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1.
Genes (Basel) ; 12(6)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072181

RESUMO

The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more than 80% of cases. The bias is explained by the existing differences in the codon composition for amino acids bearing contrasting biochemical properties. Mutation asymmetries apparently influence the biochemical features of SARS CoV-2 proteins, which may impact protein-protein interactions, fusion of viral and cellular membranes, and virion assembly.


Assuntos
COVID-19/virologia , Genoma Viral , Interações Hidrofóbicas e Hidrofílicas , Mutação , SARS-CoV-2/genética , Proteínas Virais/química , Proteínas Virais/genética , Desaminases APOBEC , Alelos , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Evolução Molecular , Variação Genética , Genótipo , Interações Hospedeiro-Patógeno , Humanos , Filogenia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
2.
Viruses ; 13(6)2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071984

RESUMO

The high sequence identity of the first SARS-CoV-2 samples collected in December 2019 at Wuhan did not foretell the emergence of novel variants in the United Kingdom, North and South America, India, or South Africa that drive the current waves of the pandemic. The viral spike receptor possesses two surface areas of high mutagenic plasticity: the supersite in its N-terminal domain (NTD) that is recognised by all anti-NTD antibodies and its receptor binding domain (RBD) where 17 residues make contact with the human Ace2 protein (angiotensin I converting enzyme 2) and many neutralising antibodies bind. While NTD mutations appear at first glance very diverse, they converge on the structure of the supersite. The mutations within the RBD, on the other hand, hone in on only a small number of key sites (K417, L452, E484, N501) that are allosteric control points enabling spike to escape neutralising antibodies while maintaining or even gaining Ace2-binding activity. The D614G mutation is the hallmark of all variants, as it promotes viral spread by increasing the number of open spike protomers in the homo-trimeric receptor complex. This review discusses the recent spike mutations as well as their evolution.


Assuntos
COVID-19/virologia , Variação Genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sítios de Ligação/genética , COVID-19/transmissão , Evolução Molecular , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química
3.
Viruses ; 13(6)2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072569

RESUMO

The COVID-19 pandemic, which began in Wuhan (Hubei, China), has been ongoing for about a year and a half. An unprecedented number of people around the world have been infected with SARS-CoV-2, the etiological agent of COVID-19. Despite the fact that the mortality rate for COVID-19 is relatively low, the total number of deaths has currently already reached more than three million and continues to increase due to high incidence. Since the beginning of the pandemic, a large number of sequences have been obtained and many genetic variants have been identified. Some of them bear significant mutations that affect biological properties of the virus. These genetic variants, currently Variants of Concern (VoC), include the so-called United Kingdom variant (20I/501Y), the Brazilian variant (20J/501Y.V3), and the South African variant (20H/501Y.V2). We describe here a novel SARS-CoV-2 variant with distinct spike protein mutations, first obtained at the end of January 2021 in northwest Russia. Therefore, it is necessary to pay attention to the dynamics of its spread among patients with COVID-19, as well as to study in detail its biological properties.


Assuntos
SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Análise Mutacional de DNA , DNA Complementar , Genoma Viral , Humanos , Modelos Moleculares , Mutação , Filogenia , Conformação Proteica , Federação Russa , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química
4.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067272

RESUMO

The COVID-19 pandemic is caused by SARS-CoV-2. Currently, most of the research efforts towards the development of vaccines and antibodies against SARS-CoV-2 were mainly focused on the spike (S) protein, which mediates virus entry into the host cell by binding to ACE2. As the virus SARS-CoV-2 continues to spread globally, variants have emerged, characterized by multiple mutations of the S glycoprotein. Herein, we employed microsecond-long molecular dynamics simulations to study the impact of the mutations of the S glycoprotein in SARS-CoV-2 Variant of Concern 202012/01 (B.1.1.7), termed the "UK variant", in comparison with the wild type, with the aim to decipher the structural basis of the reported increased infectivity and virulence. The simulations provided insights on the different dynamics of UK and wild-type S glycoprotein, regarding in particular the Receptor Binding Domain (RBD). In addition, we investigated the role of glycans in modulating the conformational transitions of the RBD. The overall results showed that the UK mutant experiences higher flexibility in the RBD with respect to wild type; this behavior might be correlated with the increased transmission reported for this variant. Our work also adds useful structural information on antigenic "hotspots" and epitopes targeted by neutralizing antibodies.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Epitopos , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Reino Unido
5.
Viruses ; 13(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067878

RESUMO

COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.


Assuntos
Enzima de Conversão de Angiotensina 2/química , COVID-19/virologia , Polissacarídeos/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Sítios de Ligação , Glicosilação , Interações entre Hospedeiro e Microrganismos , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ligação Viral
6.
PLoS One ; 16(5): e0251913, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34003827

RESUMO

Last decade has witnessed three major pandemics caused by SARS-CoV, SARS-CoV-2 and MERS-CoV that belong to Coronavirus family. Currently, there are no effective therapies available for corona virus infections. Since the three viruses belong to the same family and share many common features, we can theoretically design a drug that can be effective on all the three of them. In this study, using computational approach, we designed a peptide (Peptide 7) that can bind to the Receptor Binding Domain (RBD) of SARS-CoV, SARS-CoV-2 and MERS-CoV thereby preventing the entry of the viruses into the host cell. The peptide inhibitor was designed as a consensus peptide from three different peptides that might individually bind to the RBD of the three viruses. Docking studies and molecular dynamic simulations using Peptide 7 has shown that it binds with higher affinity than the native receptors of the RBD and forms a stable complex thereby preventing further viral-receptor interaction and inhibiting their cellular entry. This effective binding is observed for the three RBDs, despite the Peptide 7 interactions being slightly different. Hence; this peptide inhibitor can be used as a potential candidate for the development of peptide based anti-viral therapy against Corona viruses.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Peptídeos/farmacologia , Vírus da SARS/fisiologia , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
PLoS One ; 16(5): e0251368, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033650

RESUMO

COVID-19 is challenging healthcare preparedness, world economies, and livelihoods. The infection and death rates associated with this pandemic are strikingly variable in different countries. To elucidate this discrepancy, we analyzed 2431 early spread SARS-CoV-2 sequences from GISAID. We estimated continental-wise admixture proportions, assessed haplotype block estimation, and tested for the presence or absence of strains' recombination. Herein, we identified 1010 unique missense mutations and seven different SARS-CoV-2 clusters. In samples from Asia, a small haplotype block was identified, whereas samples from Europe and North America harbored large and different haplotype blocks with nonsynonymous variants. Variant frequency and linkage disequilibrium varied among continents, especially in North America. Recombination between different strains was only observed in North American and European sequences. In addition, we structurally modelled the two most common mutations, Spike_D614G and Nsp12_P314L, which suggested that these linked mutations may enhance viral entry and replication, respectively. Overall, we propose that genomic recombination between different strains may contribute to SARS-CoV-2 virulence and COVID-19 severity and may present additional challenges for current treatment regimens and countermeasures. Furthermore, our study provides a possible explanation for the substantial second wave of COVID-19 presented with higher infection and death rates in many countries.


Assuntos
Recombinação Genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Virulência/fisiologia , COVID-19/patologia , COVID-19/virologia , Bases de Dados Genéticas , Variação Genética , Haplótipos , Humanos , Desequilíbrio de Ligação , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Análise de Componente Principal , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
PLoS One ; 16(5): e0251426, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34038453

RESUMO

Two SARS-CoV-2 variants of concern showing increased transmissibility relative to the Wuhan virus have recently been identified. Although neither variant appears to cause more severe illness nor increased risk of death, the faster spread of the virus is a major threat. Using computational tools, we found that the new SARS-CoV-2 variants may acquire an increased transmissibility by increasing the propensity of its spike protein to expose the receptor binding domain via proteolysis, perhaps by neutrophil elastase and/or via reduced intramolecular interactions that contribute to the stability of the closed conformation of spike protein. This information leads to the identification of potential treatments to avert the imminent threat of these more transmittable SARS-CoV-2 variants.


Assuntos
Elastase Pancreática/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/imunologia , COVID-19/patologia , COVID-19/virologia , Humanos , Simulação de Dinâmica Molecular , Mutação , Neutrófilos/citologia , Neutrófilos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Phys Chem Chem Phys ; 23(22): 12549-12558, 2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34008647

RESUMO

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell after the receptor binding domain (RBD) of the virus spike (S) glycoprotein binds to the human angiotensin-converting enzyme 2 (hACE2). This binding requires the RBD to undergo a conformational change from a closed to an open state. In the present study, a key pair of salt bridges formed by the side chains of K537 and E619, residues at the interfaces of SD1 and SD2, respectively, was identified to promote the opening of the RBD. Mutations of K537Q and E619D reduced their side chain lengths and eliminated this pair of salt bridges; as a result, the opening of the RBD was not observed in the MD simulations. Thus, blocking the formation of this pair of salt bridges is a promising approach for treating novel coronavirus disease 2019 (COVID-19). FDA approved drug molecules were screened by their capabilities of blocking the formation of the key pair of salt bridges, achieved by their positional stabilities in the cavity containing the side chains of K537 and E619 formed in the interface between SD1 and SD2. Simeprevir, imatinib, and naldemedine were identified to possess the desired capability with the most favorable interaction energies.


Assuntos
Antivirais/farmacologia , Desenho de Fármacos , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Antivirais/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Mesilato de Imatinib/química , Mesilato de Imatinib/farmacologia , Simulação de Acoplamento Molecular , Naltrexona/análogos & derivados , Naltrexona/química , Naltrexona/farmacologia , Domínios Proteicos/efeitos dos fármacos , SARS-CoV-2/química , Simeprevir/química , Simeprevir/farmacologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Chem Asian J ; 16(12): 1634-1642, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-33949124

RESUMO

Computational drug design is increasingly becoming important with new and unforeseen diseases like COVID-19. In this study, we present a new computational de novo drug design and repurposing method and applied it to find plausible drug candidates for the receptor binding domain (RBD) of SARS-CoV-2 (COVID-19). Our study comprises three steps: atom-by-atom generation of new molecules around a receptor, structural similarity mapping to existing approved and investigational drugs, and validation of their binding strengths to the viral spike proteins based on rigorous all-atom, explicit-water well-tempered metadynamics free energy calculations. By choosing the receptor binding domain of the viral spike protein, we showed that some of our new molecules and some of the repurposable drugs have stronger binding to RBD than hACE2. To validate our approach, we also calculated the free energy of hACE2 and RBD, and found it to be in an excellent agreement with experiments. These pool of drugs will allow strategic repurposing against COVID-19 for a particular prevailing conditions.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Desenho de Fármacos , Reposicionamento de Medicamentos , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Antivirais/química , COVID-19/virologia , Simulação por Computador , Humanos , Modelos Moleculares , Conformação Proteica , Termodinâmica
11.
Molecules ; 26(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946306

RESUMO

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The "fusion core" of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one.


Assuntos
COVID-19/virologia , Mutação Puntual , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Humanos , Modelos Moleculares , Conformação Proteica , SARS-CoV-2/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
12.
J Am Chem Soc ; 143(21): 7930-7934, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34018723

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells is a complex process that involves (1) recognition of the host entry receptor, angiotensin-converting enzyme 2 (ACE2), by the SARS-CoV-2 spike protein receptor binding domain (RBD), and (2) the subsequent fusion of the viral and cell membranes. Our long-term immune-defense is the production of antibodies (Abs) that recognize the SARS-CoV-2 RBD and successfully block viral infection. Thus, to understand immunity against SARS-CoV-2, a comprehensive molecular understanding of how human SARS-CoV-2 Abs recognize the RBD is needed. Here, we report the sequence-specific backbone assignment of the SARS-CoV-2 RBD and, furthermore, demonstrate that biomolecular NMR spectroscopy chemical shift perturbation (CSP) mapping successfully and rapidly identifies the molecular epitopes of RBD-specific mAbs. By incorporating NMR-based CSP mapping with other molecular techniques to define RBD-mAb interactions and then correlating these data with neutralization efficacy, structure-based approaches for developing improved vaccines and COVID-19 mAb-based therapies will be greatly accelerated.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/química , Anticorpos Antivirais/química , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Epitopos/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade
13.
EBioMedicine ; 67: 103381, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33993052

RESUMO

BACKGROUND: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein . METHODS: SARS-CoV-2 was passaged in BALB/c mice to obtain mouse-adapted virus strain. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments determined the binding affinity of mouse-adapted SARS-CoV-2 WBP-1 RBD to mouse ACE2 and human ACE2. Finally, we tested whether TLR7/8 agonist Resiquimod (R848) could also inhibit the replication of WBP-1 in the mouse model. FINDINGS: The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, the study tentatively found that the TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge. Therefore, this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. INTERPRETATION: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies. This study also tentatively verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo. FUNDING: This research was funded by the National Key Research and Development Program of China (2020YFC0845600) and Emergency Science and Technology Project of Hubei Province (2020FCA046) and Robert A. Welch Foundation (C-1565).


Assuntos
Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , Imidazóis/administração & dosagem , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Fisiológica , Animais , Sítios de Ligação , COVID-19/metabolismo , COVID-19/prevenção & controle , Células CACO-2 , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , SARS-CoV-2/genética , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Replicação Viral/efeitos dos fármacos , Sequenciamento Completo do Genoma
14.
Science ; 372(6546): 1108-1112, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947773

RESUMO

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Afinidade de Anticorpos , COVID-19/prevenção & controle , Epitopos/imunologia , Humanos , Evasão da Resposta Imune , Imunoglobulina G/sangue , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Domínios Proteicos , Proteômica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
15.
Emerg Microbes Infect ; 10(1): 1065-1076, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34013835

RESUMO

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Hipóxia Celular/fisiologia , Heparitina Sulfato/metabolismo , Neuropilina-1/metabolismo , Glicoproteína da Espícula de Coronavírus/fisiologia , Sindecana-1/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica/efeitos dos fármacos , Heparitina Sulfato/genética , Humanos , Neuropilina-1/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sindecana-1/genética , Células Vero , Ligação Viral/efeitos dos fármacos
16.
Cell ; 184(11): 2927-2938.e11, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34010620

RESUMO

Defining long-term protective immunity to SARS-CoV-2 is one of the most pressing questions of our time and will require a detailed understanding of potential ways this virus can evolve to escape immune protection. Immune protection will most likely be mediated by antibodies that bind to the viral entry protein, spike (S). Here, we used Phage-DMS, an approach that comprehensively interrogates the effect of all possible mutations on binding to a protein of interest, to define the profile of antibody escape to the SARS-CoV-2 S protein using coronavirus disease 2019 (COVID-19) convalescent plasma. Antibody binding was common in two regions, the fusion peptide and the linker region upstream of the heptad repeat region 2. However, escape mutations were variable within these immunodominant regions. There was also individual variation in less commonly targeted epitopes. This study provides a granular view of potential antibody escape pathways and suggests there will be individual variation in antibody-mediated virus evolution.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Epitopos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Algoritmos , COVID-19/terapia , COVID-19/virologia , Linhagem Celular , Biblioteca Gênica , Humanos , Imunização Passiva , Mutação , Domínios Proteicos , SARS-CoV-2/genética , Software , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
17.
Immunity ; 54(5): 1066-1082.e5, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33951417

RESUMO

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Motivos de Aminoácidos , Linfócitos T CD4-Positivos , Criança , Convalescença , Proteínas do Nucleocapsídeo de Coronavírus/química , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Epitopos Imunodominantes/química , Masculino , Pessoa de Meia-Idade , Fenótipo , Fosfoproteínas/química , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
18.
J Mol Biol ; 433(13): 166983, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33839165

RESUMO

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.


Assuntos
Anticorpos Antivirais/química , Antígenos Virais/imunologia , COVID-19/diagnóstico , COVID-19/virologia , SARS-CoV-2/isolamento & purificação , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/isolamento & purificação , Epitopos/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Luciferases , Medições Luminescentes/métodos , Engenharia de Proteínas , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
19.
Sci Adv ; 7(22)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33888467

RESUMO

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.


Assuntos
COVID-19/imunologia , Heme/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Bilirrubina/metabolismo , Biliverdina/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos , Humanos , Soros Imunes , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade
20.
Top Curr Chem (Cham) ; 379(3): 23, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33886017

RESUMO

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still a pandemic around the world. Currently, specific antiviral drugs to control the epidemic remain deficient. Understanding the details of SARS-CoV-2 structural biology is extremely important for development of antiviral agents that will enable regulation of its life cycle. This review focuses on the structural biology and medicinal chemistry of various key proteins (Spike, ACE2, TMPRSS2, RdRp and Mpro) in the life cycle of SARS-CoV-2, as well as their inhibitors/drug candidates. Representative broad-spectrum antiviral drugs, especially those against the homologous virus SARS-CoV, are summarized with the expectation they will drive the development of effective, broad-spectrum inhibitors against coronaviruses. We are hopeful that this review will be a useful aid for discovery of novel, potent anti-SARS-CoV-2 drugs with excellent therapeutic results in the near future.


Assuntos
Antivirais/química , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Proteínas da Matriz Viral/química , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Reposicionamento de Medicamentos , Humanos , SARS-CoV-2/isolamento & purificação , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos
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