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1.
Proc Natl Acad Sci U S A ; 119(32): e2205690119, 2022 Aug 09.
Article in English | MEDLINE | ID: covidwho-1960628

ABSTRACT

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.


Subject(s)
COVID-19 , Furin , Proteolysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Amino Acid Motifs/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Furin/chemistry , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Deletion , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication/genetics
2.
Virus Res ; 318: 198845, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1946817

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents an immense global health problem. Spike (S) protein of coronavirus is the primary determinant of its entry into the host as it consists of both receptor binding and fusion domain. Besides tissue tropism, and host range, coronavirus pathogenesis are primarily controlled by the interaction of S protein with the cell receptor. Moreover, the proteolytic activation of S protein by host cell proteases plays a decisive role. The host-cell proteases have shown to be involved in the proteolysis of S protein and cleaving it into two functional subunits, S1 and S2, during the maturation process. In the present study, the interaction of the S protein of SARS-CoV-2 with different host proteases like furin, cathepsin B, and plasmin has been analyzed using molecular docking and molecular dynamics (MD) simulation. Incorporation of the furin cleavage site (R-R-A-R) in the S protein of SARS-CoV-2 has been studied by mutating the individual amino acid. MD simulation results suggest the polytropic nature of the S protein. Our analysis indicated that a single amino acid substitution in the polybasic cleavage site of S protein perturb the binding of cellular proteases. This mutation study might help to generate an attenuated SARS-CoV-2. Besides, targeting host proteases by inhibitors may result in a practical approach to stop the cellular spread of SARS-CoV-2 and develop its antiviral.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Furin/chemistry , Humans , Molecular Docking Simulation , Peptide Hydrolases/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
3.
J Mol Model ; 28(8): 224, 2022 Jul 19.
Article in English | MEDLINE | ID: covidwho-1941795

ABSTRACT

Viral-cell entry and cell-cell viral spreading processes of SARS-CoV-2 are subjected to fast evolutionary optimization because of its worldwide spreading, requiring the need for new drug developments. However, this task is still challenging, because a detailed understanding of the underlying molecular processes, mediated by the key cellular proteases TMPRSS2 and furin, is still lacking. Here, we show by large-scale atomistic calculations that binding of the ACE2 cell receptor at one of the heteromers of the SARS-CoV-2 spike leads to a release of its furin cleavage site (S1/S2), enabling an enhanced furin binding, and that this latter process promotes the binding of TMPRSS2 through the release of the TMPRSS2 cleavage site (S2') out of the ACE2-binding heteromer. Moreover, we find that, after proteolytic cleavage, improved furin binding causes that parts of the S2 subunit dissociate from the complex, suggesting that furin promotes the fusion of the S2 subunit with the cell membrane before transfer of the viral RNA. Here we show by computational means that binding of the ACE2-cell receptor at one of the heteromers of the SARS-CoV-2 spike leads to an enhanced binding of the protease furin, promoting the binding of the protease TMPRSS2. Moreover, we show that, after proteolytic cleavage, improved furin binding causes that parts of the heteromer dissociate from the spike.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Furin/chemistry , Furin/genetics , Furin/metabolism , Humans , Peptide Hydrolases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
4.
ACS Chem Biol ; 17(4): 816-821, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1852374

ABSTRACT

Inhibitors of the proprotein convertase furin might serve as broad-spectrum antiviral therapeutics. High cellular potency and antiviral activity against acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported for (3,5-dichlorophenyl)pyridine-derived furin inhibitors. Here we characterized the binding mechanism of this inhibitor class using structural, biophysical, and biochemical methods. We established a MALDI-TOF-MS-based furin activity assay, determined IC50 values, and solved X-ray structures of (3,5-dichlorophenyl)pyridine-derived compounds in complex with furin. The inhibitors induced a substantial conformational rearrangement of the active-site cleft by exposing a central buried tryptophan residue. These changes formed an extended hydrophobic surface patch where the 3,5-dichlorophenyl moiety of the inhibitors was inserted into a newly formed binding pocket. Consistent with these structural rearrangements, we observed slow off-rate binding kinetics and strong structural stabilization in surface plasmon resonance and differential scanning fluorimetry experiments, respectively. The discovered furin conformation offers new opportunities for structure-based drug discovery.


Subject(s)
Antiviral Agents , Furin , SARS-CoV-2 , Antiviral Agents/chemistry , Furin/chemistry , Proprotein Convertases , SARS-CoV-2/drug effects
5.
Int J Mol Sci ; 23(5)2022 Mar 03.
Article in English | MEDLINE | ID: covidwho-1732066

ABSTRACT

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.


Subject(s)
Amino Acid Chloromethyl Ketones/pharmacology , Enoxaparin/pharmacology , Furin/antagonists & inhibitors , Spermine/analogs & derivatives , Zeaxanthins/pharmacology , Amino Acid Chloromethyl Ketones/chemistry , Amino Acid Chloromethyl Ketones/metabolism , COVID-19/transmission , COVID-19/virology , Catalytic Domain , Cell Line, Tumor , Cell Survival/drug effects , Enoxaparin/chemistry , Enoxaparin/metabolism , Furin/chemistry , Furin/metabolism , HEK293 Cells , Humans , Molecular Docking Simulation , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Proteolysis , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spermine/chemistry , Spermine/metabolism , Spermine/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Virus Replication , Zeaxanthins/chemistry , Zeaxanthins/metabolism
6.
Comput Math Methods Med ; 2022: 9735626, 2022.
Article in English | MEDLINE | ID: covidwho-1677416

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was characterized as a pandemic by the World Health Organization (WHO) in Dec. 2019. SARS-CoV-2 binds to the cell membrane through spike proteins on its surface and infects the cell. Furin, a host-cell enzyme, possesses a binding site for the spike protein. Thus, molecules that block furin could potentially be a therapeutic solution. Defensins are antimicrobial peptides that can hypothetically inhibit furin because of their arginine-rich structure. Theta-defensins, a subclass of defensins, have attracted attention as drug candidates due to their small size, unique structure, and involvement in several defense mechanisms. Theta-defensins could be a potential treatment for COVID-19 through furin inhibition and an anti-inflammatory mechanism. Note that inflammatory events are a significant and deadly condition that could happen at the later stages of COVID-19 infection. Here, the potential of theta-defensins against SARS-CoV-2 infection was investigated through in silico approaches. Based on docking analysis results, theta-defensins can function as furin inhibitors. Additionally, a novel candidate peptide against COVID-19 with optimal properties regarding antigenicity, stability, electrostatic potential, and binding strength was proposed. Further in vitro/in vivo investigations could verify the efficiency of the designed novel peptide.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/metabolism , Defensins/pharmacology , Drug Design , Furin/antagonists & inhibitors , Animals , COVID-19/drug therapy , Catalytic Domain , Cell Membrane/virology , Computer Simulation , Data Mining , Furin/chemistry , Humans , Inflammation , Models, Molecular , Molecular Docking Simulation , Peptides/chemistry , Software , Spike Glycoprotein, Coronavirus , Static Electricity
7.
J Food Biochem ; 46(6): e14039, 2022 06.
Article in English | MEDLINE | ID: covidwho-1608330

ABSTRACT

This study aims to investigate the effects of whey proteins on SARS CoV-2 in methotrexate-induced lung tissue damage in rats. To determine the possible effects, rats were divided into four groups as control, control + whey, methotrexate (20 mg/kg, i.p.) and methotrexate + whey. Whey protein concentrate (2 g/kg, oral gavage) was administered for 10 days. Cytokine levels were measured and protein electrophoresis was carried out in serum samples. Lipid peroxidation, nitric oxide and glutathione level, and superoxide dismutase and glutathione S transferase activities were determined in lung samples. Inhibition of SARS CoV-2-targeted lung furin activity and SARS CoV-2 spike protein-angiotensin converting enzyme binding with whey protein concentrate were also measured in each group. In conclusion, whey protein concentrate improved methotrexate-induced lung damage and inhibited lung furin activity targeting SARS-CoV-2 S1/S2 site cleavage and SARS CoV-2 spike protein-angiotensin converting enzyme binding. Whey proteins are potential protective candidates that inhibit SARS CoV-2-related interactions, even in methotrexate-induced lung injury. PRACTICAL APPLICATIONS: Whey proteins have anticarcinogenic, antihypertensive, antioxidant, antibacterial, antiviral, and immunomodulating properties due to the protein, bioactive peptide, and essential amino acid content. Methotrexate is a folate antagonist and inhibits cell proliferation and purine synthesis. The combined use of whey protein concentrate and methotrexate may be an alternative in the development of new strategies to the treatment approaches against COVID-19. In addition, according to the results of this study, it is thought that the protective effect of whey proteins in healthy conditions before encountering the SARS CoV-2 may be higher than those who have never used it.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Animals , COVID-19/drug therapy , Furin/chemistry , Furin/metabolism , Lung , Methotrexate/adverse effects , Peptidyl-Dipeptidase A/chemistry , Rats , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Whey Proteins
8.
Virus Res ; 303: 198522, 2021 10 02.
Article in English | MEDLINE | ID: covidwho-1447221

ABSTRACT

The B.1.1.7 SARS-CoV-2 strain that has emerged in the UK in early December presents seven mutations and three deletions on S-protein structure that could lead to a more infective strain. The P681H mutation in the "PRRAR" furin cleavage site might affect the binding affinity to furin enzyme and hence its infectivity. Therefore, in this study, various structural bioinformatics approaches were used to model the S-protein structure with the B.1.1.7 variant amino acid substitutions and deletions. In addition to modelling the binding of furin to the cleavage site of the wild-type and the B.1.1.7 variant. Conclusively the B.1.1.7 variant resulted in dynamic stability, conformational changes and variations in binding energies in the S-protein structure, resulting in a more favourable binding of furin enzyme to the SARS-CoV-2 S-protein.


Subject(s)
Furin/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Furin/chemistry , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
9.
Cells ; 10(9)2021 09 15.
Article in English | MEDLINE | ID: covidwho-1408625

ABSTRACT

Coronavirus disease 19 (COVID-19) is caused by an enveloped, positive-sense, single-stranded RNA virus, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the realm Riboviria, order Nidovirales, family Coronaviridae, genus Betacoronavirus and the species Severe acute respiratory syndrome-related coronavirus. This viral disease is characterized by a myriad of varying symptoms, such as pyrexia, cough, hemoptysis, dyspnoea, diarrhea, muscle soreness, dysosmia, lymphopenia and dysgeusia amongst others. The virus mainly infects humans, various other mammals, avian species and some other companion livestock. SARS-CoV-2 cellular entry is primarily accomplished by molecular interaction between the virus's spike (S) protein and the host cell surface receptor, angiotensin-converting enzyme 2 (ACE2), although other host cell-associated receptors/factors, such as neuropilin 1 (NRP-1) and neuropilin 2 (NRP-2), C-type lectin receptors (CLRs), as well as proteases such as TMPRSS2 (transmembrane serine protease 2) and furin, might also play a crucial role in infection, tropism, pathogenesis and clinical outcome. Furthermore, several structural and non-structural proteins of the virus themselves are very critical in determining the clinical outcome following infection. Considering such critical role(s) of the abovementioned host cell receptors, associated proteases/factors and virus structural/non-structural proteins (NSPs), it may be quite prudent to therapeutically target them through a multipronged clinical regimen to combat the disease.


Subject(s)
COVID-19 , Host Microbial Interactions , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/pathology , COVID-19/virology , Drug Delivery Systems , Furin/chemistry , Furin/metabolism , Humans , Lectins, C-Type/chemistry , Lectins, C-Type/metabolism , Molecular Structure , Neuropilins/chemistry , Neuropilins/metabolism , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Treatment Outcome , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Internalization
10.
ACS Chem Biol ; 16(9): 1692-1700, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1366786

ABSTRACT

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin's active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin's OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.


Subject(s)
Antiviral Agents/metabolism , Furin/antagonists & inhibitors , Guanidines/metabolism , Hydrazones/metabolism , Serine Proteinase Inhibitors/metabolism , Antiviral Agents/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzyme Assays , Furin/chemistry , Furin/metabolism , Guanidines/chemistry , HEK293 Cells , Humans , Hydrazones/chemistry , Kinetics , Proprotein Convertase 5/antagonists & inhibitors , Proprotein Convertase 5/chemistry , Protein Binding , Protein Conformation , Serine Proteinase Inhibitors/chemistry , Subtilisins/antagonists & inhibitors , Subtilisins/chemistry
11.
Arch Virol ; 166(9): 2541-2549, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1309045

ABSTRACT

The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.


Subject(s)
COVID-19/epidemiology , Furin/metabolism , Polymorphism, Genetic , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Animals , Binding Sites , COVID-19/transmission , COVID-19/virology , Chiroptera/virology , Epidemiological Monitoring , Eutheria/virology , Evolution, Molecular , Furin/chemistry , Gene Expression , Humans , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Proteolysis , SARS-CoV-2/chemistry , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
12.
J Phys Chem Lett ; 11(16): 6655-6663, 2020 Aug 20.
Article in English | MEDLINE | ID: covidwho-678528

ABSTRACT

The COVID-19 pandemic is an urgent global health emergency, and the presence of Furin site in the SARS-CoV-2 spike glycoprotein alters virulence and warrants further molecular, structural, and biophysical studies. Here we report the structure of Furin in complex with SARS-CoV-2 spike glycoprotein, demonstrating how Furin binds to the S1/S2 region of spike glycoprotein and eventually cleaves the viral protein using experimental functional studies, molecular dynamics, and docking. The structural studies underline the mechanism and mode of action of Furin, which is a key process in host cell entry and a hallmark of enhanced virulence. Our whole-exome sequencing analysis shows the genetic variants/alleles in Furin were found to alter the binding affinity for viral spike glycoprotein and could vary in infectivity in humans. Unravelling the mechanisms of Furin action, binding dynamics, and the genetic variants opens the growing arena of bona fide antibodies and development of potential therapeutics targeting the blockage of Furin cleavage.


Subject(s)
Betacoronavirus/chemistry , Furin/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virulence/physiology , Amino Acid Sequence , Animals , Betacoronavirus/pathogenicity , CHO Cells , Catalytic Domain , Cricetulus , Furin/chemistry , Furin/genetics , Gene Expression/physiology , Hexosamines/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Proteolysis , SARS-CoV-2 , Serine Proteinase Inhibitors/metabolism , Spike Glycoprotein, Coronavirus/chemistry
13.
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 , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/metabolism , Betacoronavirus/ultrastructure , Binding Sites , COVID-19 , 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 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structural Homology, Protein
14.
Biophys Chem ; 264: 106420, 2020 09.
Article in English | MEDLINE | ID: covidwho-623862

ABSTRACT

One notable feature of the SARS-CoV-2 genome, the spike (S) protein of SARS-CoV-2 has a polybasic furin cleavage site (FCS) at its S1-S2 boundary through the insertion of 12 nucleotides encoding four amino acid residues PRRA. Quite intriguingly, this polybasic FCS is absent in coronaviruses of the same clade as SARS-CoV-2. Thus, with currently available experimental structural data for S protein, this short article presents a set of comprehensive structural characterization of the insertion of FCS into S protein, and argues against a hypothesis of the origin of SARS-CoV-2 from purposeful manipulation: (1), the inserted FCS is spatially located at a random coil loop region, mostly distantly solvent-exposed (instead of deeply buried), with no structural proximity to the other part of the S protein; (2), the insertion of FCS itself does not alter, neither stabilize nor de-stabilize, the three-dimensional structure of S; (3), the net result here is the insertion of a furin cleavage site into S protein, whose S1 and S2 subunits will still be strongly electrostatically bonded together from a structural and biophysical point of view, even if the polybasic FCS is actually cleaved by furin protease before or after viral cell entry.


Subject(s)
Betacoronavirus/chemistry , Furin/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Betacoronavirus/genetics , Gene Expression , Humans , Hydrogen Bonding , Models, Molecular , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Proteolysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structural Homology, Protein
15.
Mol Cell ; 78(4): 779-784.e5, 2020 05 21.
Article in English | MEDLINE | ID: covidwho-88548

ABSTRACT

The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/virology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/chemistry , Animals , Betacoronavirus/physiology , COVID-19 , Cell Line , Chlorocebus aethiops , Furin/chemistry , Furin/genetics , Furin/metabolism , Humans , Lung/metabolism , Lung/virology , Pandemics , SARS-CoV-2 , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Attachment
16.
Antiviral Res ; 176: 104742, 2020 04.
Article in English | MEDLINE | ID: covidwho-624

ABSTRACT

In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.


Subject(s)
Betacoronavirus/chemistry , Furin/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Sequence , Genome, Viral , Protein Conformation , SARS-CoV-2
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