Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 25
Filter
1.
J Immunol Methods ; 500: 113182, 2022 01.
Article in English | MEDLINE | ID: covidwho-1768318

ABSTRACT

Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/economics , COVID-19 Serological Testing/economics , Costs and Cost Analysis , Enzyme-Linked Immunosorbent Assay , Genetic Engineering , Humans , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
Nat Commun ; 13(1): 1214, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730288

ABSTRACT

The omicron variant of SARS-CoV-2 has been spreading rapidly across the globe. The virus-surface spike protein plays a critical role in the cell entry and immune evasion of SARS-CoV-2. Here we determined the 3.0 Å cryo-EM structure of the omicron spike protein ectodomain. In contrast to the original strain of SARS-CoV-2 where the receptor-binding domain (RBD) of the spike protein takes a mixture of open ("standing up") and closed ("lying down") conformations, the omicron spike molecules are predominantly in the open conformation, with one upright RBD ready for receptor binding. The open conformation of the omicron spike is stabilized by enhanced inter-domain and inter-subunit packing, which involves new mutations in the omicron strain. Moreover, the omicron spike has undergone extensive mutations in RBD regions where known neutralizing antibodies target, allowing the omicron variant to escape immune surveillance aimed at the original viral strain. The stable open conformation of the omicron spike sheds light on the cell entry and immune evasion mechanisms of the omicron variant.


Subject(s)
COVID-19/virology , SARS-CoV-2/chemistry , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cryoelectron Microscopy , Humans , Immune Evasion/genetics , Models, Molecular , Mutation , Pandemics , Protein Conformation , Protein Domains/genetics , Protein Domains/immunology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
3.
Science ; 375(6583): 864-868, 2022 02 25.
Article in English | MEDLINE | ID: covidwho-1650843

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , Immune Evasion , Receptors, Coronavirus/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Antigenic Drift and Shift , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/immunology , Broadly Neutralizing Antibodies/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Domains/genetics , Protein Interaction Domains and Motifs/genetics , Receptors, Coronavirus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
4.
Biochem Biophys Res Commun ; 592: 51-53, 2022 02 12.
Article in English | MEDLINE | ID: covidwho-1611626

ABSTRACT

Omicron is a new variant of SARS-CoV-2, which is currently infecting people around the world. Spike glycoprotein, an important molecule in pathogenesis of infection has been modeled and the interaction of its Receptor Binding Domain with human ACE-receptor has been analysed by simulation studies. Structural analysis of Omicron spike glycoprotein shows the 30 mutations to be distributed over all domains of the trimeric protein, wherein the mutant residues are seen to be participating in higher number of intra-molecular interactions including two salt bridges emanating from mutant residues thereby stabilizing their conformation, as compared to wild type. Complex of Receptor Binding Domain (RBD) with human ACE-2 receptor shows seven mutations at interacting interface comprising of two ionic interactions, eight hydrogen bonds and seven Van der Waals interactions. The number and quality of these interactions along with other binding biophysical parameters suggests more potency of RBD domain to the receptor as compared to the wild type counterpart. Results of this study explains the high transmissibility of Omicron variant of SARS-CoV-2 that is currently observed across the world.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/transmission , COVID-19/virology , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Biophysical Phenomena , COVID-19/metabolism , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , Molecular Dynamics Simulation , Mutation , Pandemics , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Structural Homology, Protein
5.
Viruses ; 13(12)2021 12 18.
Article in English | MEDLINE | ID: covidwho-1580423

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (n = 11), N501Y (n = 6), or E484K (n = 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.


Subject(s)
SARS-CoV-2/genetics , Sequence Analysis, DNA , Spike Glycoprotein, Coronavirus/genetics , COVID-19/virology , Genotype , Humans , Mutation , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/classification , Sequence Analysis, DNA/methods
6.
Comput Biol Chem ; 96: 107613, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1549716

ABSTRACT

Coronavirus Disease 2019 (COVID-19) is an ongoing global health emergency that has caused tremendous stress and loss of life worldwide. The viral spike glycoprotein is a critical molecule mediating transmission of SARS-CoV-2 by interacting with human ACE2. However, through the course of the pandemics, there has not been a thorough analysis of the spike protein mutations, and on how these mutants influence the transmission of SARS-CoV-2. Besides, cases of SARS-CoV-2 infection among pets and wild animals have been reported, so the susceptibility of these animals requires great attention to investigate, as they may also link to the renewed question of a possible intermediate host for SARS-CoV-2 before it was transmitted to humans. With over 226,000 SARS-CoV-2 sequences obtained, we found 1573 missense mutations in the spike gene, and 226 of them were within the receptor-binding domain (RBD) region that directly interacts with human ACE2. Modeling the interactions between SARS-CoV-2 spike mutants and ACE2 molecules showed that most of the 74 missense mutations in the RBD region of the interaction interface had little impact on spike binding to ACE2, whereas several within the spike RBD increased the binding affinity toward human ACE2 thus making the virus likely more contagious. On the other hand, modeling the interactions between animal ACE2 molecules and SARS-CoV-2 spike revealed that many pets and wild animals' ACE2 had a variable binding ability. Particularly, ACE2 of bamboo rat had stronger binding to SARS-CoV-2 spike protein, whereas that of mole, vole, Mus pahari, palm civet, and pangolin had a weaker binding compared to human ACE2. Our results provide structural insights into the impact on interactions of the SARS-CoV-2 spike mutants to human ACE2, and shed light on SARS-CoV-2 transmission in pets and wild animals, and possible clues to the intermediate host(s) for SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19/veterinary , COVID-19/virology , Mutation, Missense , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , Animals, Wild/genetics , Animals, Wild/virology , COVID-19/transmission , Computational Biology , Host Microbial Interactions/genetics , Host Specificity/genetics , Humans , Molecular Dynamics Simulation , Pandemics/veterinary , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/genetics , Pets/genetics , Pets/virology , Protein Interaction Domains and Motifs/genetics , Risk Factors
7.
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
8.
J Immunol Methods ; 500: 113195, 2022 01.
Article in English | MEDLINE | ID: covidwho-1536656

ABSTRACT

COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.


Subject(s)
Antibodies, Monoclonal/metabolism , COVID-19 Vaccines/immunology , COVID-19/diagnosis , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/genetics , COVID-19/immunology , COVID-19 Vaccines/genetics , Clinical Trials as Topic , Female , Genetic Engineering , Humans , Mice , Mice, Inbred BALB C , Protein Interaction Domains and Motifs/genetics , Vaccine Development , Vaccines, Subunit/genetics
9.
Phys Chem Chem Phys ; 23(46): 26451-26458, 2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1528035

ABSTRACT

Some recent SARS-CoV-2 variants appear to have increased transmissibility compared to the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on the target cells and infect them. In this study, we provide atomic-level insights into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities for the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than for the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , COVID-19/pathology , COVID-19/virology , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics
10.
Biochem Biophys Res Commun ; 586: 87-92, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1525697

ABSTRACT

There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.


Subject(s)
COVID-19/virology , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/transmission , Evolution, Molecular , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Models, Molecular , Pandemics , Protein Conformation , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
11.
PLoS One ; 16(11): e0257089, 2021.
Article in English | MEDLINE | ID: covidwho-1523422

ABSTRACT

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.


Subject(s)
Chlamydomonas reinhardtii , Protein Interaction Domains and Motifs , Recombinant Proteins , Spike Glycoprotein, Coronavirus , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Cloning, Molecular , Humans , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification
12.
PLoS Comput Biol ; 17(11): e1009560, 2021 11.
Article in English | MEDLINE | ID: covidwho-1523396

ABSTRACT

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.


Subject(s)
COVID-19/genetics , COVID-19/virology , Coronavirus Infections/genetics , Coronavirus Infections/virology , SARS-CoV-2/genetics , Adaptation, Physiological/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , CD13 Antigens/genetics , CD13 Antigens/physiology , Common Cold/genetics , Common Cold/virology , Computational Biology , Coronavirus 229E, Human/genetics , Coronavirus 229E, Human/physiology , Evolution, Molecular , Genomics , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Host Specificity/genetics , Host Specificity/physiology , Humans , Mammals/genetics , Mammals/virology , Phylogeny , Protein Interaction Domains and Motifs/genetics , Receptors, Virus/genetics , Receptors, Virus/physiology , SARS-CoV-2/physiology , Selection, Genetic , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology , Virus Internalization
13.
Nat Commun ; 12(1): 6103, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1475296

ABSTRACT

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/isolation & purification , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/ultrastructure , Spodoptera , Surface Plasmon Resonance , Virus Attachment , Virus Internalization
14.
PLoS One ; 16(9): e0257905, 2021.
Article in English | MEDLINE | ID: covidwho-1440993

ABSTRACT

SARS-CoV-2 virus, the causative agent of Covid-19, has fired up a global pandemic. The virus interacts with the human receptor angiotensin-converting enzyme 2 (ACE2) for an invasion via receptor binding domain (RBD) on its spike protein. To provide a deeper understanding of this interaction, we performed microsecond simulations of the RBD-ACE2 complex for SARS-CoV-2 and compared it with the closely related SARS-CoV discovered in 2003. We show residues in the RBD of SARS-CoV-2 that were mutated from SARS-CoV, collectively help make the RBD anchor much stronger to the N-terminal part of ACE2 than the corresponding residues on RBD of SARS-CoV. This would result in a reduced dissociation rate of SARS-CoV-2 from human receptor protein compared to SARS-CoV. The phenomenon was consistently observed in simulations beyond 500 ns and was reproducible across different force fields. Altogether, our study adds more insight into the critical dynamics of the key residues at the virus spike and human receptor binding interface and potentially aids the development of diagnostics and therapeutics to combat the pandemic efficiently.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/metabolism , SARS-CoV-2/genetics , Angiotensin-Converting Enzyme 2/genetics , Binding Sites , COVID-19/genetics , Humans , Models, Theoretical , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
15.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Article in English | MEDLINE | ID: covidwho-1307383

ABSTRACT

Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.


Subject(s)
Adaptation, Physiological/genetics , Evolution, Molecular , SARS-CoV-2/genetics , Amino Acid Substitution , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Testing , Coronavirus Nucleocapsid Proteins/genetics , Epistasis, Genetic , Genome, Viral/genetics , Humans , Immune Evasion/genetics , Mutation , Nuclear Localization Signals/genetics , Phosphoproteins/genetics , Phylogeny , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/classification , Selection, Genetic , Spike Glycoprotein, Coronavirus/genetics , Vaccination
16.
Front Immunol ; 12: 660198, 2021.
Article in English | MEDLINE | ID: covidwho-1221948

ABSTRACT

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.


Subject(s)
Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , Binding Sites/immunology , COVID-19/immunology , COVID-19/pathology , Cell Line , HEK293 Cells , Humans , Protein Domains/genetics , Protein Domains/immunology , Protein Stability , SARS-CoV-2/genetics
17.
FEBS J ; 288(17): 5148-5162, 2021 09.
Article in English | MEDLINE | ID: covidwho-1189682

ABSTRACT

Small linear motifs targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ) have been identified at the C terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A, and N showing significant interactions with dissociation constants values ranging from 3 to 82 µm. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Among the binders of the SARS-CoV-2 proteins E, 3a, or N, seven significantly affect viral replication under knock down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.


Subject(s)
COVID-19/genetics , Host-Pathogen Interactions/genetics , PDZ Domains/genetics , SARS-CoV-2/genetics , COVID-19/virology , Carrier Proteins/genetics , Coronavirus Nucleocapsid Proteins/genetics , Humans , Kinesins/genetics , Myosins/genetics , Protein Binding/genetics , Protein Interaction Domains and Motifs/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 13/genetics , SARS-CoV-2/pathogenicity , Viral Envelope Proteins/genetics , Viroporin Proteins/genetics , Virus Internalization , Virus Replication/genetics , Zonula Occludens-1 Protein/genetics
18.
Int J Mol Sci ; 22(4)2021 Feb 08.
Article in English | MEDLINE | ID: covidwho-1069830

ABSTRACT

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike-ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure-function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.


Subject(s)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Computer Simulation , Protein Interaction Domains and Motifs/genetics , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites , COVID-19/epidemiology , Humans , Protein Binding , Receptors, Virus/chemistry , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , South Africa/epidemiology , Spike Glycoprotein, Coronavirus/chemistry , United Kingdom/epidemiology
19.
Sci Signal ; 14(665)2021 01 12.
Article in English | MEDLINE | ID: covidwho-1029425

ABSTRACT

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.


Subject(s)
COVID-19/virology , Host Microbial Interactions/physiology , SARS-CoV-2/physiology , SARS-CoV-2/pathogenicity , Virus Internalization , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , COVID-19/therapy , Conserved Sequence , Host Microbial Interactions/genetics , Humans , Integrins/chemistry , Integrins/genetics , Integrins/physiology , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/physiology , Models, Biological , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/physiology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/physiology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology
20.
Nat Biotechnol ; 38(9): 1073-1078, 2020 09.
Article in English | MEDLINE | ID: covidwho-1023948

ABSTRACT

A robust serological test to detect neutralizing antibodies to SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral protection, but also vaccine efficacy during clinical trials and after large-scale vaccination. The current gold standard is the conventional virus neutralization test requiring live pathogen and a biosafety level 3 laboratory. Here, we report a SARS-CoV-2 surrogate virus neutralization test that detects total immunodominant neutralizing antibodies targeting the viral spike (S) protein receptor-binding domain in an isotype- and species-independent manner. Our simple and rapid test is based on antibody-mediated blockage of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test, which has been validated with two cohorts of patients with COVID-19 in two different countries, achieves 99.93% specificity and 95-100% sensitivity, and differentiates antibody responses to several human coronaviruses. The surrogate virus neutralization test does not require biosafety level 3 containment, making it broadly accessible to the wider community for both research and clinical applications.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/genetics , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2 , Antibodies/immunology , Antibodies/pharmacology , Betacoronavirus/genetics , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Neutralization Tests , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL