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1.
Molecules ; 27(9)2022 May 03.
Article in English | MEDLINE | ID: covidwho-1820344

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. While the development of vaccines and the emergence of antiviral therapeutics is promising, alternative strategies to combat COVID-19 (and potential future pandemics) remain an unmet need. Coronaviruses feature a unique mechanism that may present opportunities for therapeutic intervention: the RNA polymerase complex of coronaviruses is distinct in its ability to proofread and remove mismatched nucleotides during genome replication and transcription. The proofreading activity has been linked to the exonuclease (ExoN) activity of non-structural protein 14 (NSP14). Here, we review the role of NSP14, and other NSPs, in SARS-CoV-2 replication and describe the assays that have been developed to assess the ExoN function. We also review the nucleoside analogs and non-nucleoside inhibitors known to interfere with the proofreading activity of NSP14. Although not yet validated, the potential use of non-nucleoside proofreading inhibitors in combination with chain-terminating nucleosides may be a promising avenue for the development of anti-CoV agents.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Exoribonucleases/metabolism , Humans , Pandemics , RNA, Viral/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication
2.
Elife ; 112022 Mar 16.
Article in English | MEDLINE | ID: covidwho-1744665

ABSTRACT

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in the presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.


Viruses are parasites, relying on the cells they infect to make more of themselves. In doing so they change how an infected cell turns its genes on and off, forcing it to build new virus particles and turning off the immune surveillance that would allow the body to intervene. This is how SARS-CoV-2, the virus that causes COVID, survives with a genome that carries instructions to make just 29 proteins. One of these proteins, known as Nsp14, is involved in both virus reproduction and immune escape. Previous work has shown that it interacts with IMPDH2, the cellular enzyme that controls the production of the building blocks of the genetic code. The impact of this interaction is not clear. To find out more, Zaffagni et al. introduced 26 of the SARS-CoV-2 proteins into human cells one at a time. Nsp14 had the most dramatic effect, dialing around 4,000 genes up or down and changing how the cell interprets over 1,000 genes. Despite being just one protein, it mimicked the genetic changes seen during real SARS-CoV-2 infection. Blocking IMPDH2 partially reversed the effects, which suggests that the interaction of Nsp14 with the enzyme might be responsible for the effects of SARS-CoV-2 on the genes of the cell. Understanding how viral proteins affect cells can explain what happens during infection. This could lead to the discovery of new treatments designed to counteract the effects of the virus. Further work could investigate whether interfering with Nsp14 helps cells to overcome infection.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/genetics , Exoribonucleases/metabolism , Humans , RNA, Viral/metabolism , Transcriptome , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
3.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: covidwho-1684239

ABSTRACT

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.


Subject(s)
Exoribonucleases/chemistry , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Domains , RNA, Viral/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Binding Sites/genetics , COVID-19/virology , Catalytic Domain , Crystallography, X-Ray , Exoribonucleases/genetics , Exoribonucleases/metabolism , Humans , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Mutation, Missense , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
5.
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
6.
J Biol Chem ; 298(1): 101518, 2022 01.
Article in English | MEDLINE | ID: covidwho-1587356

ABSTRACT

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in kcat/Km in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Enzyme Activation , Virus Replication/drug effects
7.
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
8.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Article in English | MEDLINE | ID: covidwho-1541316

ABSTRACT

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.


Subject(s)
Exoribonucleases/chemistry , Models, Molecular , Protein Conformation , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Catalytic Domain , Conserved Sequence , Exoribonucleases/genetics , Exoribonucleases/metabolism , Microbial Viability , Nucleotide Motifs , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Proteins , Structure-Activity Relationship , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
9.
Chembiochem ; 22(24): 3410-3413, 2021 12 10.
Article in English | MEDLINE | ID: covidwho-1427071

ABSTRACT

The SARS-CoV-2 non-structural protein 14 (nsp14), known as exoribonuclease is encoded from the large polyprotein of viral genome and is a major constituent of the transcription replication complex (TRC) machinery of the viral RNA synthesis. This protein is highly conserved among the coronaviruses and is a potential target for the development of a therapeutic drug. Here, we report the SARS-CoV-2 nsp14 expression, show its structural characterization, and ss-RNA exonuclease activity through vibrational and electronic spectroscopies. The deconvolution of amide-I band in the FTIR spectrum of the protein revealed a composition of 35 % α-helix and 25 % ß-sheets. The binding between protein and RNA is evidenced from the spectral changes in the amide-I region of the nsp14, showing protein conformational changes during the binding process. A value of 20.60±3.81 mol L-1 of the binding constant (KD ) is obtained for nsp14/RNA complex. The findings reported here can motivate further studies to develop structural models for better understanding the mechanism of exonuclease enzymes for correcting the viral genome and can help in the development of drugs against SARS-CoV-2.


Subject(s)
Exoribonucleases/metabolism , RNA, Viral/metabolism , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Exoribonucleases/chemistry , Protein Binding , Protein Conformation , RNA, Viral/chemistry , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Viral Nonstructural Proteins/chemistry
10.
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
11.
Viruses ; 13(7)2021 07 15.
Article in English | MEDLINE | ID: covidwho-1335222

ABSTRACT

Viral infections cause a variety of acute and chronic human diseases, sometimes resulting in small local outbreaks, or in some cases spreading across the globe and leading to global pandemics. Understanding and exploiting virus-host interactions is instrumental for identifying host factors involved in viral replication, developing effective antiviral agents, and mitigating the severity of virus-borne infectious diseases. The diversity of CRISPR systems and CRISPR-based tools enables the specific modulation of innate immune responses and has contributed impressively to the fields of virology and immunology in a very short time. In this review, we describe the most recent advances in the use of CRISPR systems for basic and translational studies of virus-host interactions.


Subject(s)
Antiviral Agents/immunology , Antiviral Agents/pharmacology , CRISPR-Cas Systems , Virus Diseases/immunology , Animals , Exoribonucleases/metabolism , Host Microbial Interactions/immunology , Humans , Immune Evasion , Immunity, Innate , Interferons/genetics , Interferons/immunology , RNA Editing , Transcriptome , Virus Diseases/virology , Virus Internalization , Virus Replication/drug effects
12.
Antiviral Res ; 193: 105142, 2021 09.
Article in English | MEDLINE | ID: covidwho-1321985

ABSTRACT

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 µM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Methyltransferases/antagonists & inhibitors , Nucleotidyltransferases/antagonists & inhibitors , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , COVID-19/virology , Cell Line , Exoribonucleases/antagonists & inhibitors , Exoribonucleases/metabolism , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Methyltransferases/metabolism , Nucleotidyltransferases/metabolism , RNA Caps , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
13.
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
14.
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
15.
Nucleic Acids Res ; 49(9): 5382-5392, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1217861

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
16.
J Virol ; 94(16)2020 07 30.
Article in English | MEDLINE | ID: covidwho-1214962

ABSTRACT

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.


Subject(s)
Exoribonucleases/metabolism , Interferon Type I/biosynthesis , Interferons/biosynthesis , Porcine epidemic diarrhea virus/physiology , RNA Caps/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Binding Sites , Cell Line , Chlorocebus aethiops , Exoribonucleases/genetics , Gene Expression , Guanine/metabolism , Immunity, Innate , Methylation , Mutation , Porcine epidemic diarrhea virus/enzymology , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/pathogenicity , RNA, Viral/metabolism , S-Adenosylmethionine/metabolism , Swine , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication
17.
Protein Expr Purif ; 185: 105894, 2021 09.
Article in English | MEDLINE | ID: covidwho-1209890

ABSTRACT

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has led to a world-wild pandemic. The replication of SARS-CoV-2 RNA genome involves the core replication-transcription complex (RTC, nsp12-nsp7-nsp8) and the proofreading complex (nsp14-nsp10) that can correct mismatched base pairs during replication. Structures and functions of SARS-CoV-2 RTC have been actively studied, yet little is known about SARS-CoV-2 nsp14-nsp10. Here, we purified, reconstituted, and characterized the SARS-CoV-2 nsp14-nsp10 proofreading nuclease in vitro. We show that SARS-CoV-2 nsp14 is activated by nsp10, functioning as a potent RNase that can hydrolyze RNAs in the context of single- and double-stranded RNA and RNA/DNA hybrid duplex. SARS-CoV-2 nsp14-nsp10 shows a metal-dependent nuclease activity but has different metal selectivity from RTC. While RTC is activated by Ca2+, nsp14-nsp10 is completely inhibited. Importantly, the reconstituted SARS-CoV-2 nsp14-nsp10 efficiently removed the A:A mismatch at the 3'-end of the primer, enabling the stalled RTC to restart RNA replication. Our collective results confirm that SARS-CoV-2 nsp14-nsp10 functions as the RNA proofreading complex in SARS-CoV-2 replication and provide a useful foundation to understand the structure and function of SARS-CoV-2 RNA metabolism.


Subject(s)
COVID-19/virology , Exoribonucleases/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Calcium/metabolism , Enzyme Activation , Humans , Hydrolysis , Substrate Specificity
18.
SLAS Discov ; 26(6): 766-774, 2021 07.
Article in English | MEDLINE | ID: covidwho-1192708

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.


Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Exonucleases/antagonists & inhibitors , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
19.
J Med Virol ; 93(7): 4258-4264, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1173823

ABSTRACT

The recent coronavirus disease 2019 (COVID-19), causing a global pandemic with devastating effects on healthcare and social-economic systems, has no special antiviral therapies available for human coronaviruses (CoVs). The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) possesses a nonstructural protein (nsp14), with amino-terminal domain coding for proofreading exoribonuclease (ExoN) that is required for high-fidelity replication. The ability of CoVs during genome replication and transcription to proofread and exclude mismatched nucleotides has long hindered the development of anti-CoV drugs. The resistance of SARS-CoV-2 to antivirals, especially nucleoside analogs (NAs), shows the need to identify new CoV inhibition targets. Therefore, this review highlights the importance of nsp14-ExoN as a target for inhibition. Also, nucleoside analogs could be used in combination with existing anti-CoV therapeutics to target the proofreading mechanism.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Exoribonucleases/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Virus Replication/drug effects , Exoribonucleases/drug effects , Exoribonucleases/metabolism , Genome, Viral/genetics , Humans , Methyltransferases/genetics , RNA Processing, Post-Transcriptional/physiology , RNA, Viral/genetics , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/drug effects , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology
20.
SLAS Discov ; 26(6): 749-756, 2021 07.
Article in English | MEDLINE | ID: covidwho-1136206

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , Nitro Compounds/pharmacology , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiparasitic Agents/chemistry , Antiparasitic Agents/pharmacology , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Drug Repositioning , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Mass Spectrometry/methods , Methylation , Nitro Compounds/chemistry , Prescription Drugs/chemistry , Prescription Drugs/pharmacology , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Thiazoles/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
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