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1.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: covidwho-1271852

ABSTRACT

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.


Subject(s)
Coronavirus 3C Proteases/metabolism , Protein Processing, Post-Translational , Proteolysis , Torovirus Infections/embryology , Torovirus/enzymology , Animals , Coronavirus 3C Proteases/genetics , HEK293 Cells , Humans , Substrate Specificity , Swine , Torovirus/genetics , Torovirus Infections/genetics
2.
Biochem Biophys Rep ; 27: 101032, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1252494

ABSTRACT

Developing a safe and effective antiviral treatment takes a decade, however, when it comes to the coronavirus disease (COVID-19), time is a sensitive matter to slow the spread of the pandemic. Screening approved antiviral drugs against COVID-19 would speed the process of finding therapeutic treatment. The current study examines commercially approved drugs to repurpose them against COVID-19 virus main protease using structure-based in-silico screening. The main protease of the coronavirus is essential in the viral replication and is involved in polyprotein cleavage and immune regulation, making it an effective target when developing the treatment. A Number of approved antiviral drugs were tested against COVID-19 virus using molecular docking analysis by calculating the free natural affinity of the binding ligand to the active site pocket and the catalytic residues without forcing the docking of the ligand to active site. COVID-19 virus protease solved structure (PDB ID: 6LU7) is targeted by repurposed drugs. The molecular docking analysis results have shown that the binding of Remdesivir and Mycophenolic acid acyl glucuronide with the protein drug target has optimal binding features supporting that Remdesivir and Mycophenolic acid acyl glucuronide can be used as potential anti-viral treatment against COVID-19 disease.

3.
Curr Med Chem ; 2021 May 14.
Article in English | MEDLINE | ID: covidwho-1229117

ABSTRACT

Outbreaks due to Severe Acute Respiratory Syndrome-Corona virus 2 (SARS-CoV-2) initiated in Wuhan city, China, in December 2019 which continued to spread internationally, posing a pandemic threat as declared by WHO and as of March 10, 2021, confirmed cases reached 118 million along with 2.6 million deaths worldwide. In the absence of specific antiviral medication, symptomatic treatment and physical isolation remain the options to control the contagion. The recent clinical trials on antiviral drugs highlighted some promising compounds such as umifenovir (haemagglutinin-mediated fusion inhibitor), remdesivir (RdRp nucleoside inhibitor), and favipiravir (RdRp Inhibitor). WHO launched a multinational clinical trial on several promising analogs as a potential treatment to combat SARS infection. This situation urges a holistic approach to invent safe and specific drugs as a prophylactic and therapeutic cure for SARS-related-viral diseases, including COVID-19. It is significant to note that researchers worldwide have been doing their best to handle the crisis and have produced an extensive and promising literature body. It opens a scope and allows understanding the viral entry at the molecular level. A structure-based approach can reveal the molecular-level understanding of viral entry interaction. The ligand profiling and non-covalent interactions among participating amino-acid residues are critical information to delineate a structural interpretation. The structural investigation of SARS virus entry into host cells will reveal the possible strategy for designing drugs like entry inhibitors. The structure-based approach demonstrates details at the 3D molecular level. It shows specificity about SARS-CoV-2 spike interaction, which uses human angiotensin-converting enzyme 2 (ACE2) as a receptor for entry, and the human protease completes the process of viral fusion and infection. The 3D structural studies reveal the existence of two units, namely S1 and S2. S1 is called a receptor-binding domain (RBD) and responsible for interacting with the host (ACE2), and the S2 unit participates in the fusion of viral and cellular membranes. TMPRSS2 mediates the cleavage at S1/S2 subunit interface in S-protein of SARS CoV-2, leading to viral fusion. Conformational difference associated with S1 binding alters ACE2 interaction and inhibits viral fusion. Overall, the detailed 3D structural studies help understand the 3D structural basis of interaction between viruses with host factors and available scope for the new drug discovery process targeting SARS-related virus entry into the host cell.

4.
mBio ; 12(3)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1225698

ABSTRACT

The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.


Subject(s)
Bronchi/virology , Furin/metabolism , Respiratory Mucosa/virology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Transcriptome , Animals , Bronchi/cytology , Cells, Cultured , Chlorocebus aethiops , Epithelial Cells/virology , Humans , RNA-Seq , Respiratory Mucosa/cytology , Sequence Deletion , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
5.
Int J Biol Sci ; 17(6): 1555-1564, 2021.
Article in English | MEDLINE | ID: covidwho-1206436

ABSTRACT

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the novel lineage B betacoroanvirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant mortality, morbidity, and socioeconomic disruptions worldwide. Effective antivirals are urgently needed for COVID-19. The main protease (Mpro) of SARS-CoV-2 is an attractive antiviral target because of its essential role in the cleavage of the viral polypeptide. In this study, we performed an in silico structure-based screening of a large chemical library to identify potential SARS-CoV-2 Mpro inhibitors. Among 8,820 compounds in the library, our screening identified trichostatin A, a histone deacetylase inhibitor and an antifungal compound, as an inhibitor of SARS-CoV-2 Mpro activity and replication. The half maximal effective concentration of trichostatin A against SARS-CoV-2 replication was 1.5 to 2.7µM, which was markedly below its 50% effective cytotoxic concentration (75.7µM) and peak serum concentration (132µM). Further drug compound optimization to develop more stable analogues with longer half-lives should be performed. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of SARS-CoV-2.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , Animals , Caco-2 Cells , Chlorocebus aethiops , Computer Simulation , Drug Discovery , Drug Evaluation, Preclinical , Humans , Molecular Docking Simulation , Molecular Structure , Protease Inhibitors/chemistry , Vero Cells
6.
Cell Chem Biol ; 28(6): 855-865.e9, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1201399

ABSTRACT

The COVID-19 pandemic has been disastrous to society and effective drugs are urgently needed. The papain-like protease domain (PLpro) of SARS-CoV-2 (SCoV2) is indispensable for viral replication and represents a putative target for pharmacological intervention. In this work, we describe the development of a potent and selective SCoV2 PLpro inhibitor, 19. The inhibitor not only effectively blocks substrate cleavage and immunosuppressive function imparted by PLpro, but also markedly mitigates SCoV2 replication in human cells, with a submicromolar IC50. We further present a convenient and sensitive activity probe, 7, and complementary assays to readily evaluate SCoV2 PLpro inhibitors in vitro or in cells. In addition, we disclose the co-crystal structure of SCoV2 PLpro in complex with a prototype inhibitor, which illuminates their detailed binding mode. Overall, these findings provide promising leads and important tools for drug discovery aiming to target SCoV2 PLpro.


Subject(s)
Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Delivery Systems/methods , Drug Development/methods , Protease Inhibitors/administration & dosage , SARS-CoV-2/drug effects , A549 Cells , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Antiviral Agents/metabolism , COVID-19/drug therapy , COVID-19/enzymology , Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Dose-Response Relationship, Drug , HEK293 Cells , HeLa Cells , Humans , Mice , Molecular Docking Simulation/methods , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology
7.
Cells ; 10(4)2021 04 06.
Article in English | MEDLINE | ID: covidwho-1178117

ABSTRACT

Coronavirus belongs to the family of Coronaviridae, comprising single-stranded, positive-sense RNA genome (+ ssRNA) of around 26 to 32 kilobases, and has been known to cause infection to a myriad of mammalian hosts, such as humans, cats, bats, civets, dogs, and camels with varied consequences in terms of death and debilitation. Strikingly, novel coronavirus (2019-nCoV), later renamed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and found to be the causative agent of coronavirus disease-19 (COVID-19), shows 88% of sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, 79% with SARS-CoV and 50% with MERS-CoV, respectively. Despite key amino acid residual variability, there is an incredible structural similarity between the receptor binding domain (RBD) of spike protein (S) of SARS-CoV-2 and SARS-CoV. During infection, spike protein of SARS-CoV-2 compared to SARS-CoV displays 10-20 times greater affinity for its cognate host cell receptor, angiotensin-converting enzyme 2 (ACE2), leading proteolytic cleavage of S protein by transmembrane protease serine 2 (TMPRSS2). Following cellular entry, the ORF-1a and ORF-1ab, located downstream to 5' end of + ssRNA genome, undergo translation, thereby forming two large polyproteins, pp1a and pp1ab. These polyproteins, following protease-induced cleavage and molecular assembly, form functional viral RNA polymerase, also referred to as replicase. Thereafter, uninterrupted orchestrated replication-transcription molecular events lead to the synthesis of multiple nested sets of subgenomic mRNAs (sgRNAs), which are finally translated to several structural and accessory proteins participating in structure formation and various molecular functions of virus, respectively. These multiple structural proteins assemble and encapsulate genomic RNA (gRNA), resulting in numerous viral progenies, which eventually exit the host cell, and spread infection to rest of the body. In this review, we primarily focus on genomic organization, structural and non-structural protein components, and potential prospective molecular targets for development of therapeutic drugs, convalescent plasm therapy, and a myriad of potential vaccines to tackle SARS-CoV-2 infection.


Subject(s)
COVID-19/therapy , COVID-19/virology , Drug Discovery , SARS-CoV-2/physiology , Viral Nonstructural Proteins/metabolism , Viral Structural Proteins/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , COVID-19/drug therapy , COVID-19/metabolism , Drug Design , Humans , Immunization, Passive , Molecular Targeted Therapy , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Structural Proteins/chemistry , Viral Structural Proteins/genetics , Virus Internalization/drug effects , Virus Replication/drug effects
8.
Nat Commun ; 12(1): 743, 2021 02 02.
Article in English | MEDLINE | ID: covidwho-1061105

ABSTRACT

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.


Subject(s)
Papain/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Humans , Mutation , Polyproteins/metabolism , Substrate Specificity , Virus Replication/drug effects
9.
Viruses ; 13(2)2021 01 24.
Article in English | MEDLINE | ID: covidwho-1052508

ABSTRACT

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.


Subject(s)
Antiviral Agents/metabolism , Coronavirus 3C Proteases/antagonists & inhibitors , Luciferases/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Cell Survival/drug effects , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Lentivirus/genetics , Luciferases/genetics , Protease Inhibitors/pharmacology
10.
Viruses ; 13(2)2021 01 24.
Article in English | MEDLINE | ID: covidwho-1045366

ABSTRACT

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.


Subject(s)
Antiviral Agents/metabolism , Coronavirus 3C Proteases/antagonists & inhibitors , Luciferases/metabolism , Protease Inhibitors/metabolism , SARS-CoV-2/enzymology , Antiviral Agents/pharmacology , Cell Survival/drug effects , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/metabolism , Drug Evaluation, Preclinical , HEK293 Cells , Humans , Lentivirus/genetics , Luciferases/genetics , Protease Inhibitors/pharmacology
11.
Emerg Microbes Infect ; 10(1): 178-195, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-998195

ABSTRACT

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Interferon Regulatory Factor-3/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Amino Acid Sequence , Animals , COVID-19/pathology , Cell Line , Chiroptera/virology , Coronavirus 3C Proteases/genetics , Coronavirus Papain-Like Proteases/genetics , HEK293 Cells , Humans , Mice , SARS-CoV-2/enzymology , SARS-CoV-2/genetics
12.
J Immunol Res ; 2020: 9465398, 2020.
Article in English | MEDLINE | ID: covidwho-879699

ABSTRACT

This new decade has started with a global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), precipitating a worldwide health crisis and economic downturn. Scientists and clinicians have been racing against time to find therapies for COVID-19. Repurposing approved drugs, developing vaccines and employing passive immunization are three major therapeutic approaches to fighting COVID-19. Chicken immunoglobulin Y (IgY) has the potential to be used as neutralizing antibody against respiratory infections, and its advantages include high avidity, low risk of adverse immune responses, and easy local delivery by intranasal administration. In this study, we raised antibody against the spike (S) protein of SARS-CoV-2 in chickens and extracted IgY (called IgY-S) from egg yolk. IgY-S exhibited high immunoreactivity against SARS-CoV-2 S, and by epitope mapping, we found five linear epitopes of IgY-S in SARS-CoV-2 S, two of which are cross-reactive with SARS-CoV S. Notably, epitope SIIAYTMSL, one of the identified epitopes, partially overlaps the S1/S2 cleavage region in SARS-CoV-2 S and is located on the surface of S trimer in 3D structure, close to the S1/S2 cleavage site. Thus, antibody binding at this location could physically block the access of proteolytic enzymes to S1/S2 cleavage site and thereby impede S1/S2 proteolytic cleavage, which is crucial to subsequent virus-cell membrane fusion and viral cell entry. Therefore, the feasibility of using IgY-S or epitope SIIAYTMS-specific IgY as neutralizing antibody for preventing or treating SARS-CoV-2 infection is worth exploring.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/therapy , Epitope Mapping , Immunoglobulins/isolation & purification , Pneumonia, Viral/therapy , Administration, Intranasal , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/administration & dosage , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , COVID-19 , Chickens , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cross Reactions , Feasibility Studies , Humans , Immunization, Passive/methods , Immunoglobulins/administration & dosage , Immunoglobulins/immunology , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
13.
Nat Commun ; 11(1): 4282, 2020 08 27.
Article in English | MEDLINE | ID: covidwho-733525

ABSTRACT

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus, Feline/drug effects , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , A549 Cells , Animals , Antiviral Agents/chemistry , Betacoronavirus/enzymology , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus, Feline/enzymology , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cytopathogenic Effect, Viral/drug effects , Drug Repositioning , Humans , Inhibitory Concentration 50 , Molecular Structure , Prodrugs , Protease Inhibitors/chemistry , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2 , Sulfonic Acids , Vero Cells , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects
14.
J Med Virol ; 92(6): 602-611, 2020 06.
Article in English | MEDLINE | ID: covidwho-153847

ABSTRACT

To investigate the evolutionary history of the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China, a total of 70 genomes of virus strains from China and elsewhere with sampling dates between 24 December 2019 and 3 February 2020 were analyzed. To explore the potential intermediate animal host of the SARS-CoV-2 virus, we reanalyzed virome data sets from pangolins and representative SARS-related coronaviruses isolates from bats, with particular attention paid to the spike glycoprotein gene. We performed phylogenetic, split network, transmission network, likelihood-mapping, and comparative analyses of the genomes. Based on Bayesian time-scaled phylogenetic analysis using the tip-dating method, we estimated the time to the most recent common ancestor and evolutionary rate of SARS-CoV-2, which ranged from 22 to 24 November 2019 and 1.19 to 1.31 × 10-3 substitutions per site per year, respectively. Our results also revealed that the BetaCoV/bat/Yunnan/RaTG13/2013 virus was more similar to the SARS-CoV-2 virus than the coronavirus obtained from the two pangolin samples (SRR10168377 and SRR10168378). We also identified a unique peptide (PRRA) insertion in the human SARS-CoV-2 virus, which may be involved in the proteolytic cleavage of the spike protein by cellular proteases, and thus could impact host range and transmissibility. Interestingly, the coronavirus carried by pangolins did not have the RRAR motif. Therefore, we concluded that the human SARS-CoV-2 virus, which is responsible for the recent outbreak of COVID-19, did not come directly from pangolins.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Genome, Viral , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Betacoronavirus/classification , Betacoronavirus/pathogenicity , COVID-19 , Chiroptera/virology , Coronavirus Infections/virology , Eutheria/virology , Evolution, Molecular , Host Specificity , Humans , Phylogeny , Pneumonia, Viral/virology , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/metabolism
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