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1.
Chembiochem ; 23(2): e202100514, 2022 01 19.
Article in English | MEDLINE | ID: covidwho-1653182

ABSTRACT

In addition to a membrane anchor, the transmembrane domain (TMD) of single-pass transmembrane proteins (SPTMPs) recently has shown essential roles in the cross-membrane activity or receptor assembly/clustering. However, these small TMD peptides are generally hydrophobic and dynamic, difficult to be expressed and purified. Here, we have integrated the power of TrpLE fusion protein and a sequence-specific nickel-assisted cleavage (SNAC)-tag to produce small TMD peptides in a highly efficient way under mild conditions, which uses Ni2+ as the cleavage reagent, avoiding the usage of toxic cyanogen bromide (CNBr). Furthermore, this method simplifies the downstream protein purification and reconstitution. Two representative TMDs, including the Spike-TMD from severe acute respiratory syndrome coronavirus 2 (SARS2), were successfully produced with high-quality nuclear magnetic resonance (NMR) spectra. Therefore, our study provides a more efficient and practical approach for general structural characterization of the small TM proteins.


Subject(s)
Nickel/chemistry , Peptides/metabolism , Recombinant Fusion Proteins/metabolism , COVID-19/pathology , COVID-19/virology , Catalysis , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Peptides/isolation & purification , Proteolysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
2.
Int J Mol Sci ; 23(3)2022 Jan 18.
Article in English | MEDLINE | ID: covidwho-1625435

ABSTRACT

Spike protein of SARS-CoV-2 contains a single-span transmembrane (TM) domain and plays roles in receptor binding, viral attachment and viral entry to the host cells. The TM domain of spike protein is critical for viral infectivity. Herein, the TM domain of spike protein of SARS-CoV-2 was reconstituted in detergent micelles and subjected to structural analysis using solution NMR spectroscopy. The results demonstrate that the TM domain of the protein forms a helical structure in detergent micelles. An unstructured linker is identified between the TM helix and heptapeptide repeat 2 region. The linker is due to the proline residue at position 1213. Side chains of the three tryptophan residues preceding to and within the TM helix important for the function of S-protein might adopt multiple conformations which may be critical for their function. The side chain of W1212 was shown to be exposed to solvent and the side chains of residues W1214 and W1217 are buried in micelles. Relaxation study shows that the TM helix is rigid in solution while several residues have exchanges. The secondary structure and dynamics of the TM domain in this study provide insights into the function of the TM domain of spike protein.


Subject(s)
Detergents/pharmacology , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , COVID-19/virology , Cell Membrane/metabolism , Cross-Linking Reagents/pharmacology , Detergents/chemistry , Humans , Magnetic Resonance Spectroscopy , Micelles , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Domains/drug effects , Protein Structure, Secondary/drug effects , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/drug effects , Spike Glycoprotein, Coronavirus/metabolism
3.
Chembiochem ; 23(2): e202100514, 2022 01 19.
Article in English | MEDLINE | ID: covidwho-1549172

ABSTRACT

In addition to a membrane anchor, the transmembrane domain (TMD) of single-pass transmembrane proteins (SPTMPs) recently has shown essential roles in the cross-membrane activity or receptor assembly/clustering. However, these small TMD peptides are generally hydrophobic and dynamic, difficult to be expressed and purified. Here, we have integrated the power of TrpLE fusion protein and a sequence-specific nickel-assisted cleavage (SNAC)-tag to produce small TMD peptides in a highly efficient way under mild conditions, which uses Ni2+ as the cleavage reagent, avoiding the usage of toxic cyanogen bromide (CNBr). Furthermore, this method simplifies the downstream protein purification and reconstitution. Two representative TMDs, including the Spike-TMD from severe acute respiratory syndrome coronavirus 2 (SARS2), were successfully produced with high-quality nuclear magnetic resonance (NMR) spectra. Therefore, our study provides a more efficient and practical approach for general structural characterization of the small TM proteins.


Subject(s)
Nickel/chemistry , Peptides/metabolism , Recombinant Fusion Proteins/metabolism , COVID-19/pathology , COVID-19/virology , Catalysis , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Peptides/isolation & purification , Proteolysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
Angew Chem Int Ed Engl ; 60(48): 25428-25435, 2021 11 22.
Article in English | MEDLINE | ID: covidwho-1490696

ABSTRACT

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/chemistry , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Cysteine Proteinase Inhibitors/chemistry , Drug Evaluation, Preclinical , Microbial Sensitivity Tests , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Multimerization , SARS-CoV-2/chemistry , Small Molecule Libraries/chemistry , Vero Cells
5.
Eur Biophys J ; 50(8): 1129-1137, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1460302

ABSTRACT

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many viruses use their own IDPs to "hack" these processes to deactivate host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational preferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2, whose C-terminal region (CtR) is predicted to be disordered, interacts with human proteins that regulate translation initiation and endosome vesicle sorting. Molecules that block these interactions could be valuable leads for drug development. The 13Cß and backbone 13CO, 1HN, 13Cα, and 15N nuclei of Nsp2's 45-residue CtR were assigned and used to characterize its structure and dynamics in three contexts; namely: (1) retaining an N-terminal His tag, (2) without the His tag and with an adventitious internal cleavage, and (3) lacking both the His tag and the internal cleavage. Two five-residue segments adopting a minor extended population were identified. Overall, the dynamic behavior is midway between a completely rigid and a fully flexible chain. Whereas the presence of an N-terminal His tag and internal cleavage stiffen and loosen, respectively, neighboring residues, they do not affect the tendency of two regions to populate extended conformations.


Subject(s)
Intrinsically Disordered Proteins , SARS-CoV-2 , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation
6.
Biomol NMR Assign ; 15(2): 255-260, 2021 10.
Article in English | MEDLINE | ID: covidwho-1453890

ABSTRACT

The nucleoprotein (N) from SARS-CoV-2 is an essential cofactor of the viral replication transcription complex and as such represents an important target for viral inhibition. It has also been shown to colocalize to the transcriptase-replicase complex, where many copies of N decorate the viral genome, thereby protecting it from the host immune system. N has also been shown to phase separate upon interaction with viral RNA. N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45-175 and 264-365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1-44 and N3, spanning residues 176-263). Our assignment provides the basis for the identification of inhibitors and functional and interaction studies of this essential protein.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Nucleoproteins/chemistry , SARS-CoV-2 , Viral Proteins/chemistry , Models, Molecular , Protein Domains , Protein Structure, Secondary
7.
Biomol NMR Assign ; 15(2): 467-474, 2021 10.
Article in English | MEDLINE | ID: covidwho-1442185

ABSTRACT

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7-33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.


Subject(s)
5' Untranslated Regions , Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2/genetics , Nucleic Acid Conformation , RNA, Spliced Leader
8.
Biomol NMR Assign ; 15(2): 335-340, 2021 10.
Article in English | MEDLINE | ID: covidwho-1442184

ABSTRACT

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.


Subject(s)
5' Untranslated Regions , Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2/genetics , Inverted Repeat Sequences/genetics
9.
Biomol NMR Assign ; 15(2): 287-295, 2021 10.
Article in English | MEDLINE | ID: covidwho-1442183

ABSTRACT

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5' untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Models, Molecular , Protein Domains
10.
Angew Chem Int Ed Engl ; 60(21): 11884-11891, 2021 05 17.
Article in English | MEDLINE | ID: covidwho-1384108

ABSTRACT

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Protons , RNA, Viral/analysis , SARS-CoV-2/chemistry , Magnetic Phenomena , RNA, Viral/chemistry
11.
J Biol Chem ; 297(3): 101018, 2021 09.
Article in English | MEDLINE | ID: covidwho-1380706

ABSTRACT

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.


Subject(s)
COVID-19/virology , Pyrimidine Nucleotides/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Binding , RNA/metabolism , SARS-CoV-2/physiology , Virus Replication
12.
FEBS Lett ; 595(17): 2248-2256, 2021 09.
Article in English | MEDLINE | ID: covidwho-1326724

ABSTRACT

The endoplasmic reticulum transmembrane protein vesicle-associated membrane protein-associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT-like motifs. In this study, we investigated the interactions of eight peptides containing FFAT-like motifs with the VAP-A MSP domain (VAP-AMSP ) by solution NMR. Six of eight peptides are specifically bound to VAP-A. Furthermore, we found that the RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT-like motif which specifically binds to VAP-AMSP as well as other FFAT-like motifs. Our results will contribute to the discovery of new VAP interactors.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Peptides/chemistry , SARS-CoV-2/enzymology , Vesicular Transport Proteins/chemistry , Amino Acid Motifs , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Nuclear Magnetic Resonance, Biomolecular , Peptides/metabolism , Protein Binding , SARS-CoV-2/metabolism , Vesicular Transport Proteins/metabolism
13.
Biochem Biophys Res Commun ; 563: 92-97, 2021 07 23.
Article in English | MEDLINE | ID: covidwho-1248814

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has the characteristic accessory protein ORF8. Although clinical reports indicate that ORF8 variant strains (Δ382 and L84S variants) are less likely to cause severe illness, functional differences between wild-type and variant ORF8 are unknown. Furthermore, the physicochemical properties of the ORF8 protein have not been analyzed. In this study, the physicochemical properties of the wild-type ORF8 and its L84S variant were analyzed and compared. Using the tobacco BY-2 cell production system, which has been successfully used to produce the wild-type ORF8 protein with a single conformation, was used to successfully produce the ORF8 L84S variant protein at the same level as wild-type ORF8. The produced proteins were purified, and their temperature and pH dependencies were examined using nuclear magnetic resonance spectra. Our data suggested that the wild-type and L84S variant ORF8 structures are highly stable over a wide temperature range. Both proteins displayed an aggregated conformation at higher temperature that reverted when the temperature was decreased to room temperature. Moreover, ORF8 precipitated at acidic pH and this precipitation was reversed when the solution pH was shifted to neutral. Interestingly, the L84S variant exhibited greater solubility than wild-type ORF8 under acidic conditions. Thus, the finding indicated that conformational stability and reversibility of ORF8 are key properties related to function in oppressive environments.


Subject(s)
COVID-19/virology , SARS-CoV-2/chemistry , Viral Proteins/chemistry , COVID-19/metabolism , COVID-19/pathology , Humans , Molecular Conformation , Mutation , Nuclear Magnetic Resonance, Biomolecular/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Structure-Activity Relationship , Viral Proteins/genetics , Viral Proteins/metabolism
14.
J Am Chem Soc ; 143(21): 7930-7934, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1237972

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells is a complex process that involves (1) recognition of the host entry receptor, angiotensin-converting enzyme 2 (ACE2), by the SARS-CoV-2 spike protein receptor binding domain (RBD), and (2) the subsequent fusion of the viral and cell membranes. Our long-term immune-defense is the production of antibodies (Abs) that recognize the SARS-CoV-2 RBD and successfully block viral infection. Thus, to understand immunity against SARS-CoV-2, a comprehensive molecular understanding of how human SARS-CoV-2 Abs recognize the RBD is needed. Here, we report the sequence-specific backbone assignment of the SARS-CoV-2 RBD and, furthermore, demonstrate that biomolecular NMR spectroscopy chemical shift perturbation (CSP) mapping successfully and rapidly identifies the molecular epitopes of RBD-specific mAbs. By incorporating NMR-based CSP mapping with other molecular techniques to define RBD-mAb interactions and then correlating these data with neutralization efficacy, structure-based approaches for developing improved vaccines and COVID-19 mAb-based therapies will be greatly accelerated.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Viral/chemistry , COVID-19/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , Binding Sites , Epitopes/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Domains , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
15.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
16.
Future Microbiol ; 16: 577-588, 2021 05.
Article in English | MEDLINE | ID: covidwho-1223836

ABSTRACT

Aim: To understand the pathological progress of COVID-19 and to explore the potential biomarkers. Background: The COVID-19 pandemic is ongoing. There is metabolomics research about COVID-19 indicating the rich information of metabolomics is worthy of further data mining. Methods: We applied bioinformatics technology to reanalyze the published metabolomics data of COVID-19. Results: Benzoate, ß-alanine and 4-chlorobenzoic acid were first reported to be used as potential biomarkers to distinguish COVID-19 patients from healthy individuals; taurochenodeoxycholic acid 3-sulfate, glucuronate and N,N,N-trimethyl-alanylproline betaine TMAP are the top classifiers in the receiver operating characteristic curve of COVID-severe and COVID-nonsevere patients. Conclusion: These unique metabolites suggest an underlying immunoregulatory treatment strategy for COVID-19.


Subject(s)
COVID-19/blood , COVID-19/diagnosis , Metabolome/physiology , Metabolomics , Benzoates/blood , Biomarkers/blood , COVID-19/immunology , Chlorobenzoates/blood , Chromatography, Liquid , Computational Biology , Glucuronic Acid/blood , Humans , Mass Spectrometry , Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2/immunology , Taurochenodeoxycholic Acid/analogs & derivatives , Taurochenodeoxycholic Acid/blood , beta-Alanine/blood
17.
Biomol NMR Assign ; 15(2): 341-345, 2021 10.
Article in English | MEDLINE | ID: covidwho-1220536

ABSTRACT

During the past 17 years, the coronaviruses have become a global public emergency, with the first appearance in 2012 in Saudi Arabia of the Middle East respiratory syndrome. Among the structural proteins encoded in the viral genome, the nucleocapsid protein is the most abundant in infected cells. It is a multifunctional phosphoprotein involved in the capsid formation, in the modulation and regulation of the viral life cycle. The N-terminal domain of N protein specifically interacts with transcriptional regulatory sequence (TRS) and is involved in the discontinuous transcription through the melting activity of double-stranded TRS (dsTRS).


Subject(s)
Middle East Respiratory Syndrome Coronavirus , Nuclear Magnetic Resonance, Biomolecular , Nucleocapsid Proteins/chemistry , Models, Molecular , Protein Domains
18.
Protein J ; 39(6): 600-618, 2020 12.
Article in English | MEDLINE | ID: covidwho-1188139

ABSTRACT

Many research teams all over the world focus their research on the SARS-CoV-2, the new coronavirus that causes the so-called COVID-19 disease. Most of the studies identify the main protease or 3C-like protease (Mpro/3CLpro) as a valid target for large-spectrum inhibitors. Also, the interaction of the human receptor angiotensin-converting enzyme 2 (ACE2) with the viral surface glycoprotein (S) is studied in depth. Structural studies tried to identify the residues responsible for enhancement/weaken virus-ACE2 interactions or the cross-reactivity of the neutralizing antibodies. Although the understanding of the immune system and the hyper-inflammatory process in COVID-19 are crucial for managing the immediate and the long-term consequences of the disease, not many X-ray/NMR/cryo-EM crystals are available. In addition to 3CLpro, the crystal structures of other nonstructural proteins offer valuable information for elucidating some aspects of the SARS-CoV-2 infection. Thus, the structural analysis of the SARS-CoV-2 is currently mainly focused on three directions-finding Mpro/3CLpro inhibitors, the virus-host cell invasion, and the virus-neutralizing antibody interaction.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/chemistry , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Papain-Like Proteases/chemistry , Coronavirus RNA-Dependent RNA Polymerase/chemistry , SARS-CoV-2/chemistry , Amino Acid Sequence , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Cryoelectron Microscopy , Crystallography, X-Ray , Drug Discovery , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/chemistry , Protein Conformation , Protein Kinase Inhibitors/pharmacology , SARS-CoV-2/drug effects
19.
Biomol NMR Assign ; 15(2): 287-295, 2021 10.
Article in English | MEDLINE | ID: covidwho-1155327

ABSTRACT

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5' untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Models, Molecular , Protein Domains
20.
Biomol NMR Assign ; 15(2): 235-241, 2021 10.
Article in English | MEDLINE | ID: covidwho-1146127

ABSTRACT

As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , RNA-Binding Proteins/chemistry , Viral Nonstructural Proteins/chemistry , Hydrogen-Ion Concentration , Models, Molecular , Protein Structure, Secondary
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