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1.
Protein J ; 39(3): 198-216, 2020 06.
Article in English | MEDLINE | ID: covidwho-1718840

ABSTRACT

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Nucleocapsid Proteins , Drug Discovery/methods , Genome, Viral , Host-Pathogen Interactions/drug effects , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Polyproteins , Protein Interaction Maps/drug effects , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins
3.
Nucleic Acids Res ; 50(3): 1484-1500, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1624985

ABSTRACT

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/metabolism , Genome, Viral/genetics , Genomic Instability , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Exoribonucleases/antagonists & inhibitors , Genome, Viral/drug effects , Genomic Instability/drug effects , Genomic Instability/genetics , HIV Integrase Inhibitors/pharmacology , Isoindoles/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/metabolism , Organoselenium Compounds/pharmacology , RNA, Viral/biosynthesis , RNA, Viral/genetics , Raltegravir Potassium/pharmacology , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Virus Replication/drug effects , Virus Replication/genetics
4.
Int J Mol Sci ; 23(1)2021 Dec 28.
Article in English | MEDLINE | ID: covidwho-1580696

ABSTRACT

The inhibition of key enzymes that may contain the viral replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have assumed central importance in drug discovery projects. Nonstructural proteins (nsps) are essential for RNA capping and coronavirus replication since it protects the virus from host innate immune restriction. In particular, nonstructural protein 16 (nsp16) in complex with nsp10 is a Cap-0 binding enzyme. The heterodimer formed by nsp16-nsp10 methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs and thus it is one of the enzymes that is a potential target for antiviral therapy. In this study, we have evaluated the mechanism of the 2'-O methylation of the viral mRNA cap using hybrid quantum mechanics/molecular mechanics (QM/MM) approach. It was found that the calculated free energy barriers obtained at M062X/6-31+G(d,p) is in agreement with experimental observations. Overall, we provide a detailed molecular analysis of the catalytic mechanism involving the 2'-O methylation of the viral mRNA cap and, as expected, the results demonstrate that the TS stabilization is critical for the catalysis.


Subject(s)
Methyltransferases/metabolism , RNA Caps/chemistry , RNA Caps/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Biocatalysis , Biomechanical Phenomena , Methylation , Methyltransferases/chemistry , Molecular Dynamics Simulation , Quantum Theory , RNA Processing, Post-Transcriptional , Viral Nonstructural Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry
5.
Cell Death Differ ; 29(2): 285-292, 2022 02.
Article in English | MEDLINE | ID: covidwho-1550276

ABSTRACT

The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Exoribonucleases/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , A549 Cells , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Antiviral Agents/pharmacology , Humans , SARS-CoV-2/enzymology , Virus Replication/drug effects
6.
Molecules ; 26(20)2021 Oct 12.
Article in English | MEDLINE | ID: covidwho-1518621

ABSTRACT

In continuation of our previous effort, different in silico selection methods were applied to 310 naturally isolated metabolites that exhibited antiviral potentialities before. The applied selection methods aimed to pick the most relevant inhibitor of SARS-CoV-2 nsp10. At first, a structural similarity study against the co-crystallized ligand, S-Adenosyl Methionine (SAM), of SARS-CoV-2 nonstructural protein (nsp10) (PDB ID: 6W4H) was carried out. The similarity analysis culled 30 candidates. Secondly, a fingerprint study against SAM preferred compounds 44, 48, 85, 102, 105, 182, 220, 221, 282, 284, 285, 301, and 302. The docking studies picked 48, 182, 220, 221, and 284. While the ADMET analysis expected the likeness of the five candidates to be drugs, the toxicity study preferred compounds 48 and 182. Finally, a density-functional theory (DFT) study suggested vidarabine (182) to be the most relevant SARS-Cov-2 nsp10 inhibitor.


Subject(s)
Antiviral Agents/chemistry , Biological Products/chemistry , SARS-CoV-2/metabolism , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Binding Sites , Biological Products/metabolism , Biological Products/therapeutic use , COVID-19/drug therapy , COVID-19/pathology , Density Functional Theory , Humans , Ligands , Molecular Docking Simulation , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/isolation & purification , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/therapeutic use , Vidarabine/chemistry , Vidarabine/metabolism , Vidarabine/therapeutic use , Viral Regulatory and Accessory Proteins/metabolism
7.
Viruses ; 13(11)2021 10 27.
Article in English | MEDLINE | ID: covidwho-1488757

ABSTRACT

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.


Subject(s)
Immunomodulation , Ion Channels/metabolism , Viroporin Proteins/metabolism , Virus Diseases/drug therapy , Viruses/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Autophagy , Host-Pathogen Interactions , Human Immunodeficiency Virus Proteins/chemistry , Human Immunodeficiency Virus Proteins/metabolism , Immune Evasion , Inflammasomes/immunology , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Viroporin Proteins/chemistry , Virus Diseases/immunology , Virus Diseases/virology , Viruses/drug effects , Viruses/immunology , Viruses/pathogenicity
8.
Molecules ; 26(20)2021 Oct 12.
Article in English | MEDLINE | ID: covidwho-1463775

ABSTRACT

In continuation of our previous effort, different in silico selection methods were applied to 310 naturally isolated metabolites that exhibited antiviral potentialities before. The applied selection methods aimed to pick the most relevant inhibitor of SARS-CoV-2 nsp10. At first, a structural similarity study against the co-crystallized ligand, S-Adenosyl Methionine (SAM), of SARS-CoV-2 nonstructural protein (nsp10) (PDB ID: 6W4H) was carried out. The similarity analysis culled 30 candidates. Secondly, a fingerprint study against SAM preferred compounds 44, 48, 85, 102, 105, 182, 220, 221, 282, 284, 285, 301, and 302. The docking studies picked 48, 182, 220, 221, and 284. While the ADMET analysis expected the likeness of the five candidates to be drugs, the toxicity study preferred compounds 48 and 182. Finally, a density-functional theory (DFT) study suggested vidarabine (182) to be the most relevant SARS-Cov-2 nsp10 inhibitor.


Subject(s)
Antiviral Agents/chemistry , Biological Products/chemistry , SARS-CoV-2/metabolism , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Binding Sites , Biological Products/metabolism , Biological Products/therapeutic use , COVID-19/drug therapy , COVID-19/pathology , Density Functional Theory , Humans , Ligands , Molecular Docking Simulation , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/isolation & purification , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/therapeutic use , Vidarabine/chemistry , Vidarabine/metabolism , Vidarabine/therapeutic use , Viral Regulatory and Accessory Proteins/metabolism
9.
Sci Rep ; 11(1): 18851, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434149

ABSTRACT

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.


Subject(s)
COVID-19/drug therapy , Ozone/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Microscopy, Electron, Transmission , Protein Structure, Secondary/drug effects , Protein Structure, Tertiary/drug effects , SARS-CoV-2/ultrastructure , Time Factors , Viral Envelope/chemistry , Viral Envelope/drug effects , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Zinc/chemistry , Zinc/metabolism
10.
Viruses ; 13(9)2021 08 30.
Article in English | MEDLINE | ID: covidwho-1390783

ABSTRACT

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.


Subject(s)
COVID-19/virology , Methyltransferases/metabolism , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Chromatography, Liquid , Gene Expression Regulation, Viral , Humans , Mass Spectrometry , Methylation , Methyltransferases/genetics , RNA Caps , RNA, Viral/genetics , SARS-CoV-2/genetics , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics
12.
Nucleic Acids Res ; 49(9): 5382-5392, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1387965

ABSTRACT

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.


Subject(s)
Biocatalysis , Exoribonucleases/chemistry , Exoribonucleases/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Exoribonucleases/genetics , Guanine , Methyltransferases/chemistry , Methyltransferases/deficiency , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Protein Domains/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics
13.
Cell Death Dis ; 12(8): 788, 2021 08 12.
Article in English | MEDLINE | ID: covidwho-1356553

ABSTRACT

In the last months, many studies have clearly described several mechanisms of SARS-CoV-2 infection at cell and tissue level, but the mechanisms of interaction between host and SARS-CoV-2, determining the grade of COVID-19 severity, are still unknown. We provide a network analysis on protein-protein interactions (PPI) between viral and host proteins to better identify host biological responses, induced by both whole proteome of SARS-CoV-2 and specific viral proteins. A host-virus interactome was inferred, applying an explorative algorithm (Random Walk with Restart, RWR) triggered by 28 proteins of SARS-CoV-2. The analysis of PPI allowed to estimate the distribution of SARS-CoV-2 proteins in the host cell. Interactome built around one single viral protein allowed to define a different response, underlining as ORF8 and ORF3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. Finally, the network-based approach highlighted a possible direct action of ORF3a and NS7b to enhancing Bradykinin Storm. This network-based representation of SARS-CoV-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. We identified possible host responses induced by specific proteins of SARS-CoV-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe COVID-19 patients.


Subject(s)
COVID-19/metabolism , COVID-19/virology , SARS-CoV-2/metabolism , Host Microbial Interactions , Immunity/immunology , Protein Interaction Maps/physiology , Proteome , Proteomics/methods , SARS-CoV-2/pathogenicity , Severity of Illness Index , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism
14.
Viruses ; 13(8)2021 07 29.
Article in English | MEDLINE | ID: covidwho-1335227

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.


Subject(s)
COVID-19/virology , Methyltransferases/metabolism , RNA Caps/genetics , RNA, Viral/genetics , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Antibodies, Monoclonal/analysis , Humans , Methyltransferases/analysis , Methyltransferases/genetics , Protein Transport , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Nonstructural Proteins/analysis , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/analysis , Viral Regulatory and Accessory Proteins/genetics
15.
Front Immunol ; 12: 708264, 2021.
Article in English | MEDLINE | ID: covidwho-1325532

ABSTRACT

There are still many unanswered questions concerning viral SARS-CoV-2 pathogenesis in COVID-19. Accessory proteins in SARS-CoV-2 consist of eleven viral proteins whose roles during infection are still not completely understood. Here, a review on the current knowledge of SARS-CoV-2 accessory proteins is summarized updating new research that could be critical in understanding SARS-CoV-2 interaction with the host. Some accessory proteins such as ORF3b, ORF6, ORF7a and ORF8 have been shown to be important IFN-I antagonists inducing an impairment in the host immune response. In addition, ORF3a is involved in apoptosis whereas others like ORF9b and ORF9c interact with cellular organelles leading to suppression of the antiviral response in infected cells. However, possible roles of ORF7b and ORF10 are still awaiting to be described. Also, ORF3d has been reassigned. Relevant information on the knowns and the unknowns in these proteins is analyzed, which could be crucial for further understanding of SARS-CoV-2 pathogenesis and to design strategies counteracting their actions evading immune responses in COVID-19.


Subject(s)
COVID-19/immunology , SARS-CoV-2/pathogenicity , Viral Regulatory and Accessory Proteins/immunology , COVID-19/pathology , Coronavirus/metabolism , Coronavirus/pathogenicity , Humans , Immune Evasion , Immunity , Interferons/antagonists & inhibitors , SARS-CoV-2/metabolism , Viral Regulatory and Accessory Proteins/metabolism
16.
Int J Mol Sci ; 21(19)2020 Oct 06.
Article in English | MEDLINE | ID: covidwho-1298151

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.


Subject(s)
Molecular Dynamics Simulation , Viral Regulatory and Accessory Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Protein Binding , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , Sequence Homology , Viral Regulatory and Accessory Proteins/metabolism , Zinc Fingers
17.
Biochem J ; 478(13): 2445-2464, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1290093

ABSTRACT

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/antagonists & inhibitors , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Animals , Aurintricarboxylic Acid/pharmacology , Chlorocebus aethiops , Enzyme Assays , Exoribonucleases/metabolism , Fluorescence , High-Throughput Screening Assays , Patulin/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism
18.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1289949

ABSTRACT

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/antagonists & inhibitors , Methyltransferases/antagonists & inhibitors , RNA Caps/metabolism , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/chemistry , Chlorobenzenes/pharmacology , Chlorocebus aethiops , Enzyme Assays , Exoribonucleases/genetics , Exoribonucleases/isolation & purification , Exoribonucleases/metabolism , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Indazoles/pharmacology , Indenes/pharmacology , Indoles/pharmacology , Methyltransferases/genetics , Methyltransferases/isolation & purification , Methyltransferases/metabolism , Nitriles/pharmacology , Phenothiazines/pharmacology , Purines/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Substrate Specificity , Trifluperidol/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/isolation & purification , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/isolation & purification , Viral Regulatory and Accessory Proteins/metabolism
19.
Viruses ; 13(6)2021 06 13.
Article in English | MEDLINE | ID: covidwho-1270126

ABSTRACT

Coronavirus accessory proteins are a unique set of proteins whose genes are interspersed among or within the genes encoding structural proteins. Different coronavirus genera, or even different species within the same coronavirus genus, encode varying amounts of accessory proteins, leading to genus- or species-specificity. Though accessory proteins are dispensable for the replication of coronavirus in vitro, they play important roles in regulating innate immunity, viral proliferation, and pathogenicity. The function of accessory proteins on virus infection and pathogenesis is an area of particular interest. In this review, we summarize the current knowledge on accessory proteins of several representative coronaviruses that infect humans or animals, including the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with an emphasis on their roles in interaction between virus and host, mainly involving stress response, innate immunity, autophagy, and apoptosis. The cross-talking among these pathways is also discussed.


Subject(s)
Immunity, Innate , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Viral Regulatory and Accessory Proteins/metabolism , COVID-19/immunology , COVID-19/virology , Host-Pathogen Interactions , Humans , Immune Evasion , Open Reading Frames , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Regulatory and Accessory Proteins/genetics , Virus Replication
20.
Nat Commun ; 12(1): 3287, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1253936

ABSTRACT

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Subject(s)
Magnesium/chemistry , RNA Caps/metabolism , RNA, Viral/metabolism , SARS-CoV-2/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Amino Acid Sequence , Binding Sites , Biocatalysis , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Viral , Humans , Magnesium/metabolism , Methylation , Methyltransferases , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA Caps/chemistry , RNA Caps/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics
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