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1.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-624792

ABSTRACT

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Subject(s)
Actinobacteria/genetics , Genome, Bacterial , Protein Interaction Domains and Motifs/drug effects , Small Molecule Libraries/pharmacology , Tacrolimus Binding Protein 1A/chemistry , Tacrolimus Binding Protein 1A/metabolism , Actinobacteria/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Autoantigens/genetics , Autoantigens/metabolism , Calcineurin/genetics , Calcineurin/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Evolution, Molecular , HEK293 Cells , Humans , Models, Molecular , Protein Conformation , Sequence Homology , Sirolimus/chemistry , Sirolimus/metabolism , Small Molecule Libraries/chemistry , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
2.
Cell Rep ; 31(11): 107772, 2020 06 16.
Article in English | MEDLINE | ID: covidwho-626232

ABSTRACT

ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-ß-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.


Subject(s)
Cytokines/metabolism , Signal Transduction , Ubiquitins/metabolism , Animals , HEK293 Cells , Humans , Influenza, Human/immunology , Influenza, Human/metabolism , Interferon-gamma/immunology , Interferon-gamma/metabolism , Jurkat Cells , Mice , Mice, Inbred C57BL , Mycobacterium Infections/immunology , Mycobacterium Infections/metabolism , Pathogen-Associated Molecular Pattern Molecules , Typhoid Fever/immunology , Typhoid Fever/metabolism , Viral Nonstructural Proteins/metabolism
3.
J Exp Med ; 217(12)2020 12 07.
Article in English | MEDLINE | ID: covidwho-726090

ABSTRACT

Type I interferons (IFN-I) are a major antiviral defense and are critical for the activation of the adaptive immune system. However, early viral clearance by IFN-I could limit antigen availability, which could in turn impinge upon the priming of the adaptive immune system. In this study, we hypothesized that transient IFN-I blockade could increase antigen presentation after acute viral infection. To test this hypothesis, we infected mice with viruses coadministered with a single dose of IFN-I receptor-blocking antibody to induce a short-term blockade of the IFN-I pathway. This resulted in a transient "spike" in antigen levels, followed by rapid antigen clearance. Interestingly, short-term IFN-I blockade after coronavirus, flavivirus, rhabdovirus, or arenavirus infection induced a long-lasting enhancement of immunological memory that conferred improved protection upon subsequent reinfections. Short-term IFN-I blockade also improved the efficacy of viral vaccines. These findings demonstrate a novel mechanism by which IFN-I regulate immunological memory and provide insights for rational vaccine design.


Subject(s)
Immunogenicity, Vaccine/immunology , Interferon Type I/antagonists & inhibitors , Interferon-alpha/immunology , Receptor, Interferon alpha-beta/immunology , Viral Vaccines/immunology , Zika Virus Infection/immunology , Zika Virus/immunology , Animals , Antibodies, Blocking/immunology , Antibodies, Blocking/pharmacology , Antibodies, Viral/immunology , Antigen Presentation/immunology , CD8-Positive T-Lymphocytes/metabolism , Dendritic Cells/immunology , Disease Models, Animal , Gene Expression/immunology , HEK293 Cells , Humans , Immunologic Memory , Interferon-alpha/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Receptor, Interferon alpha-beta/genetics , Transfection , Zika Virus Infection/virology
4.
Nat Commun ; 11(1): 4207, 2020 08 21.
Article in English | MEDLINE | ID: covidwho-724410

ABSTRACT

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/administration & dosage , Adenoviridae/genetics , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Coronavirus Infections/immunology , Dose-Response Relationship, Immunologic , Female , HEK293 Cells , Humans , Immunity, Cellular , Macaca mulatta , Male , Mice , Mice, Inbred BALB C , Pneumonia, Viral/immunology , Respiratory System/pathology , Respiratory System/virology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/administration & dosage
5.
Nat Commun ; 11(1): 4198, 2020 08 21.
Article in English | MEDLINE | ID: covidwho-724360

ABSTRACT

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Immunoglobulin A/immunology , Peptidyl-Dipeptidase A/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Chlorocebus aethiops , Cross Reactions , Epitopes , HEK293 Cells , Humans , Immunoglobulin A/metabolism , Immunoglobulin A, Secretory/immunology , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Interaction Domains and Motifs , SARS Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
6.
Nat Commun ; 11(1): 4081, 2020 08 14.
Article in English | MEDLINE | ID: covidwho-717117

ABSTRACT

The unprecedented coronavirus disease 2019 (COVID-19) epidemic has created a worldwide public health emergency, and there is an urgent need to develop an effective vaccine to control this severe infectious disease. Here, we find that a single vaccination with a replication-defective human type 5 adenovirus encoding the SARS-CoV-2 spike protein (Ad5-nCoV) protect mice completely against mouse-adapted SARS-CoV-2 infection in the upper and lower respiratory tracts. Additionally, a single vaccination with Ad5-nCoV protects ferrets from wild-type SARS-CoV-2 infection in the upper respiratory tract. This study suggests that the mucosal vaccination may provide a desirable protective efficacy and this delivery mode is worth further investigation in human clinical trials.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Animals , Antibodies, Viral/immunology , Coronavirus Infections/immunology , Disease Models, Animal , Drug Design , Female , Genetic Vectors , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/genetics
7.
Emerg Microbes Infect ; 9(1): 1567-1579, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-707709

ABSTRACT

Diverse SARS-like coronaviruses (SL-CoVs) have been identified from bats and other animal species. Like SARS-CoV, some bat SL-CoVs, such as WIV1, also use angiotensin converting enzyme 2 (ACE2) from human and bat as entry receptor. However, whether these viruses can also use the ACE2 of other animal species as their receptor remains to be determined. We report herein that WIV1 has a broader tropism to ACE2 orthologs than SARS-CoV isolate Tor2. Among the 9 ACE2 orthologs examined, human ACE2 exhibited the highest efficiency to mediate the infection of WIV1 pseudotyped virus. Our findings thus imply that WIV1 has the potential to infect a wide range of wild animals and may directly jump to humans. We also showed that cell entry of WIV1 could be restricted by interferon-induced transmembrane proteins (IFITMs). However, WIV1 could exploit the airway protease TMPRSS2 to partially evade the IFITM3 restriction. Interestingly, we also found that amphotericin B could enhance the infectious entry of SARS-CoVs and SL-CoVs by evading IFITM3-mediated restriction. Collectively, our findings further underscore the risk of exposure to animal SL-CoVs and highlight the vulnerability of patients who take amphotericin B to infection by SL-CoVs, including the most recently emerging (SARS-CoV-2).


Subject(s)
Betacoronavirus/physiology , Chiroptera/virology , Membrane Proteins/metabolism , Peptidyl-Dipeptidase A/metabolism , RNA-Binding Proteins/metabolism , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Virus Internalization , Animals , Betacoronavirus/classification , HEK293 Cells , Humans , Rats , SARS Virus/physiology , Viverridae
8.
Mol Syst Biol ; 16(7): e9628, 2020 07.
Article in English | MEDLINE | ID: covidwho-707164

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has is a global health challenge. Angiotensin-converting enzyme 2 (ACE2) is the host receptor for SARS-CoV-2 entry. Recent studies have suggested that patients with hypertension and diabetes treated with ACE inhibitors (ACEIs) or angiotensin receptor blockers have a higher risk of COVID-19 infection as these drugs could upregulate ACE2, motivating the study of ACE2 modulation by drugs in current clinical use. Here, we mined published datasets to determine the effects of hundreds of clinically approved drugs on ACE2 expression. We find that ACEIs are enriched for ACE2-upregulating drugs, while antineoplastic agents are enriched for ACE2-downregulating drugs. Vorinostat and isotretinoin are the top ACE2 up/downregulators, respectively, in cell lines. Dexamethasone, a corticosteroid used in treating severe acute respiratory syndrome and COVID-19, significantly upregulates ACE2 both in vitro and in vivo. Further top ACE2 regulators in vivo or in primary cells include erlotinib and bleomycin in the lung and vancomycin, cisplatin, and probenecid in the kidney. Our study provides leads for future work studying ACE2 expression modulators.


Subject(s)
Angiotensin Receptor Antagonists/pharmacology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , A549 Cells , Betacoronavirus , Bleomycin/pharmacology , Dexamethasone/pharmacology , Drug Design , Drug Evaluation, Preclinical , Erlotinib Hydrochloride/pharmacology , Fluphenazine/pharmacology , HEK293 Cells , Humans , Kidney/drug effects , Lung/drug effects , MCF-7 Cells , Pandemics , Peptidyl-Dipeptidase A , Systems Biology , Up-Regulation , Vemurafenib/pharmacology
9.
J Exp Med ; 217(11)2020 11 02.
Article in English | MEDLINE | ID: covidwho-697830

ABSTRACT

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , Betacoronavirus/immunology , Coronavirus Infections/immunology , Immunoassay/methods , Pneumonia, Viral/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/genetics , Cell Line , Chimera/genetics , Chimera/immunology , Chlorocebus aethiops , Coronavirus Infections/virology , HEK293 Cells , HIV-1/genetics , HIV-1/immunology , Humans , Neutralization Tests/methods , Pandemics , Pneumonia, Viral/virology , Recombination, Genetic , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/immunology
10.
Biochem Biophys Res Commun ; 529(2): 251-256, 2020 08 20.
Article in English | MEDLINE | ID: covidwho-692859

ABSTRACT

The nucleocapsid protein is significant in the formation of viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins. Here, we report for the first time that the 11S proteasomal activator PA28γ regulates the intracellular abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identified proteasome activator PA28γ as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction, nCoV N could be degraded by PA28γ-20S in vitro degradation assay. This was also demonstrated by blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28γ-knockout cells was greater than in PA28γ-wildtype cells. Notably, immunofluorescence staining revealed that knockout of the PA28γ gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression of PA28γ enhanced proteolysis of nCoV N compared to that in PA28γ-N151Y cells containing a dominant-negative PA28γ mutation, which reduced this process. These results suggest that PA28γ binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-19.


Subject(s)
Autoantigens/metabolism , Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Nucleocapsid Proteins/metabolism , Pneumonia, Viral/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Coronavirus Infections/virology , HEK293 Cells , Humans , In Vitro Techniques , Pandemics , Pneumonia, Viral/virology , Protein Binding , Protein Stability , Protein Transport
11.
Life Sci Alliance ; 3(9)2020 09.
Article in English | MEDLINE | ID: covidwho-675904

ABSTRACT

The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.


Subject(s)
Alveolar Epithelial Cells/virology , Betacoronavirus/physiology , Furin/genetics , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Alveolar Epithelial Cells/cytology , Animals , Binding Sites , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Internalization , Virus Replication
12.
Biochem Biophys Res Commun ; 530(1): 4-9, 2020 09 10.
Article in English | MEDLINE | ID: covidwho-663155

ABSTRACT

COVID-19 has become one of the worst epidemic in the world, currently already more than four million people have been infected, which probably co-exist with human beings, and has a significant impact on the global economy and political order. In the process of fighting against the epidemic in China, the clinical value of a variety of herbal medicines has been recognized and written into the clinical application guide. However, their effective molecular mechanism and potential targets are still not clear. Pathology and pharmacology research will gradually attract attention in the post-epidemic outbreak term. Here, we constructed a COVID-19 protein microarray of potential therapy targets, which contains the main drug targets to the SARS-CoV-2 virus and the anti-virus, anti-inflammatory cellar targets of the host. Series of quality controls test has been carried out, which showed that it could be applied for drug target screening of bio-active natural products. The establishment of this microarray will provide a useful tool for the study of the molecular pharmacology of natural products.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Drugs, Chinese Herbal/pharmacology , Pneumonia, Viral/drug therapy , Proteins/metabolism , Viral Proteins/metabolism , Betacoronavirus/metabolism , Biological Products/pharmacology , Chlorogenic Acid/pharmacology , Coronavirus Infections/metabolism , Diterpenes/pharmacology , Drug Discovery , Glucosides/pharmacology , HEK293 Cells , Humans , Molecular Docking Simulation , Molecular Targeted Therapy , Pandemics , Pneumonia, Viral/metabolism , Protein Array Analysis , Stilbenes/pharmacology
13.
Nature ; 584(7821): 353-363, 2020 08.
Article in English | MEDLINE | ID: covidwho-643609

ABSTRACT

Antibody-dependent enhancement (ADE) of disease is a general concern for the development of vaccines and antibody therapies because the mechanisms that underlie antibody protection against any virus have a theoretical potential to amplify the infection or trigger harmful immunopathology. This possibility requires careful consideration at this critical point in the pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we review observations relevant to the risks of ADE of disease, and their potential implications for SARS-CoV-2 infection. At present, there are no known clinical findings, immunological assays or biomarkers that can differentiate any severe viral infection from immune-enhanced disease, whether by measuring antibodies, T cells or intrinsic host responses. In vitro systems and animal models do not predict the risk of ADE of disease, in part because protective and potentially detrimental antibody-mediated mechanisms are the same and designing animal models depends on understanding how antiviral host responses may become harmful in humans. The implications of our lack of knowledge are twofold. First, comprehensive studies are urgently needed to define clinical correlates of protective immunity against SARS-CoV-2. Second, because ADE of disease cannot be reliably predicted after either vaccination or treatment with antibodies-regardless of what virus is the causative agent-it will be essential to depend on careful analysis of safety in humans as immune interventions for COVID-19 move forward.


Subject(s)
Antibodies, Viral/adverse effects , Antibodies, Viral/immunology , Antibody-Dependent Enhancement/immunology , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , Coronavirus Infections/immunology , Coronavirus Infections/virology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Animals , Antibodies, Neutralizing/adverse effects , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Coronavirus Infections/prevention & control , Dengue Virus/immunology , Disease Models, Animal , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Macaca mulatta , Mice , Middle East Respiratory Syndrome Coronavirus/immunology , Orthomyxoviridae/immunology , Pandemics , Rats , SARS Virus/immunology , Viral Vaccines/adverse effects , Viral Vaccines/immunology
14.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Article in English | MEDLINE | ID: covidwho-640223

ABSTRACT

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Subject(s)
Betacoronavirus/genetics , Host-Pathogen Interactions/genetics , Peptidyl-Dipeptidase A/chemistry , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Animals , Betacoronavirus/metabolism , Betacoronavirus/ultrastructure , Binding Sites , Chiroptera/virology , Coronavirus Infections/virology , Cryoelectron Microscopy , Evolution, Molecular , Furin/chemistry , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Stability , Proteolysis , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structural Homology, Protein
15.
Cell ; 182(3): 685-712.e19, 2020 08 06.
Article in English | MEDLINE | ID: covidwho-624826

ABSTRACT

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Drug Evaluation, Preclinical/methods , Pneumonia, Viral/metabolism , Proteomics/methods , A549 Cells , Animals , Antiviral Agents/pharmacology , Caco-2 Cells , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/metabolism , Chlorocebus aethiops , Coronavirus Infections/virology , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , HEK293 Cells , Host-Pathogen Interactions , Humans , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Phosphorylation , Pneumonia, Viral/virology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism
16.
Cell ; 182(3): 722-733.e11, 2020 08 06.
Article in English | MEDLINE | ID: covidwho-628738

ABSTRACT

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Middle East Respiratory Syndrome Coronavirus/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , SARS Virus/immunology , Universal Design , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/chemistry , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Infections/virology , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Middle East Respiratory Syndrome Coronavirus/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Binding , Protein Interaction Domains and Motifs/immunology , Receptors, Virus/metabolism , SARS Virus/chemistry , Sf9 Cells , Specific Pathogen-Free Organisms , Spodoptera , Transfection , Vaccination/methods , Vero Cells , Viral Vaccines
17.
J Immunol ; 205(4): 915-922, 2020 08 15.
Article in English | MEDLINE | ID: covidwho-616100

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Pneumonia, Viral/drug therapy , Pneumonia, Viral/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , Chlorocebus aethiops , Coronavirus Infections/virology , Disease Models, Animal , Epitope Mapping , Female , HEK293 Cells , Humans , Immunodominant Epitopes/immunology , Mice , Mice, Inbred C57BL , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Transfection , Vero Cells
18.
Vaccine ; 38(35): 5653-5658, 2020 07 31.
Article in English | MEDLINE | ID: covidwho-612504

ABSTRACT

The COVID-19 outbreak has become a global pandemic responsible for over 2,000,000 confirmed cases and over 126,000 deaths worldwide. In this study, we examined the immunogenicity of CHO-expressed recombinant SARS-CoV-2 S1-Fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a COVID-19 vaccine. We demonstrate that the S1-Fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day 7. Strong virus neutralizing activity was observed on day 14 in rabbits immunized with the S1-Fc fusion protein using a pseudovirus neutralization assay. Most importantly, in <20 days and three injections of the S1-Fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered COVID-19 patient in a live SARS-CoV-2 infection assay. Our data strongly suggests that the CHO-expressed SARS-CoV-2 S1-Fc recombinant protein could be a strong candidate for vaccine development against COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/immunology , Immunoglobulin Fc Fragments/chemistry , Macaca/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Animals , CHO Cells , Coronavirus Infections/prevention & control , Coronavirus Infections/therapy , Cricetulus , Female , HEK293 Cells , Humans , Immunization, Passive , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Mice , Pandemics , Rabbits
19.
Virus Res ; 286: 198074, 2020 09.
Article in English | MEDLINE | ID: covidwho-611212

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Subject(s)
Betacoronavirus/genetics , Host-Pathogen Interactions/genetics , Interferon-beta/genetics , NF-kappa B/genetics , Nucleocapsid Proteins/genetics , Viral Proteins/genetics , Betacoronavirus/immunology , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Interferon-beta/immunology , Luciferases/genetics , Luciferases/metabolism , NF-kappa B/immunology , Nucleocapsid Proteins/immunology , Plasmids/chemistry , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Response Elements , Sendai virus/genetics , Sendai virus/immunology , Signal Transduction , Transfection/methods , Viral Proteins/immunology
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