Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2

The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain named MASCp36 that causes acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was ~100 PFU in aged, male BALB/c mice. Deep sequencing identified three amino acid mutations, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, which significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of mACE2 in complex with the RBD of MASCp36 at 3.7-angstrom resolution elucidates molecular basis for the receptor-binding switch driven by amino acid substitutions. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in mice.

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Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-25903-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shihui Sun ◽

Hongjing Gu ◽

Lei Cao ◽

Qi Chen ◽

Qing Ye ◽

...

Keyword(s):

Binding Affinity ◽

Receptor Binding ◽

Structural Basis ◽

Rapid Adaptation ◽

Cryo Electron Microscopy ◽

Electron Microscopy Analysis ◽

Microscopy Analysis ◽

And Gender ◽

Related Mortality

AbstractThere is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.

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Genetic Variation and Evolution of the 2019 Novel Coronavirus

Public Health Genomics ◽

10.1159/000513530 ◽

2021 ◽

pp. 1-13

Author(s):

Salvatore Dimonte ◽

Muhammed Babakir-Mina ◽

Taib Hama-Soor ◽

Salar Ali

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Rapid Evolution ◽

Single Amino Acid ◽

Angiotensin Converting Enzyme 2 ◽

New Type ◽

Amino Acid Mutations ◽

Host Infection ◽

Human Coronaviruses ◽

Novel Coronavirus

Introduction: SARS-CoV-2 is a new type of coronavirus causing a pandemic severe acute respiratory syndrome (SARS-2). Coronaviruses are very diverting genetically and mutate so often periodically. The natural selection of viral mutations may cause host infection selectivity and infectivity. Methods: This study was aimed to indicate the diversity between human and animal coronaviruses through finding the rate of mutation in each of the spike, nucleocapsid, envelope, and membrane proteins. Results: The mutation rate is abundant in all 4 structural proteins. The most number of statistically significant amino acid mutations were found in spike receptor-binding domain (RBD) which may be because it is responsible for a corresponding receptor binding in a broad range of hosts and host selectivity to infect. Among 17 previously known amino acids which are important for binding of spike to angiotensin-converting enzyme 2 (ACE2) receptor, all of them are conservative among human coronaviruses, but only 3 of them significantly are mutated in animal coronaviruses. A single amino acid aspartate-454, that causes dissociation of the RBD of the spike and ACE2, and F486 which gives the strength of binding with ACE2 remain intact in all coronaviruses. Discussion/Conclusion: Observations of this study provided evidence of the genetic diversity and rapid evolution of SARS-CoV-2 as well as other human and animal coronaviruses.

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Investigation of serum homocysteine levels in relation to age and sex

Journal of the Hellenic Veterinary Medical Society ◽

10.12681/jhvms.22225 ◽

2020 ◽

Vol 70 (4) ◽

pp. 1811

Author(s):

N.H. AKSOY

Keyword(s):

Risk Factor ◽

Amino Acid ◽

Metabolic Diseases ◽

Reference Value ◽

Methionine Metabolism ◽

Age And Gender ◽

Serum Homocysteine ◽

Healthy Sheep ◽

And Gender ◽

Age And Sex

Homocysteine is a non-proteinogenic and a derived amino acid in the methionine metabolism and is a risk factor for cardiovascular and many other metabolic diseases. In this study, the purpose was to determine serum homocysteine levels in healthy sheep based on differences in age and gender. 220 healthy Akkaraman sheep, composed of both females (n=55 lambs and 55 ewes) and males (n=55 lambs and 55 rams), were used as animal samples. The measurements of serum homocysteine concentrations were performed with ELISA-HCY kit. The levels of serum homocysteine of sheep were detected in ewes, female lambs, rams and male lambs as 2,91±0,50; 2,99±0,42; 11,22±3,10; 6,43±1,26 μmol/L, respectively. The primary intent of this study was to investigation and characterization the serum Hcy concentrations in healthy sheep broken down by different ages in both genders As a result, the serum homocysteine values that can constitute a reference value for healthy breeds of sheep were determined in this study.

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Hemagglutination Inhibition (HAI) Antibody Landscapes after Vaccination with diverse H7 hemagglutinin (HA) proteins

10.1101/2021.01.25.428062 ◽

2021 ◽

Author(s):

Hyesun Jang ◽

Ted M Ross

Keyword(s):

Amino Acid ◽

Hemagglutination Inhibition ◽

Lethal Dose ◽

Antigenic Site ◽

Glycosylation Site ◽

Amino Acid Sequences ◽

Serum Samples ◽

Influenza Hemagglutinin ◽

Amino Acid Mutations ◽

Antigenic Profile

AbstractBackgroundA systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-blocking antibodies against co-circulating H7 influenza strains.MethodA panel of nine H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013.ResultsVLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain.ConclusionThis study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.

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Ten emerging SARS-CoV-2 spike variants exhibit variable infectivity, animal tropism, and antibody neutralization

Communications Biology ◽

10.1038/s42003-021-02728-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Li Zhang ◽

Zhimin Cui ◽

Qianqian Li ◽

Bo Wang ◽

Yuanling Yu ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

Receptor Binding ◽

Immune Escape ◽

Cathepsin L ◽

Single Amino Acid ◽

Receptor Binding Domain ◽

Therapeutic Antibodies ◽

Amino Acid Mutations ◽

Better Than

AbstractEmerging mutations in SARS-CoV-2 cause several waves of COVID-19 pandemic. Here we investigate the infectivity and antigenicity of ten emerging SARS-CoV-2 variants—B.1.1.298, B.1.1.7(Alpha), B.1.351(Beta), P.1(Gamma), P.2(Zeta), B.1.429(Epsilon), B.1.525(Eta), B.1.526-1(Iota), B.1.526-2(Iota), B.1.1.318—and seven corresponding single amino acid mutations in the receptor-binding domain using SARS-CoV-2 pseudovirus. The results indicate that the pseudovirus of most of the SARS-CoV-2 variants (except B.1.1.298) display slightly increased infectivity in human and monkey cell lines, especially B.1.351, B.1.525 and B.1.526 in Calu-3 cells. The K417N/T, N501Y, or E484K-carrying variants exhibit significantly increased abilities to infect mouse ACE2-overexpressing cells. The activities of furin, TMPRSS2, and cathepsin L are increased against most of the variants. RBD amino acid mutations comprising K417T/N, L452R, Y453F, S477N, E484K, and N501Y cause significant immune escape from 11 of 13 monoclonal antibodies. However, the resistance to neutralization by convalescent serum or vaccines elicited serum is mainly caused by the E484K mutation. The convalescent serum from B.1.1.7- and B.1.351-infected patients neutralized the variants themselves better than other SARS-CoV-2 variants. Our study provides insights regarding therapeutic antibodies and vaccines, and highlights the importance of E484K mutation.

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A single de novo substitution in SARS-CoV-2 spike informs enhanced adherence to human ACE2.

10.1101/2021.07.16.452441 ◽

2021 ◽

Author(s):

Elena Erausquin ◽

Jacinto Lopez-Sagaseta

Keyword(s):

Amino Acid ◽

Cell Membrane ◽

Receptor Binding ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Structural Basis ◽

Receptor Binding Domain ◽

To Come ◽

Single Substitution

SARS-CoV-2 initiates colonization of host cells by binding to cell membrane ACE2 receptor. This binding is mediated by the viral spike receptor binding domain (RBD). The COVID-19 pandemic has brought devastating consequences at a clinical, social and economical levels. Therefore, anticipation of potential novel SARS-causing species or SARS-CoV-2 variants with enhanced binding to ACE2 is key in the prevention of future threats to come. We have characterized a de novo single substitution, Q498Y, in SARS-CoV-2 RBD that confers stronger adherence to ACE2. While the SARS-CoV-2 beta variant, which includes three simultaneous amino acid replacements, induces a 4-fold stronger affinity, a single Q498Y substitution results in 2.5-fold tighter binding, compared to the Wuhan-Hu-1 SARS-CoV-2 2019 strain. Additionally, we crystallized RBDQ498Y complexed with ACE2 and provide here the structural basis for this enhanced affinity. These studies inform a rationale for prevention of potential SARS-causing viruses to come.

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Complete functional mapping of infection- and vaccine-elicited antibodies against the fusion peptide of HIV

10.1101/307587 ◽

2018 ◽

Cited By ~ 1

Author(s):

Adam S. Dingens ◽

Priyamvada Acharya ◽

Hugh K. Haddox ◽

Reda Rawi ◽

Kai Xu ◽

...

Keyword(s):

Amino Acid ◽

Animal Models ◽

Fusion Peptide ◽

Antibody Responses ◽

Viral Escape ◽

Single Amino Acid ◽

Structural Basis ◽

Major Goal ◽

Amino Acid Mutations ◽

Hiv 1

AbstractEliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape provide a template to improve the breadth or potency of future vaccine-induced antibodies against Env’s fusion peptide.Author summaryA major goal of HIV-1 vaccine design is to elicit antibodies that neutralize diverse strains of HIV-1. Recently, some of us elicited such antibodies in animal models using immunogens based on the epitope of a broad antibody (VRC34.01) isolated from an infected individual. Further improving these vaccine-elicited antibody responses will require a detailed understanding of how the resulting antibodies target HIV’s envelope protein (Env). Here, we used mutational antigenic profiling to precisely map the epitope of two vaccine-elicited antibodies and the template VRC34.01 antibody. We did this by quantifying the effect of all possible amino acid mutations to Env on antibody neutralization. Although all antibodies target a similar region of Env, we found clear differences in the functional interaction of Env with the vaccine- and infection-elicited antibodies. We combined these functional data with structural analyses to identify antibody–Env interactions that could improve the breadth of vaccine-elicited antibodies, and thereby help to refine vaccination schemes to achieve broader responses.

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