Coronavirus Nucleocapsid Protein Research Articles

A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294–314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8–63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-β) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-β production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Intrinsic factors behind long COVID: IV. Hypothetical roles of the SARS-CoV-2 nucleocapsid protein and its liquid-liquid phase separation.

When the SARS-CoV-2virus infects humans, it leads to a condition called COVID-19that has a wide spectrum of clinical manifestations, from no symptoms to acute respiratory distress syndrome. The virus initiates damage by attaching to the ACE-2protein on the surface of endothelial cells that line the blood vessels and using these cells as hosts for replication. Reactive oxygen species levels are increased during viral replication, which leads to oxidative stress. About three-fifths(~60%) of the people who get infected with the virus eradicate it from their body after 28 days and recover their normal activity. However, a large fraction (~40%) of the people who are infected with the virus suffer from various symptoms (anosmia and/or ageusia, fatigue, cough, myalgia, cognitive impairment, insomnia, dyspnea, and tachycardia) beyond 12 weeks and are diagnosed with a syndrome called long COVID. Long-term clinical studies in a group of people who contracted SARS-CoV-2have been contrasted with a noninfected matched group of people. A subset of infected people can be distinguished by a set of cytokine markers to have persistent, low-grade inflammation and often self-report two or more bothersome symptoms. No medication can alleviate their symptoms efficiently. Coronavirus nucleocapsid proteins have been investigated extensively as potential drug targets due to their key roles in virus replication, among which is their ability to bind their respective genomic RNAs for incorporation into emerging virions. This review highlights basic studies of the nucleocapsid protein and its ability to undergo liquid-liquid phase separation. We hypothesize that this ability of the nucleocapsid protein for phase separation may contribute to long COVID. This hypothesis unlocks new investigation angles and could potentially open novel avenues for a better understanding of long COVID and treating this condition.

Targeting protein-protein interaction interfaces with antiviral N protein inhibitor in SARS-CoV-2

Coronaviruses not only pose significant global public health threats but also cause extensive damage to livestock-based industries. Previous studies have shown that 5-benzyloxygramine (P3) targets the Middle East respiratory syndrome coronavirus (MERS-CoV) nucleocapsid (N) protein N-terminal domain (N-NTD), inducing non-native protein-protein interactions (PPIs) that impair N protein function. Moreover, P3 exhibits broad-spectrum antiviral activity against CoVs. The sequence similarity of N proteins is relatively low among CoVs, further exhibiting notable variations in the hydrophobic residue responsible for non-native PPIs in the N-NTD. Therefore, to ascertain the mechanism by which P3 demonstrates broad-spectrum anti-CoV activity, we determined the crystal structure of the SARS-CoV-2 N-NTD:P3 complex. We found that P3 was positioned in the dimeric N-NTD via hydrophobic contacts. Compared with the interfaces in MERS-CoV N-NTD, P3 had a reversed orientation in SARS-CoV-2 N-NTD. The Phe residue in the MERS-CoV N-NTD:P3 complex stabilized both P3 moieties. However, in the SARS-CoV-2 N-NTD:P3 complex, the Ile residue formed only one interaction with the P3 benzene ring. Moreover, the pocket in the SARS-CoV-2 N-NTD:P3 complex was more hydrophobic, favoring the insertion of the P3 benzene ring into the complex. Nevertheless, hydrophobic interactions remained the primary stabilizing force in both complexes. These findings suggested that despite the differences in the sequence, P3 can accommodate a hydrophobic pocket in N-NTD to mediate a non-native PPI, enabling its effectiveness against various CoVs.

Detection of SARS-CoV-2 N protein using AgNPs-modified aligned silicon nanowires BioSERS chip.

The SARS-CoV-2 (COVID-19) pandemic had a strong impact on societies and economies worldwide and tests for high-performance detection of SARS-CoV-2 biomarkers are still needed for potential future outbreaks of the disease. In this paper, we present the different steps for the design of an aptamer-based surface-enhanced Raman scattering (BioSERS) sensing chip capable of detecting the coronavirus nucleocapsid protein (N protein) in spiked phosphate-buffered solutions and real samples of human blood serum. Optimization of the preparation steps in terms of the aptamer concentration used for the functionalization of the silver nanoparticles, time for affixing the aptamer, incubation time with target protein, and insulation of the silver active surface with cysteamine, led to a sensitive BioSERS chip, which was able to detect the N protein in the range from 1 to 75 ng mL-1 in spiked phosphate-buffered solutions with a detection limit of 1 ng mL-1 within 30 min. Furthermore, the BioSERS chip was used to detect the target protein in scarcely spiked human serum. This study demonstrates the possibility of a clinical application that can improve the detection limit and accuracy of the currently commercialized SARS-CoV-2 immunodiagnostic kit. Additionally, the system is modular and can be applied to detect other proteins by only changing the aptamer.

Development of an EBOV MiniG plus system as an advanced tool for anti-Ebola virus drug screening

The incidence of zoonotic diseases, such as coronavirus disease 2019 and Ebola virus disease, is increasing worldwide. However, drug and vaccine development for zoonotic diseases has been hampered because the experiments involving live viruses are limited to high-containment laboratories. The Ebola virus minigenome system enables researchers to study the Ebola virus under BSL-2 conditions. Here, we found that the addition of the nucleocapsid protein of human coronaviruses, such as severe acute respiratory syndrome coronavirus 2, can increase the ratio of green fluorescent protein-positive cells by 1.5–2 folds in the Ebola virus minigenome system. Further analysis showed that the nucleocapsid protein acts as an activator of the Ebola virus minigenome system. Here, we developed an EBOV MiniG Plus system based on the Ebola virus minigenome system by adding the SARS-CoV-2 nucleocapsid protein. By evaluating the antiviral effect of remdesivir and rupintrivir, we demonstrated that compared to that of the traditional Ebola virus minigenome system, significant concentration-dependent activity was observed in the EBOV MiniG Plus system. Taken together, these results demonstrate the utility of adding nucleocapsid protein to the Ebola virus minigenome system to create a powerful platform for screening antiviral drugs against the Ebola virus.

Novel Fluorescent Benzothiazolyl-Coumarin Hybrids as Anti-SARS-COVID-2 Agents Supported by Molecular Docking Studies: Design, Synthesis, X-ray Crystal Structures, DFT, and TD-DFT/PCM Calculations.

This study revealed the design and preparation of new 3-(benzo[d]thiazol-2-yl)-2H-chromen-2-one derivatives 9a-h. The structures of the synthesized products were elucidated by their spectroscopic data and X-ray crystallography for compounds 9a and 9d. The prepared new compounds were measured for their fluorescence, and a good result indicated that the emission efficiency was decreased by increasing the electron-withdrawing groups from the unsubstituted compound 9a to the highly substituted derivative 9h (2 Br heavy atoms). On the other hand, the B3LYP/6-311G** theoretical level of theory was used to optimize the quantum mechanical calculations of the geometrical characteristics and energy of the novel compounds 9a-h under study. The electronic transition was investigated using the TD-DFT/PCM B3LYP approach, which uses time-dependent density functional calculations. Moreover, the compounds exhibited nonlinear optical properties (NLO) and a small HOMO-LUMO energy gap, which makes them easy to polarize. Furthermore, the acquired infrared spectra were compared with the expected harmonic vibrations of the substances 9a-h. On the other hand, binding energy analyses of compounds 9a-h with human corona virus nucleocapsid protein Nl63 (PDB ID: 5epw) were predicted using molecular docking and virtual screening tools. The results showed a promising binding and how these potent compounds were inhibiting the COVID-19 virus. Compound 9h was the most active anti-COVID-19 agent among all the synthesized benzothiazolyl-coumarin derivatives, as it forms five bonds. The presence of the two bromine atoms in its structure was responsible for the potent activity.

A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus–host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug–host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N–N interaction, N oligomerization, and N–viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N–cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins.

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

Structural insights into ribonucleoprotein dissociation by nucleocapsid protein interacting with non-structural protein 3 in SARS-CoV-2

The coronavirus nucleocapsid (N) protein interacts with non-structural protein 3 (Nsp3) to facilitate viral RNA synthesis and stabilization. However, structural information on the N-Nsp3 complex is limited. Here, we report a 2.6 Å crystal structure of the N-terminal domain (NTD) of the N protein in complex with the ubiquitin-like domain 1 (Ubl1) of Nsp3 in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One NTD and two Ubl1s formed a stable heterotrimer. We performed mutational analysis to reveal the key residues for this interaction. We confirmed the colocalization of SARS-CoV-2 N and Nsp3 in Huh-7 cells. N-Ubl1 interaction also exists in SARS-CoV and Middle East respiratory syndrome coronavirus. We found that SARS-CoV-2 Ubl1 competes with RNA to bind N protein in a dose-dependent manner. Based on our results, we propose a model for viral ribonucleoprotein dissociation through N protein binding to Ubl1 of Nsp3.

Targeting intracellular Neu1 for coronavirus infection treatment

There are currently no effective therapies for COVID-19 or antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and vaccines appear less effective against new SARS-CoV-2 variants; thus, there is an urgent need to understand better the virulence mechanisms of SARS-CoV-2 and the host response to develop therapeutic agents. Herein, we show that host Neu1 regulates coronavirus replication by controlling sialylation on coronavirus nucleocapsid protein. Coronavirus nucleocapsid proteins in COVID-19 patients and in coronavirus HCoV-OC43-infected cells were heavily sialylated; this sialylation controlled the RNA-binding activity and replication of coronavirus. Neu1 overexpression increased HCoV-OC43 replication, whereas Neu1 knockdown reduced HCoV-OC43 replication. Moreover, a newly developed Neu1 inhibitor, Neu5Ac2en-OAcOMe, selectively targeted intracellular sialidase, which dramatically reduced HCoV-OC43 and SARS-CoV-2 replication invitro and rescued mice from HCoV-OC43 infection-induced death. Our findings suggest Neu1 inhibitors could be used to limit SARS-CoV-2 replication in patients with COVID-19, making Neu1 a potential therapeutic target for COVID-19 and future coronavirus pandemics.

SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

The coronavirus nucleocapsid (N)protein is known to bind to nucleic acids and facilitate viral genome encapsulation. Here we report that the N protein can mediate RNA or DNA entering neighboring cells through ACE2-independent, receptor (STEAP2)-mediated endocytosis, and achieve gene expression. The effect is more pronounced for the N protein of wild-type SARS-CoV-2 than that of the Omicron variant and other human coronaviruses. This effect is enhanced by RANTES (CCL5), a chemokine induced by N protein, and lactate, a metabolite produced in hypoxia, to cause more damage. These findings might explain the clinical observations in SARS-CoV-2-infected cases. Moreover, the N protein-mediated function can be inhibited by N protein-specific monoclonal antibodies or p38 mitogen-activated protein kinase inhibitors. Since the N-protein-mediated nucleic acid endocytosis involves a receptor commonly expressed in many types of cells, our findings suggest that N protein may have an additional role in SARS-CoV-2 pathogenesis.

Synthesis of Novel 1,3,4-Oxadiazole-Derived α-Aminophosphonates/α-Aminophosphonic Acids and Evaluation of Their In Vitro Antiviral Activity against the Avian Coronavirus Infectious Bronchitis Virus.

An efficient and simple approach has been developed for the synthesis of eight dialkyl/aryl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl]phosphonates through the Pudovik-type reaction of dialkyl/arylphosphite with imines, obtained from 5-phenyl-1,3,4-oxadiazol-2-amine and aromatic aldehydes, under microwave irradiation. Five of them were hydrolyzed to lead to the corresponding phosphonic acids. Selected synthesized compounds were screened for their in vitro antiviral activity against the avian bronchitis virus (IBV). In the MTT cytotoxicity assay, the dose-response curve showed that all test compounds were safe in the range concentration of 540-1599 µM. The direct contact of novel synthesized compounds with IBV showed that the diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethoxyphenyl)methyl]phosphonate (5f) (at 33 µM) and the [(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl] phosphonic acid (6a) (at 1.23 µM) strongly inhibited the IBV infectivity, indicating their high virucidal activity. However, virus titers from IBV-infected Vero cells remained unchanged in response to treatment with the lowest non-cytotoxic concentrations of synthesized compounds suggesting their incapacity to inhibit the virus replication inside the host cell. Lack of antiviral activity might presumably be ascribed to their polarity that hampers their diffusion across the lipophilic cytoplasmic membrane. Therefore, the interactions of 5f and 6a were analyzed against the main coronavirus protease, papain-like protease, and nucleocapsid protein by molecular docking methods. Nevertheless, the novel 1,3,4-oxadiazole-based α-aminophosphonic acids and α-amino-phosphonates hold potential for developing new hygienic virucidal products for domestic, chemical, and medical uses.

Comparison of a Prototype SARS-CoV-2 Lateral Flow IMMUNOASSAY with the BinaxNOWTM COVID-19 Antigen CARD

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. From the onset of the pandemic, rapid antigen tests have quickly proved themselves to be an accurate and accessible diagnostic platform. The initial (and still most commonly used antigen tests) for COVID-19 diagnosis were constructed using monoclonal antibodies (mAbs) specific to severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP). These mAbs are able to bind SARS-CoV-2 NP due to high homology between the two viruses. However, since first being identified in 2019, SARS-CoV-2 has continuously mutated, and a multitude of variants have appeared. These mutations have an elevated risk of leading to possible diagnostic escape when using tests produced with SARS-CoV-derived mAbs. Here, we established a library of 18 mAbs specific to SARS-CoV-2 NP and used two of these mAbs (1CV7 and 1CV14) to generate a prototype antigen-detection lateral flow immunoassay (LFI). A side-by-side analysis of the 1CV7/1CV14 LFI and the commercially available BinaxNOWTM COVID-19 Antigen CARD was performed. Results indicated the 1CV7/1CV14 LFI outperformed the BinaxNOWTM test in the detection of BA.2, BA.2.12.1, and BA.5 Omicron sub-variants when testing remnant RT-PCR positive patient nasopharyngeal swabs diluted in viral transport media.

Coronaviruses exploit a host cysteine-aspartic protease for replication.

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2(refs.1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1(ref.4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

Analysis of a crucial interaction between the coronavirus nucleocapsid protein and the major membrane-bound subunit of the viral replicase-transcriptase complex

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.

Targeting the coronavirus nucleocapsid protein through GSK-3 inhibition

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

Structural and conformational analysis of SARS CoV 2 N-CTD revealing monomeric and dimeric active sites during the RNA-binding and stabilization: Insights towards potential inhibitors for N-CTD

The advent of SARS-CoV-2 has become a universal health issue with no appropriate cure available to date. The coronavirus nucleocapsid (N) protein combines viral genomic RNA into a ribonucleoprotein and protects the viral genome from the host's nucleases. Structurally, the N protein comprises two independent domains: the N-terminal domain (NTD) for RNA-binding and C-terminal domain (CTD) involved in RNA-binding, protein dimerization, and nucleocapsid stabilization. The present study explains the structural aspects associated with the involvement of nucleocapsid C-terminal domain in the subunit assembly that helps the RNA binding and further stabilizing the virus assembly by protecting RNA from the hosts exonucleases degradation. The molecular dynamics (MD) simulations of the N-CTD and RNA complex suggests two active sites (site I: a monomer) and (site II: a dimer) with structural stability (RMSD: ~2 Å), Cα fluctuations (RMSF: ~3 Å) and strong protein-ligand interactions were estimated through the SiteMap module of Schrodinger. Virtual screening of 2456 FDA-approved drugs using structure-based docking identified top two leads distinctively against Site-I (monomer): Ceftaroline fosamil (MM-GBSA = −47.12 kcal/mol) and Cefoperazone (−45.84 kcal/mol); and against Site-II (dimer): Boceprevir, (an antiviral protease inhibitor, −106.78 kcal/mol) and Ceftaroline fosamil (−99.55 kcal/mol). The DCCM and PCA of drugs Ceftaroline fosamil (PC1+PC2 = 71.9%) and Boceprevir (PC1 +PC2 = 61.6%) show significant correlated residue motions which suggests highly induced conformational changes in the N-CTD dimer. Therefore, we propose N-CTD as a druggable target with two active binding sites (monomer and dimer) involved in specific RNA binding and stability. The RNA binding site with Ceftaroline fosamil binding can prevent viral assembly and can act as an antiviral for coronavirus.

Epitope mapping of severe acute respiratory syndrome-related coronavirus nucleocapsid protein with a rabbit monoclonal antibody

The emergency SARS-CoV-2, a member of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV), is still greatly harming the health of mankind. SARS-CoV-2-specific monoclonal antibodies (MAbs), which can identify SARS-CoV-2 from common human coronaviruses, are considered to extensively apply to developing rapid and reliable antigen assays. In this study we generated a rabbit MAb (RAb) detecting SARS-CoV-2 nucleocapsid protein (NP), which has cross-reaction with SARS-CoV-1 NP, but not with NPs of MERS and common human CoVs (OC43, NL63, 229E, and HKU1). With truncated NP fragments and synthesized peptides, the linear epitope detected by RAb was mapped in peptide N4-8, 393–407 amino acid residue (TLLPAADLDDFSKQL) of SARS-CoV-2 NP. This epitope N4-8 was highly conserved in SARSr-CoVs, including SARS-CoV-2, SARS-CoV-1, and bat CoV RaTG13 strain. However, the corresponding peptide of bat SARSr-CoV BtKY72 strain could not be recognized by RAb, which indicates amino acid D399 may be critical for N4-8 epitope detected by RAb. The present study will be conducive to developing reliable diagnosis for SARS-CoV-2 and gaining insights into the function of the SARS-CoV-2 N protein.

Phylogenic analysis of coronavirus genome and molecular studies on potential anti-COVID-19 agents from selected FDA-approved drugs

The emergence of 2019 novel Coronavirus (COVID-19 or 2019-nCoV) has caused significant global morbidity and mortality with no consensus specific treatment. We tested the hypothesis that FDA-approved antiretrovirals, antibiotics, and antimalarials will effectively inhibit COVID-19 two major drug targets, coronavirus nucleocapsid protein (NP) and hemagglutinin-esterase (HE). To test this hypothesis, we carried out a phylogenic analysis of coronavirus genome to understand the origins of NP and HE, and also modeled the proteins before molecular docking, druglikeness, toxicity assessment, molecular dynamics simulation (MDS) and ligand-based pharmacophore modeling of the selected FDA-approved drugs. Our models for NP and HE had over 95% identity with templates 5EPW and 3CL5 respectively in the PDB database, with majority of the amino acids occupying acceptable regions. The active sites of the proteins contained conserved residues that were involved in ligand binding. Lopinavir and ritonavir possessed greater binding affinities for NP and HE relative to remdesivir, while levofloxacin and hydroxychloroquine were the most notable among the other classes of drugs. The Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of gyration (Rg), and binding energy values obtained after 100 ns of MDS revealed good stability of these compounds in the binding sites of the proteins while important pharmacophore features were also identified. The study showed that COVID-19 likely originated from bat, owing to the over 90% genomic similarity observed, and that lopinavir, levofloxacin, and hydroxychloroquine might serve as potential anti-COVID-19 lead molecules for additional optimization and drug development for the treatment of COVID-19. Communicated by Ramaswamy H. Sarma