemic outbreak of the 21st century, has gripped more than 1.8 million people to death and infected almost eighty six million. As it is a new variant of SARS, there is no approved drug or vaccine available against this virus. This study aims to predict some promising cytotoxic T lymphocyte epitopes in the SARS-CoV-2 proteome utilizing immunoinformatic approaches. Firstly, we identified 21 epitopes from 7 different proteins of SARS-CoV-2 inducing immune response and checked for allergenicity and conservancy. Based on these factors, we selected the top three epitopes, namely KAYNVTQAF, ATSRTLSYY, and LTALRLCAY showing functional interactions with the maximum number of MHC alleles and no allergenicity. Secondly, the 3D model of selected epitopes and HLA-A*29:02 were built and Molecular Docking simulation was performed. Most interestingly, the best two epitopes predicted by docking are part of two different structural proteins of SARS-CoV-2, namely Membrane Glycoprotein (ATSRTLSYY) and Nucleocapsid Phosphoprotein (KAYNVTQAF), which are generally target of choice for vaccine designing. Upon Molecular Docking, interactions between selected epitopes and HLA-A*29:02 were further validated by 50 ns Molecular Dynamics (MD) simulation. Analysis of RMSD, Rg, SASA, number of hydrogen bonds, RMSF, MM-PBSA, PCA, and DCCM from MD suggested that ATSRTLSYY is the most stable and promising epitope than KAYNVTQAF epitope. Moreover, we also identified B-cell epitopes for each of the antigenic proteins of SARS CoV-2. Findings of our work will be a good resource for wet lab experiments and will lessen the timeline for vaccine construction.Communicated by Ramaswamy H. Sarma.

17.
Arch Microbiol ; 203(1): 59-66, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1064447

ABSTRACT

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) belongs to the single-stranded positive-sense RNA family. The virus contains a large genome that encodes four structural proteins, small envelope (E), matrix (M), nucleocapsid phosphoprotein (N), spike (S), and 16 nonstructural proteins (nsp1-16) that together, ensure replication of the virus in the host cell. Among these proteins, the interactions of N and Nsp3 are essential that links the viral genome for processing. The N proteins reside at CoV RNA synthesis sites known as the replication-transcription complexes (RTCs). The N-terminal of N has RNA-binding domain (N-NTD), capturing the RNA genome while the C-terminal domain (N-CTD) anchors the viral Nsp3, a component of RTCs. Although the structural information has been recently released, the residues involved in contacts between N-CTD with Nsp3 are still unknown. To find the residues involved in interactions between two proteins, three-dimensional structures of both proteins were retrieved and docked using HADDOCK. Residues at N-CTD were detected in interaction with L499, R500, K501, V502, P503, T504, D505, N506, Y507, I508, T509, K529, K530K532, S533 of Nsp3 and N-NTD to synthesize SARS-CoV-2 RNA. The interaction between Nsp3 and CTD of N protein may be a potential drug target. The current study provides information for better understanding the interaction between N protein and Nsp3 that could be a possible target for future inhibitors.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus Papain-Like Proteases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , COVID-19/drug therapy , Computer Simulation , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Papain-Like Proteases/genetics , Crystallography, X-Ray , Drug Design , Genome, Viral , Humans , Molecular Docking Simulation , Nucleocapsid/metabolism , Protein Binding/physiology , RNA-Binding Proteins/metabolism , Viral Nonstructural Proteins/genetics
18.
Diagn Microbiol Infect Dis ; 100(1): 115334, 2021 May.
Article in English | MEDLINE | ID: covidwho-1062310

ABSTRACT

Several real-time RT-PCR assays have received Emergency Use Authorization from the United States Food and Drug Administration. The BD MAX™ SARS-CoV-2 assay, run by the BD MAX™ system, is a qualitative test that detects the SARS-CoV-2 specific nucleocapsid phosphoprotein gene regions, N1 and N2. The human RNase P gene is used as the endogenous nucleic acid extraction control. The Cepheid Xpert® Xpress SARS-CoV-2 assay, run by the GeneXpert system, detects the pan-sarbecovirus E gene and the N2 region of the N gene. We evaluated the performance characteristics of the BD and Cepheid assays using matched patient samples. We also analyzed comparative Ct values for both assays using 183 positive samples tested at this facility. In addition, we mitigated reporting false positive results without relying on interpretive software. We found that both systems showed comparable sensitivity. We found an approximately 3.5% false positive rate from the BD MAX™ system results.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Real-Time Polymerase Chain Reaction/methods , Coronavirus Envelope Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , False Positive Reactions , Humans , Phosphoproteins/genetics
19.
Gene Rep ; 21: 100886, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1023577

ABSTRACT

Since December 2019, a severe pandemic of pneumonia, COVID-19 associated with a novel coronavirus (SARS-CoV-2), have emerged in Wuhan, China and spreading throughout the world. As RNA viruses have a high mutation rate therefore we wanted to identify whether this virus is also prone to mutations. For this reason we selected four major structural (Spike protein (S), Envelope protein (E), Membrane glycoprotein (M), Nucleocapsid phosphoprotein (N)) and ORF8 protein of 100 different SARS-CoV-2 isolates of fifteen countries from NCBI database and compared these to the reference sequence, Wuhan NC_045512.2, which was the first isolate of SARS-CoV-2 that was sequenced. By multiple sequence alignment of amino acids, we observed substitutions and deletion in S protein at 13 different sites in the isolates of five countries (China, USA, Finland, India and Australia) as compared to the reference sequence. Similarly, alignment of N protein revealed substitutions at three different sites in isolates of China, Spain and Japan. M protein exhibits substitution only in one isolates from USA, however, no mutation was observed in E protein of any isolate. Interestingly, in ORF8 substitution of Leucine, a nonpolar to Serine a polar amino acid at same position (aa84 L to S) in 23 isolates of five countries i.e. China, USA, Spain, Taiwan and India were observed, which may affect the conformation of peptides. Thus, we observed several mutations in the isolates thereafter the first sequencing of SARS-CoV-2 isolate, NC_045512.2, which suggested that this virus might be a threat to the whole world and therefore further studies are needed to characterize how these mutations in different proteins affect the functionality and pathogenesis of SARS-CoV-2.

20.
PeerJ ; 9: e10666, 2021.
Article in English | MEDLINE | ID: covidwho-1006829

ABSTRACT

SARS-CoV-2 genome encodes four structural proteins that include the spike glycoprotein, membrane protein, envelope protein and nucleocapsid phosphoprotein (N-protein). The N-protein interacts with viral genomic RNA and helps in packaging. As SARS-CoV-2 spread to almost all countries worldwide within 2-3 months, it also acquired mutations in its RNA genome. Therefore, this study was conducted with an aim to identify the variations present in N-protein of SARS-CoV-2. Here, we analysed 4,163 reported sequence of N-protein from United States of America (USA) and compared them with the first reported sequence from Wuhan, China. Our study identified 107 mutations that reside all over the N-protein. Further, we show the high rate of mutations in intrinsically disordered regions (IDRs) of N-protein. Our study show 45% residues of IDR2 harbour mutations. The RNA-binding domain (RBD) and dimerization domain of N-protein also have mutations at key residues. We further measured the effect of these mutations on N-protein stability and dynamicity and our data reveals that multiple mutations can cause considerable alterations. Altogether, our data strongly suggests that N-protein is one of the mutational hotspot proteins of SARS-CoV-2 that is changing rapidly and these mutations can potentially interferes with various aspects of N-protein functions including its interaction with RNA, oligomerization and signalling events.

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