Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 21
Filter
1.
J Med Chem ; 64(19): 14887-14894, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1428719

ABSTRACT

Antiviral treatments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been extensively pursued to conquer the pandemic. To inhibit the viral entry to the host cell, we designed and obtained three peptide sequences via quartz crystal microbalance measurement screening, which showed high affinity at nanomole to the S1 subunit of the spike protein and wild-type SARS-CoV-2 pseudovirus. Circular dichroism spectroscopy measurements revealed significant conformation changes of the S1 protein upon encounter with the three peptides. The peptides were able to effectively block the infection of a pseudovirus to 50% by inhibiting the host cell lines binding with the S1 protein, evidenced by the results from Western blotting and pseudovirus luciferase assay. Moreover, the combination of the three peptides could increase the inhibitory rate to 75%. In conclusion, the three chemically synthetic neutralizing peptides and their combinations hold promising potential as effective therapeutics in the prevention and treatment of COVID-19.


Subject(s)
Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cell Survival/drug effects , Circular Dichroism , Humans , Neutralization Tests , Peptides/chemistry , Peptides/pharmacology , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization/drug effects
2.
Nat Commun ; 12(1): 141, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-1387322

ABSTRACT

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.


Subject(s)
CD13 Antigens/metabolism , Coronavirus 229E, Human/physiology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Cell Line, Tumor , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cryoelectron Microscopy , Humans , Models, Molecular , Protein Conformation, alpha-Helical , Protein Multimerization/physiology , Protein Structure, Quaternary , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure
3.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-1387194
4.
Cell Res ; 31(10): 1047-1060, 2021 10.
Article in English | MEDLINE | ID: covidwho-1380899

ABSTRACT

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike's S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike's receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~103 times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 106 times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Tensile Strength , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19/therapy , COVID-19/virology , Humans , Hydrogen-Ion Concentration , Immunization, Passive , Molecular Dynamics Simulation , Protein Binding , Protein Domains/immunology , Protein Subunits/chemistry , Protein Subunits/immunology , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization
5.
Int J Mol Sci ; 22(16)2021 Aug 22.
Article in English | MEDLINE | ID: covidwho-1367850

ABSTRACT

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1ß, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Endothelial Cells/metabolism , Hemoglobins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/virology , Cell Hypoxia , Cell Survival , Cells, Cultured , Endothelial Cells/virology , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Humans , Protein Subunits/metabolism , Pulmonary Artery/cytology , Pulmonary Artery/pathology , Reactive Oxygen Species/metabolism , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity
6.
J Am Chem Soc ; 143(33): 12930-12934, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1358340

ABSTRACT

The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1-ms-long molecular dynamics simulation performed by D. E. Shaw's research group shows that it readily undergoes a symmetry-breaking event on passing from the solid state to aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analyzing this long simulation, we uncover a previously unrecognized role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling simulations performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.


Subject(s)
SARS-CoV-2/enzymology , Viral Matrix Proteins/chemistry , Water/chemistry , COVID-19/pathology , COVID-19/virology , Crystallography, X-Ray , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/metabolism
7.
Int J Mol Sci ; 22(15)2021 Jul 30.
Article in English | MEDLINE | ID: covidwho-1350316

ABSTRACT

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, ß-amyloid (Aß) levels in the blood are increased; however, the impact of elevated Aß levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aß1-42, but not Aß1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aß1-42. Furthermore, Aß1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aß1-42 show that the clearance of Aß1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aß antibody. In conclusion, these findings suggest that the binding of Aß1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aß1-42 in the blood is beneficial to the fight against COVID-19 and AD.


Subject(s)
Amyloid beta-Peptides/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Peptide Fragments/metabolism , SARS-CoV-2/enzymology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Alzheimer Disease/complications , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Animals , COVID-19/complications , COVID-19/metabolism , Chlorocebus aethiops , Humans , Interleukin-6/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/chemistry , Protein Subunits/chemistry , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Internalization
8.
J Am Chem Soc ; 143(33): 13205-13211, 2021 08 25.
Article in English | MEDLINE | ID: covidwho-1349637

ABSTRACT

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.


Subject(s)
Micelles , Peptides/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , COVID-19/pathology , COVID-19/virology , Humans , Hydrophobic and Hydrophilic Interactions , Peptides/chemistry , Phospholipids/chemistry , Phospholipids/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
9.
Microbiol Spectr ; 9(1): e0003021, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1341308

ABSTRACT

Monitoring and strategic response to variants in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent a considerable challenge in the current pandemic and for future viral outbreaks. Mutations/deletions of the virion's prefusion Spike protein may have significant impact on vaccines and therapeutics that utilize this key structural protein in their mitigation strategies. In this study, we have demonstrated how dominant energetic landscape mappings ("glue points") based on ab inito all-atom force fields coupled with phylogenetic sequence alignment information can identify key residue mutations and deletions associated with variants. We also found several examples of excellent homology of stabilizing residue glue points across the lineages of betacoronavirus Spike proteins that we have called "sequence homologous glue points." SARS-CoV-2 demonstrates the least number of stabilizing glue points associated with interchain interactions among Down-state protomers across lineages. Additionally, we computationally studied variants among the trimeric Spike protein of SARS-CoV-2 using all-atom molecular dynamics to ascertain structural and energetic changes among variants. We examined both a theoretically based triple mutant and the UK or B.1.1.7 variant. For the theoretical triple mutant, we demonstrated through alanine substitutions that three key residues could cause the transition of Down-to-Up protomer states, where the transition is characterized by the "arm" length of the receptor-binding domain (RBD) rather than the hinge angle. For the B.1.1.7 variant, we demonstrated the critical importance of mutations D614G and N501Y on the structure and binding, respectively, of the Spike protein. We note that these same two key mutations are also found in the South African B.1.351 variant. IMPORTANCE Viral variants represent a major challenge to monitoring viral outbreaks and formulating strategic health care responses. Variants represent transmitting viruses that have specific mutations and deletions associated with their genome. In the case of SARS-CoV-2 and other related viruses (betacoronaviruses), many of these mutations and deletions are associated with the Spike protein that the virus uses to infect cells. Here, we have analyzed both SARS-CoV-2 variants and related viruses, such as Middle Eastern respiratory syndrome coronavirus (MERS-CoV), in order to understand not only differences, but also key similarities between them. Understanding similarities can be as important as differences in determining key functional features of a class of viruses, such as the betacoronaviruses. We have used both phylogenetic analysis, which traces genetic similarities and differences, along with independent biophysics analysis, which adds function or behavior, in order to determine possible functional differences and hence possible transmission and infection differences among variants and lineages.


Subject(s)
Protein Subunits/genetics , Protein Subunits/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Base Sequence , COVID-19/virology , Humans , Molecular Dynamics Simulation , Mutation , Phylogeny , Protein Binding , Protein Conformation , SARS-CoV-2/classification , Sequence Alignment , Spike Glycoprotein, Coronavirus/classification , United Kingdom
10.
Int J Mol Sci ; 22(15)2021 Jul 30.
Article in English | MEDLINE | ID: covidwho-1335101

ABSTRACT

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, ß-amyloid (Aß) levels in the blood are increased; however, the impact of elevated Aß levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aß1-42, but not Aß1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aß1-42. Furthermore, Aß1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aß1-42 show that the clearance of Aß1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aß antibody. In conclusion, these findings suggest that the binding of Aß1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aß1-42 in the blood is beneficial to the fight against COVID-19 and AD.


Subject(s)
Amyloid beta-Peptides/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Peptide Fragments/metabolism , SARS-CoV-2/enzymology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Alzheimer Disease/complications , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Animals , COVID-19/complications , COVID-19/metabolism , Chlorocebus aethiops , Humans , Interleukin-6/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/chemistry , Protein Subunits/chemistry , Protein Subunits/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells , Virus Internalization
11.
Int J Mol Sci ; 21(20)2020 Oct 09.
Article in English | MEDLINE | ID: covidwho-1298152

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels responsible for rapid neural and neuromuscular signal transmission. Although it is well documented that 16 subunits are encoded by the human genome, their presence in airway epithelial cells (AECs) remains poorly understood, and contribution to pathology is mainly discussed in the context of cancer. We analysed nAChR subunit expression in the human lungs of smokers and non-smokers using transcriptomic data for whole-lung tissues, isolated large AECs, and isolated small AECs. We identified differential expressions of nAChRs in terms of detection and repartition in the three modalities. Smoking-associated alterations were also unveiled. Then, we identified an nAChR transcriptomic print at the single-cell level. Finally, we reported the localizations of detectable nAChRs in bronchi and large bronchioles. Thus, we compiled the first complete atlas of pulmonary nAChR subunits to open new avenues to further unravel the involvement of these receptors in lung homeostasis and respiratory diseases.


Subject(s)
Lung/metabolism , Protein Subunits/metabolism , Receptors, Nicotinic/metabolism , Adult , Age Factors , Cell Cycle , Epithelial Cells/metabolism , Gene Expression Regulation , Humans , Protein Subunits/chemistry , Protein Subunits/genetics , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Signal Detection, Psychological , Smoking , Transcription, Genetic
12.
Science ; 372(6541): 525-530, 2021 04 30.
Article in English | MEDLINE | ID: covidwho-1138286

ABSTRACT

Substitution for aspartic acid (D) by glycine (G) at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. Here, we report cryo-electron microscopy structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations that differ primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer-effectively increasing the number of functional spikes and enhancing infectivity-and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.


Subject(s)
SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , COVID-19/virology , Cryoelectron Microscopy , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
13.
Sci Rep ; 11(1): 4257, 2021 02 19.
Article in English | MEDLINE | ID: covidwho-1091461

ABSTRACT

The worldwide CoVid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. Existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. Here, we are using in silico modelling of possible SARS-CoV-2 drug targets, as deposited on the Protein Databank (PDB), and ascertain their dynamics, flexibility and rigidity. For example, for the SARS-CoV-2 spike protein-using its complete homo-trimer configuration with 2905 residues-our method identifies a large-scale opening and closing of the S1 subunit through movement of the S[Formula: see text] domain. We compute the full structural information of this process, allowing for docking studies with possible drug structures. In a dedicated database, we present similarly detailed results for the further, nearly 300, thus far resolved SARS-CoV-2-related protein structures in the PDB.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Development/methods , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Antiviral Agents/therapeutic use , Binding Sites , COVID-19/epidemiology , COVID-19/virology , Crystallography, X-Ray , Humans , Models, Molecular , Pandemics/prevention & control , Protein Binding , Protein Domains/drug effects , Protein Multimerization/drug effects , Protein Subunits/drug effects , Protein Subunits/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/drug effects , Spike Glycoprotein, Coronavirus/ultrastructure
14.
Eur J Med Chem ; 215: 113242, 2021 Apr 05.
Article in English | MEDLINE | ID: covidwho-1086914

ABSTRACT

Currently, SARS-CoV-2 virus is an emerging pathogen that has posed a serious threat to public health worldwide. However, no agents have been approved to treat SARS-CoV-2 infections to date, underscoring the great need for effective and practical therapies for SARS-CoV-2 outbreaks. We reported that a focused screen of OA saponins identified 3-O-ß-chacotriosyl OA benzyl ester 2 as a novel small molecule inhibitor of SARS-CoV-2 virus entry, via binding to SARS-CoV-2 glycoprotein (S). We performed structure-activity relationship profiling of 2 and discovered C-17-COOH of OA was an important modification site that improved both inhibitor potency toward SARS-CoV-2 and selectivity index. Then optimization from hit to lead resulted in a potent fusion inhibitor 12f displaying strong inhibition against infectious SARS-CoV-2 with an IC50 value of 0.97 µM in vitro. Mechanism studies confirmed that inhibition of SARS-CoV-2 viral entry of 12f was mediated by the direct interaction with SARS-CoV-2 S2 subunit to block membrane fusion. These 3-O-ß-chacotriosyl OA amide saponins are suitable for further optimization as SARS-CoV-2 entry inhibitors with the potential to be developed as therapeutic agents for the treatment of SARS-CoV-2 virus infections.


Subject(s)
Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , Saponins/pharmacology , Triterpenes/pharmacology , Virus Internalization/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Chlorocebus aethiops , Drug Discovery , HEK293 Cells , Humans , Microbial Sensitivity Tests , Molecular Structure , Protein Binding , Protein Subunits/metabolism , Saponins/chemical synthesis , Saponins/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Triterpenes/chemical synthesis , Triterpenes/metabolism , Vero Cells
15.
Electrophoresis ; 42(6): 687-692, 2021 03.
Article in English | MEDLINE | ID: covidwho-1059406

ABSTRACT

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor-binding domain (RBD) with His- and Fc-tag, the S1 subunit with His-tag, the S1/S2 subunits with His-tag and the human angiotensin-converting enzyme 2 (hACE2) with His-tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24-9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36-9.88 (8.91) and for the S1/His it is 7.30-8.37 (7.80). The pI range of the S1/S2/His is 4.41-5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19-6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS-CoV-2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19/virology , Isoelectric Focusing/methods , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/metabolism , Humans , Isoelectric Point , Protein Interaction Domains and Motifs , Protein Subunits/chemistry , Protein Subunits/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
16.
Cell Rep ; 34(2): 108630, 2021 01 12.
Article in English | MEDLINE | ID: covidwho-987231

ABSTRACT

The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.


Subject(s)
Peptide Hydrolases/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/pathology , COVID-19/virology , Cryoelectron Microscopy , Humans , Immunogenicity, Vaccine , Molecular Dynamics Simulation , Mutation , Protein Domains , Protein Stability , Protein Structure, Quaternary , Protein Subunits/metabolism , Proteolysis , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
17.
Ann Diagn Pathol ; 51: 151682, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-987026

ABSTRACT

Neurologic complications of symptomatic COVID-19 are common. Brain tissues from 13 autopsies of people who died of COVID-19 were examined. Cultured endothelial and neuronal cells were incubated with and wild type mice were injected IV with different spike subunits. In situ analyses were used to detect SARS-CoV-2 proteins and the host response. In 13/13 brains from fatal COVID-19, pseudovirions (spike, envelope, and membrane proteins without viral RNA) were present in the endothelia of microvessels ranging from 0 to 14 positive cells/200× field (mean 4.3). The pseudovirions strongly co-localized with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons demonstrated increased NMDAR2 and neuronal NOS plus decreased MFSD2a and SHIP1 proteins. Tail vein injection of the full length S1 spike subunit in mice led to neurologic signs (increased thirst, stressed behavior) not evident in those injected with the S2 subunit. The S1 subunit localized to the endothelia of microvessels in the mice brain and showed co-localization with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons showed increased neuronal NOS and decreased MFSD2a. It is concluded that ACE2+ endothelial damage is a central part of SARS-CoV2 pathology and may be induced by the spike protein alone. Thus, the diagnostic pathologist can use either hematoxylin and eosin stain or immunohistochemistry for caspase 3 and ACE2 to document the endothelial cell damage of COVID-19.


Subject(s)
COVID-19/virology , Endothelial Cells/virology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adult , Aged , Aged, 80 and over , Animals , Autopsy/methods , Disease Models, Animal , Endothelial Cells/metabolism , Female , Humans , Male , Mice , Microvessels/metabolism , Microvessels/virology , Middle Aged , Protein Subunits/metabolism , RNA, Viral/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
18.
PLoS One ; 15(11): e0241168, 2020.
Article in English | MEDLINE | ID: covidwho-917991

ABSTRACT

The SARS-CoV-2 virion responsible for the current world-wide pandemic COVID-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD) within its distal, prefusion S1 domain. The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions (charge, partial charge, and van der Waals) of the SARS-CoV-2 Spike protein in its static prefusion state based on two recent and independent experimental structure publications. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences (different so-called "glue" points) between residues in the Up and Down state protomers. The S2 proximal, fusion domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer's intrachain, S1-S2 interactions. However, the S1 domain interactions across neighboring protomers, which include the RBD-NTD cross chain interactions, showed significant energetic differences between Up-Down and Down-Down neighboring protomers. This included, for example, a key RBD residue ARG357 in the Up-Down interaction and a three residue sequence ALA520-PRO521-ALA522, associated with a turn structure in the RBD of the Up state protomer, acting as a stabilizing interaction with the NTD of its neighbor protomer. Additionally, our intra chain dominant energy mappings within each protomer, identified a significant "glue" point or possible "latch" for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the amino acid R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-CoV-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. We conducted preliminary molecular dynamics simulations across 0.1 µ seconds to see if this latch provided structural stability and indeed found that a single point mutation (Q564G) resulted in the latch releasing transforming the protomer from the Down to the Up state conformation. Full trimeric Spike protein studies of the same mutation across all protomers, however, did not exhibit latch release demonstrating the critical importance of interchain interactions across the S1 domain, including RBD-NTD neighboring chain interactions. Therapies aimed at disrupting these noncovalent interactions could be a viable route for the physico-chemical mitigation of this deadly virion.


Subject(s)
Betacoronavirus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Point Mutation , Protein Binding , Protein Domains , Protein Stability , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics
19.
Neurobiol Dis ; 146: 105131, 2020 12.
Article in English | MEDLINE | ID: covidwho-872391

ABSTRACT

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Blood-Brain Barrier/metabolism , Capillary Permeability/physiology , Endothelial Cells/metabolism , Inflammation/metabolism , Matrix Metalloproteinases/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/physiology , Blood-Brain Barrier/drug effects , COVID-19 , Capillary Permeability/drug effects , Cell Adhesion Molecules/drug effects , Cell Adhesion Molecules/metabolism , Cell Survival/drug effects , Dementia/metabolism , Electric Impedance , Endothelial Cells/drug effects , Frontal Lobe/metabolism , Humans , Hypertension/metabolism , In Vitro Techniques , Intercellular Junctions/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Lab-On-A-Chip Devices , Matrix Metalloproteinases/drug effects , Primary Cell Culture , Protein Domains , Protein Subunits/metabolism , Protein Subunits/pharmacology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Spike Glycoprotein, Coronavirus/pharmacology
20.
Sci Adv ; 6(42)2020 10.
Article in English | MEDLINE | ID: covidwho-781066

ABSTRACT

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.


Subject(s)
Betacoronavirus/immunology , Peptidyl-Dipeptidase A/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Cell Surface Display Techniques/methods , Coronavirus Infections/pathology , Coronavirus Infections/virology , Dimerization , Humans , Kinetics , Pandemics , Peptides/chemistry , Peptides/immunology , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Domains/immunology , Protein Subunits/chemistry , Protein Subunits/immunology , Protein Subunits/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...