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1.
PLoS One ; 17(1): e0262657, 2022.
Article in English | MEDLINE | ID: covidwho-1639087

ABSTRACT

BACKGROUND: Tests for SARS-CoV-2 immunity are needed to help assess responses to vaccination, which can be heterogeneous and may wane over time. The plaque reduction neutralization test (PRNT) is considered the gold standard for measuring serum neutralizing antibodies but requires high level biosafety, live viral cultures and days to complete. We hypothesized that competitive enzyme linked immunoassays (ELISAs) based on SARS-CoV-2 spike protein's receptor binding domain (RBD) attachment to its host receptor, the angiotensin converting enzyme 2 receptor (ACE2r), would correlate with PRNT, given the central role of RBD-ACE2r interactions in infection and published studies to date, and enable evaluation of vaccine responses. METHODS AND RESULTS: Configuration and development of a competitive ELISA with plate-bound RBD and soluble biotinylated ACE2r was accomplished using pairs of pre/post vaccine serum. When the competitive ELISA was used to evaluate N = 32 samples from COVID-19 patients previously tested by PRNT, excellent correlation in IC50 results were observed (rs = .83, p < 0.0001). When the competitive ELISA was used to evaluate N = 42 vaccinated individuals and an additional N = 13 unvaccinated recovered COVID-19 patients, significant differences in RBD-ACE2r inhibitory activity were associated with prior history of COVID-19 and type of vaccine received. In longitudinal analyses pre and up to 200 days post vaccine, surrogate neutralizing activity increased markedly after primary and booster vaccine doses, but fell substantially, up to <12% maximal levels within 6 months. CONCLUSIONS: A competitive ELISA based on inhibition of RBD-ACE2r attachment correlates well with PRNT, quantifies significantly higher activity among vaccine recipients with prior COVID (vs. those without), and highlights marked declines in surrogate neutralizing activity over a 6 month period post vaccination. The findings raise concern about the duration of vaccine responses and potential need for booster shots.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Vaccines, Synthetic/immunology , /immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Vaccines, Synthetic/administration & dosage , /administration & dosage
2.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1626013

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell-cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2' fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2' cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2' cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Membrane Fusion , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Mice , Protein Binding , Proteolysis , Virus Internalization
3.
MAbs ; 14(1): 2021601, 2022.
Article in English | MEDLINE | ID: covidwho-1625321

ABSTRACT

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.


Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Affinity , Antibody Specificity , Antigen-Antibody Reactions , Antigens, Viral/chemistry , Antigens, Viral/genetics , Broadly Neutralizing Antibodies/genetics , Broadly Neutralizing Antibodies/metabolism , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , Humans , Immunoglobulin Fragments/immunology , Molecular Docking Simulation , Monte Carlo Method , Neutralization Tests , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Domains , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
4.
Commun Biol ; 5(1): 1421, 2022 01 06.
Article in English | MEDLINE | ID: covidwho-1612213

ABSTRACT

As the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic continues to spread, several variants of the virus, with mutations distributed all over the viral genome, are emerging. While most of the variants present mutations having little to no effects at the phenotypic level, some of these variants are spreading at a rate that suggests they may present a selective advantage. In particular, these rapidly spreading variants present specific mutations on the spike protein. These observations call for an urgent need to characterize the effects of these variants' mutations on phenotype features like contagiousness and antigenicity. With this aim, we performed molecular dynamics simulations on a selected set of possible spike variants in order to assess the stabilizing effect of particular amino acid substitutions on the molecular complex. We specifically focused on the mutations that are both characteristic of the top three most worrying variants at the moment, i.e the English, South African, and Amazonian ones, and that occur at the molecular interface between SARS-CoV-2 spike protein and its human ACE2 receptor. We characterize these variants' effect in terms of (i) residue mobility, (ii) compactness, studying the network of interactions at the interface, and (iii) variation of shape complementarity via expanding the molecular surfaces in the Zernike basis. Overall, our analyses highlighted greater stability of the three variant complexes with respect to both the wild type and two negative control systems, especially for the English and Amazonian variants. In addition, in the three variants, we investigate the effects a not-yet observed mutation in position 501 could provoke on complex stability. We found that a phenylalanine mutation behaves similarly to the English variant and may cooperate in further increasing the stability of the South African one, hinting at the need for careful surveillance for the emergence of these mutations in the population. Ultimately, we show that the proposed observables describe key features for the stability of the ACE2-spike complex and can help to monitor further possible spike variants.


Subject(s)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Mutation , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Molecular Dynamics Simulation , Protein Binding
5.
Biochemistry (Mosc) ; 86(7): 800-817, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1594970

ABSTRACT

COVID-19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its infectious agent, SARS-CoV-2, differs from other coronaviruses in a number of structural features that make this virus more pathogenic and transmissible. In this review, we discuss some important characteristics of the main SARS-CoV-2 surface antigen, the spike (S) protein, such as (i) ability of the receptor-binding domain (RBD) to switch between the "standing-up" position (open pre-fusion conformation) for receptor binding and the "lying-down" position (closed pre-fusion conformation) for immune system evasion; (ii) advantage of a high binding affinity of the RBD open conformation to the human angiotensin-converting enzyme 2 (ACE2) receptor for efficient cell entry; and (iii) S protein preliminary activation by the intracellular furin-like proteases for facilitation of the virus spreading across different cell types. We describe interactions between the S protein and cellular receptors, co-receptors, and antagonists, as well as a hypothetical mechanism of the homotrimeric spike structure destabilization that triggers the fusion of the viral envelope with the cell membrane at physiological pH and mediates the viral nucleocapsid entry into the cytoplasm. The transition of the S protein pre-fusion conformation to the post-fusion one on the surface of virions after their treatment with some reagents, such as ß-propiolactone, is essential, especially in relation to the vaccine production. We also compare the COVID-19 pathogenesis with that of severe outbreaks of "avian" influenza caused by the A/H5 and A/H7 highly pathogenic viruses and discuss the structural similarities between the SARS-CoV-2 S protein and hemagglutinins of those highly pathogenic strains. Finally, we touch on the prospective and currently used COVID-19 antiviral and anti-pathogenetic therapeutics, as well as recently approved conventional and innovative COVID-19 vaccines and their molecular and immunological features.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , COVID-19/genetics , COVID-19/metabolism , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza A virus/metabolism , Influenza, Human/epidemiology , Influenza, Human/genetics , Influenza, Human/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
6.
PLoS Biol ; 19(12): e3001510, 2021 12.
Article in English | MEDLINE | ID: covidwho-1592147

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse-one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Disease Resistance/genetics , Epistasis, Genetic , SARS-CoV-2/physiology , Amino Acids , Angiotensin II/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , COVID-19/enzymology , COVID-19/genetics , Dogs , Evolution, Molecular , Gene Frequency , Humans , Hydrolysis , Mice , Mutation , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment
7.
Elife ; 102021 12 20.
Article in English | MEDLINE | ID: covidwho-1592091

ABSTRACT

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Molecular Dynamics Simulation , Polysaccharides/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , COVID-19/prevention & control , COVID-19/virology , Glycosylation , Humans , Polysaccharides/chemistry , Protein Binding , Protein Engineering , Receptors, Virus/chemistry , Receptors, Virus/genetics , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
8.
Biochem Biophys Res Commun ; 590: 34-41, 2022 01 29.
Article in English | MEDLINE | ID: covidwho-1588232

ABSTRACT

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to more than 270 million infections and 5.3 million of deaths worldwide. Several major variants of SARS-CoV-2 have emerged and posed challenges in controlling the pandemic. The recently occurred Omicron variant raised serious concerns about reducing the efficacy of vaccines and neutralization antibodies due to its vast mutations. We have modelled the complex structure of the human ACE2 protein and the receptor binding domain (RBD) of Omicron Spike protein (S-protein), and conducted atomistic molecular dynamics simulations to study the binding interactions. The analysis shows that the Omicron RBD binds more strongly to the human ACE2 protein than the original strain. The mutations at the ACE2-RBD interface enhance the tight binding by increasing hydrogen bonding interaction and enlarging buried solvent accessible surface area.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , Host-Pathogen Interactions , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
9.
Nat Commun ; 12(1): 7345, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1585860

ABSTRACT

The emergence of SARS-CoV-2 Kappa and Beta variants with enhanced transmissibility and resistance to neutralizing antibodies has created new challenges for the control of the ongoing COVID-19 pandemic. Understanding the structural nature of Kappa and Beta spike (S) proteins and their association with ACE2 is of significant importance. Here we present two cryo-EM structures for each of the Kappa and Beta spikes in the open and open-prone transition states. Compared with wild-type (WT) or G614 spikes, the two variant spikes appear more untwisted/open especially for Beta, and display a considerable population shift towards the open state as well as more pronounced conformational dynamics. Moreover, we capture four conformational states of the S-trimer/ACE2 complex for each of the two variants, revealing an enlarged conformational landscape for the Kappa and Beta S-ACE2 complexes and pronounced population shift towards the three RBDs up conformation. These results implicate that the mutations in Kappa and Beta may modify the kinetics of receptor binding and viral fusion to improve virus fitness. Combined with biochemical analysis, our structural study shows that the two variants are enabled to efficiently interact with ACE2 receptor despite their sensitive ACE2 binding surface is modified to escape recognition by some potent neutralizing MAbs. Our findings shed new light on the pathogenicity and immune evasion mechanism of the Beta and Kappa variants.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Cryoelectron Microscopy , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19 , Humans , Kinetics , Molecular Conformation , Mutation , Protein Binding
10.
Sci Rep ; 11(1): 24336, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1585788

ABSTRACT

ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Bile Ducts/metabolism , Bile Ducts/virology , Binding Sites , COVID-19/pathology , COVID-19/virology , Humans , Lung/metabolism , Lung/virology , Microscopy, Fluorescence, Multiphoton , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization
11.
Mol Med ; 27(1): 162, 2021 12 27.
Article in English | MEDLINE | ID: covidwho-1582120

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel type b coronavirus responsible for the COVID-19 pandemic. With over 224 million confirmed infections with this virus and more than 4.6 million people dead because of it, it is critically important to define the immunological processes occurring in the human response to this virus and pathogenetic mechanisms of its deadly manifestation. This perspective focuses on the contribution of the recently discovered interaction of SARS-CoV-2 Spike protein with neuropilin 1 (NRP1) receptor, NRP1 as a virus entry receptor for SARS-CoV-2, its role in different physiologic and pathologic conditions, and the potential to target the Spike-NRP1 interaction to combat virus infectivity and severe disease manifestations.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Neuropilin-1/chemistry , Neuropilin-1/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , COVID-19/etiology , Comorbidity , Diabetes Mellitus/epidemiology , Diabetes Mellitus/virology , Female , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Humans , Infant , Molecular Targeted Therapy/methods , Neuropilin-1/immunology , Pregnancy , Pregnancy Complications, Infectious/drug therapy , Pregnancy Complications, Infectious/virology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism
12.
Molecules ; 27(1)2022 Jan 01.
Article in English | MEDLINE | ID: covidwho-1580563

ABSTRACT

Before entering the cell, the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) binds to the human angiotensin-converting enzyme 2 (hACE2) receptor. Hence, this RBD is a critical target for the development of antiviral agents. Recent studies have discovered that SARS-CoV-2 variants with mutations in the RBD have spread globally. The purpose of this in silico study was to determine the potential of a fruit bromelain-derived peptide. DYGAVNEVK. to inhibit the entry of various SARS-CoV-2 variants into human cells by targeting the hACE binding site within the RBD. Molecular docking analysis revealed that DYGAVNEVK interacts with several critical RBD binding residues responsible for the adhesion of the RBD to hACE2. Moreover, 100 ns MD simulations revealed stable interactions between DYGAVNEVK and RBD variants derived from the trajectory of root-mean-square deviation (RMSD), radius of gyration (Rg), and root-mean-square fluctuation (RMSF) analysis, as well as free binding energy calculations. Overall, our computational results indicate that DYGAVNEVK warrants further investigation as a candidate for preventing SARS-CoV-2 due to its interaction with the RBD of SARS-CoV-2 variants.


Subject(s)
Angiotensin-Converting Enzyme 2 , Bromelains , Computer Simulation , Protein Interaction Domains and Motifs , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Bromelains/chemistry , Bromelains/pharmacology , COVID-19/drug therapy , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptides/chemistry , Peptides/pharmacology , Protein Binding , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry
13.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
14.
Biochem J ; 478(19): 3671-3684, 2021 10 15.
Article in English | MEDLINE | ID: covidwho-1557441

ABSTRACT

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. Recombinant ACE2 from human, hamster, horseshoe bat, cat, ferret, and cow were evaluated for RBD binding. A gradient of binding affinities were seen where human and hamster ACE2 were similarly in the low nanomolar range, followed by cat and cow. Surprisingly, horseshoe bat (Rhinolophus sinicus) and ferret (Mustela putorius) ACE2s had poor binding activity compared with the other species' ACE2. The residue differences and binding properties between the species' variants provide a framework for understanding ACE2-RBD binding and virus tropism.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/epidemiology , COVID-19/metabolism , Cats , Dogs , Humans , Mice , Protein Binding , Protein Domains , SARS-CoV-2/metabolism , Species Specificity , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism
15.
Cell Rep ; 37(12): 110156, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1549680

ABSTRACT

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Beta (B.1.351) and Gamma (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the Alpha (B.1.1.7) variant that enhances affinity of the spike protein for its receptor, angiotensin-converting enzyme 2 (ACE2). Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Epsilon variants (B.1.427/429) lack the N501Y mutation yet exhibit antibody evasion. We have engineered spike proteins to express these receptor binding domain (RBD) VoC mutations either in isolation or in different combinations and analyze the effects using biochemical assays and cryoelectron microscopy (cryo-EM) structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , Cryoelectron Microscopy , Humans , Molecular Dynamics Simulation , Mutation , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/classification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
16.
Phys Chem Chem Phys ; 23(27): 14873-14888, 2021 Jul 14.
Article in English | MEDLINE | ID: covidwho-1541260

ABSTRACT

The COVID-19 disease caused by the virus SARS-CoV-2, first detected in December 2019, is still emerging through virus mutations. Although almost under control in some countries due to effective vaccines that are mitigating the worldwide pandemic, the urgency to develop additional vaccines and therapeutic treatments is imperative. In this work, the natural polyphenols corilagin and 1,3,6-tri-O-galloy-ß-d-glucose (TGG) are investigated to determine the structural basis of inhibitor interactions as potential candidates to inhibit SARS-CoV-2 viral entry into target cells. First, the therapeutic potential of the ligands are assessed on the ACE2/wild-type RBD. We first use molecular docking followed by molecular dynamics, to take into account the conformational flexibility that plays a significant role in ligand binding and that cannot be captured using only docking, and then analyze more precisely the affinity of these ligands using MMPBSA binding free energy. We show that both ligands bind to the ACE2/wild-type RBD interface with good affinities which might prevent the ACE2/RBD association. Second, we confirm the potency of these ligands to block the ACE2/RBD association using a combination of surface plasmon resonance and biochemical inhibition assays. These experiments confirm that TGG and, to a lesser extent, corilagin, inhibit the binding of RBD to ACE2. Both experiments and simulations show that the ligands interact preferentially with RBD, while weak binding is observed with ACE2, hence, avoiding potential physiological side-effects induced by the inhibition of ACE2. In addition to the wild-type RBD, we also study numerically three RBD mutations (E484K, N501Y and E484K/N501Y) found in the main SARS-CoV-2 variants of concerns. We find that corilagin could be as effective for RBD/E484K but less effective for the RBD/N501Y and RBD/E484K-N501Y mutants, while TGG strongly binds at relevant locations to all three mutants, demonstrating the significant interest of these molecules as potential inhibitors for variants of SARS-CoV-2.


Subject(s)
Antiviral Agents/chemistry , Gallic Acid/analogs & derivatives , Glucose/analogs & derivatives , Glucosides/chemistry , Hydrolyzable Tannins/chemistry , SARS-CoV-2/drug effects , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , Gallic Acid/chemistry , Glucose/chemistry , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Binding/drug effects , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
18.
Sci Rep ; 11(1): 22202, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1514421

ABSTRACT

SARS-CoV-2 is responsible for COVID-19 pandemic, causing large numbers of cases and deaths. It initiates entry into human cells by binding to the peptidase domain of angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain of S1 subunit of spike protein (SARS-CoV-2-RBD). Employing neutralizing antibodies to prevent binding between SARS-CoV-2-RBD and ACE2 is an effective COVID-19 therapeutic solution. Previous studies found that CC12.3 is a highly potent neutralizing antibody that was isolated from a SARS-CoV-2 infected patient, and its Fab fragment (Fab CC12.3) bound to SARS-CoV-2-RBD with comparable binding affinity to ACE2. To enhance its binding affinity, we employed computational protein design to redesign all CDRs of Fab CC12.3 and molecular dynamics (MD) to validate their predicted binding affinities by the MM-GBSA method. MD results show that the predicted binding affinities of the three best designed Fabs CC12.3 (CC12.3-D02, CC12.3-D05, and CC12.3-D08) are better than those of Fab CC12.3 and ACE2. Additionally, our results suggest that enhanced binding affinities of CC12.3-D02, CC12.3-D05, and CC12.3-D08 are caused by increased SARS-CoV-2-RBD binding interactions of CDRs L1 and L3. This study redesigned neutralizing antibodies with better predicted binding affinities to SARS-CoV-2-RBD than Fab CC12.3 and ACE2. They are promising candidates as neutralizing antibodies against SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/metabolism , COVID-19/metabolism , Immunoglobulin Fab Fragments/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/chemistry , Binding Sites , Humans , Immunoglobulin Fab Fragments/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Domains , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry
19.
Sci Rep ; 11(1): 21601, 2021 11 08.
Article in English | MEDLINE | ID: covidwho-1506097

ABSTRACT

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide, with implications on the spread of the pandemic. Characterizing the cross-reactivity of antibodies against these VOCs is necessary to understand the humoral response of non-hospitalized individuals previously infected with SARS-CoV-2, a population that remains understudied. Thirty-two SARS-CoV-2-positive (PCR-confirmed) and non-hospitalized Canadian adults were enrolled 14-21 days post-diagnosis in 2020, before the emergence of the B.1.351 (also known as Beta), B.1.617.2 (Delta) and P.1 (Gamma) VOCs. Sera were collected 4 and 16 weeks post-diagnosis. Antibody levels and pseudo-neutralization of the ectodomain of SARS-CoV-2 spike protein/human ACE-2 receptor interaction were analyzed with native, B.1.351, B.1.617.2 and P.1 variant spike proteins. Despite a lower response observed for the variant spike proteins, we report evidence of a sustained humoral response against native, B.1.351, B.1.617.2 and P.1 variant spike proteins among non-hospitalized Canadian adults. Furthermore, this response inhibited the interaction between the spike proteins from the different VOCs and ACE-2 receptor for ≥ 16 weeks post-diagnosis, except for individuals aged 18-49 years who showed no inhibition of the interaction between B.1.617.1 or B.1.617.2 spike and ACE-2. Interestingly, the affinity (KD) measured between the spike proteins (native, B.1.351, B.1.617.2 and P.1) and antibodies elicited in sera of infected and vaccinated (BNT162b2 and ChAdOx1 nCoV-19) individuals was invariant. Relative to sera from vaccine-naïve (and previously infected) individuals, sera from vaccinated individuals had higher antibody levels (as measured with label-free SPR) and more efficiently inhibited the spike-ACE-2 interactions, even among individuals aged 18-49 years, showing the effectiveness of vaccination.


Subject(s)
Antibodies, Viral/chemistry , COVID-19 Vaccines , COVID-19/blood , COVID-19/immunology , Spike Glycoprotein, Coronavirus , Adolescent , Adult , Aged , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/immunology , Area Under Curve , COVID-19 Nucleic Acid Testing , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G , Kinetics , Middle Aged , Polymerase Chain Reaction , Protein Binding , SARS-CoV-2 , Vaccination , Young Adult
20.
Sci Rep ; 11(1): 21735, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1504063

ABSTRACT

The COVID19 pandemic, caused by SARS-CoV-2, has infected more than 200 million people worldwide. Due to the rapid spreading of SARS-CoV-2 and its impact, it is paramount to find effective treatments against it. Human neutralizing antibodies are an effective method to fight viral infection. However, the recent discovery of new strains that substantially change the S-protein sequence has raised concern about vaccines and antibodies' effectiveness. Here, using molecular simulations, we investigated the binding mechanisms between the S-protein and several antibodies. Multiple mutations were included to understand the strategies for antibody escape in new variants. We found that the combination of mutations K417N, E484K, L452R, and T478K produced higher binding energy to ACE2 than the wild type, suggesting higher efficiency to enter host cells. The mutations' effect depends on the antibody class. While Class I enhances the binding avidity in the presence of N501Y mutation, class II antibodies showed a sharp decline in the binding affinity. Our simulations suggest that Class I antibodies will remain effective against the new strains. In contrast, Class II antibodies will have less affinity to the S-protein, potentially affecting these antibodies' efficiency.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , COVID-19/immunology , COVID-19/virology , Mutation , SARS-CoV-2/genetics , Antibodies, Viral/immunology , Cluster Analysis , Computational Biology , Computer Simulation , Humans , Hydrogen Bonding , Molecular Conformation , Molecular Dynamics Simulation , Protein Binding , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism
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