Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy

The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.

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Asumsi masyarakat mengenai keselamatan pasien pada masa covid 19 di rumah sakit

10.31219/osf.io/6c7r8 ◽

2020 ◽

Author(s):

Raudatun Hasanah

Keyword(s):

Electron Microscopy ◽

Membrane Protein ◽

World Health Organization ◽

Envelope Protein ◽

Protein S ◽

Spike Protein ◽

World Health ◽

Cryo Electron Microscopy ◽

Health Organization

Rumah sakit sebagai sarana pelayanan kesehatan pada dasarnya adalah untuk menyelamatkan pasien, keselamatan pasien merupakan prioritas bagi pelaksanaan lima isu penting tentang keselamatan di rumah sakit, karena masalah keselamatan pasien berkaitan erat dengan kualitas dan citra rumah sakit itu sendiri. Perkembangan ilmu pengetahuan dan tekhnologi yang sedemikian pesat menyebabkan pelayanan kesehatan di rumah sakit menjadi sangat kompleks sehingga jika tidak dilakukan dengan benar dan hati-hati akan berpotensi untuk terjadinya Insiden Keselamatan Pasien (IKP) yang terdiri dari Kejadian Tidak Diharapkan (KTD), Kejadian Nyaris Cedera (KNC), Kejadian Tidak Cedera (KTC) dan Kondisi Potensial Cedera (KPC) (Depkes,2006).Setiap rumah sakit sudah diwajibkan untuk melakukan gerakan keselamatan pasien, apalagi PERSI sudah menerbitkan buku tentang pelaksanan keselamatan pasien di rumah sakit dan dalam waktu mendatang keselamatan pasien termasuk dalam penilaian akreditasi rumah sakit. Pada prinsipnya pelaksaan keselamatan pasien sesuai dengan standar departemen kesehatan, namun didalam pelaksanaan keselamatan pasien hampir sama pada setiap rumah sakit.Diawal tahun ini keselamatan pasien harus semakin ditingkatkan disebabkan munculnya virus covid 19 yang awalnya terjadi di Wuhan, provinsi Hubei, China dan dikaitkan dengan pasar binatang. Dalam rentang waktu satu bulan terjadi peningkatan kasus yang signifikan dan meluas ke beberapa provinsi di China, bahkan ke Jepang, Thailand dan Korea Selatan.satu Penyebaran penyakit yang begitu cepat serta meluas ke beberapa negara menyebabkan World Health Organization (WHO) akhirnya mengumumkan COVID-19 sebagai pandemi pada 12 Maret 2020. Virus corona berbentuk bulat dengan diameter sekitar 125 nm seperti yang digambarkan dalam penelitian menggunakan cryo-electron microscopy. Partikel virus corona mengandung empat protein struktural utama, yaitu protein S (spike protein) yang berbentuk seperti paku, protein M (membrane protein), protein E (envelope protein), dan protein N (nucleocapside protein). Protein S (~150 kda), protein M (~25– 30 kda), protein E (~8–12 kda) sedangkan protein N terdapat di dalam nukleokapsid.Virus corona merupakan zoonosis, sehingga terdapat kemungkinkan virus berasal dari hewan dan ditularkan ke manusia. Pada COVID-19 belum diketahui dengan pasti proses penularan dari Perkembangan data selanjutnya menunjukkan penularan antar manusia (human to human), yaitu diprediksi melalui droplet dan kontak dengan virus yang dikeluarkan dalam droplet. Penularan ini terjadi umumnya melalui droplet dan kontak dengan virus kemudian virus dapat masuk ke dalam mukosa yang terbuka. Suatu analisis mencoba mengukur laju penularan berdasarkan masa inkubasi, gejala dan durasi antara gejala dengan pasien yang diisolasi. Analisis tersebut mendapatkan hasil penularan dari 1 pasien ke sekitar 3 orang di sekitarnya, tetapi kemungkinan penularan di masa inkubasi menyebabkan masa kontak pasien ke orang sekitar lebih lama sehingga risiko jumlah kontak tertular dari 1 pasien mungkin dapat lebih besar.

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Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Science ◽

10.1126/science.abe6230 ◽

2021 ◽

Vol 371 (6530) ◽

pp. eabe6230 ◽

Cited By ~ 5

Author(s):

Paul-Albert Koenig ◽

Hrishikesh Das ◽

Hejun Liu ◽

Beate M. Kümmerer ◽

Florian N. Gohr ◽

...

Keyword(s):

Electron Microscopy ◽

Receptor Binding ◽

Conformational Changes ◽

Passive Immunization ◽

Spike Protein ◽

Receptor Binding Domain ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Escape Mutants

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

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Structural basis for antibody resistance to SARS-CoV-2 omicron variant

10.1101/2021.12.21.473620 ◽

2021 ◽

Author(s):

Gabriele Cerutti ◽

Yicheng Guo ◽

Liu Lihong ◽

Zhening Zhang ◽

Liyuan Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Structural Basis ◽

Receptor Binding Domain ◽

Functional Studies ◽

Antibody Recognition ◽

Cryo Electron Microscopy ◽

Antibody Resistance

The recently reported B.1.1.529 Omicron variant of SARS-CoV-2 includes 34 mutations in the spike protein relative to the Wuhan strain that initiated the COVID-19 pandemic, including 15 mutations in the receptor binding domain (RBD). Functional studies have shown omicron to substantially escape the activity of many SARS-CoV-2-neutralizing antibodies. Here we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of the Omicron spike protein ectodomain. The structure depicts a spike that is exclusively in the 1-RBD-up conformation with increased mobility and inter-protomer asymmetry. Many mutations cause steric clashes and/or altered interactions at antibody binding surfaces, whereas others mediate changes of the spike structure in local regions to interfere with antibody recognition. Overall, the structure of the omicron spike reveals how mutations alter its conformation and explains its extraordinary ability to evade neutralizing antibodies.

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Predicted pH-dependent stability of SARS-CoV-2 spike protein trimer from interfacial acidic groups

10.1101/2021.06.06.447235 ◽

2021 ◽

Author(s):

Vanessa R Lobo ◽

Jim Warwicker

Keyword(s):

Electron Microscopy ◽

Receptor Binding ◽

Ph Dependence ◽

Spike Protein ◽

Ph Effects ◽

Large Set ◽

Acidic Ph ◽

Receptor Binding Domain ◽

Cryo Electron Microscopy ◽

Ph Dependent

Transition between receptor binding domain (RBD) up and down forms of the SARS-CoV-2 spike protein trimer is coupled to receptor binding and is one route by which variants can alter viral properties. It is becoming apparent that key roles in the transition are played by pH and a more compact closed form, termed locked. Calculations of pH-dependence are made for a large set of spike trimers, including locked form trimer structures that have recently become available. Several acidic sidechains become sufficiently buried in the locked form to give a predicted pH-dependence in the mild acidic range, with stabilisation of the locked form as pH reduces from 7.5 to 5, consistent with emerging characterisation by cryo-electron microscopy. The calculated pH effects in pre-fusion spike trimers are modulated mainly by aspartic acid residues, rather than the more familiar histidine role at mild acidic pH. These acidic sidechains are generally surface located and weakly interacting when not in a locked conformation. In this model, their replacement (perhaps with asparagine) would remove the pH-dependent destabilisation of locked spike trimer conformations, and increase their recovery at neutral pH. This would provide an alternative or supplement to the insertion of disulphide linkages for stabilising spike protein trimers, with potential relevance for vaccine design.

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Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

Nature Communications ◽

10.1038/s41467-021-27388-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dukas Jurėnas ◽

Leonardo Talachia Rosa ◽

Martial Rey ◽

Julia Chamot-Rooke ◽

Rémi Fronzes ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Spike Protein ◽

Modular Organization ◽

Cross Linking ◽

Multidomain Proteins ◽

Type Vi Secretion ◽

Cryo Electron Microscopy ◽

Dependent Growth ◽

Contact Dependent Growth Inhibition

AbstractBacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

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Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

Science ◽

10.1126/science.abd0826 ◽

2020 ◽

Vol 369 (6510) ◽

pp. 1501-1505 ◽

Cited By ~ 50

Author(s):

Ching-Lin Hsieh ◽

Jory A. Goldsmith ◽

Jeffrey M. Schaub ◽

Andrea M. DiVenere ◽

Hung-Che Kuo ◽

...

Keyword(s):

Electron Microscopy ◽

Room Temperature ◽

Spike Protein ◽

High Yield ◽

Stability Testing ◽

Freeze Thaw ◽

Cryo Electron Microscopy ◽

Yield Production ◽

Serological Diagnostics ◽

High Yield Production

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo–electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants

10.1101/2021.09.02.458774 ◽

2021 ◽

Author(s):

James W. Saville ◽

Dhiraj Mannar ◽

Xing Zhu ◽

Shanti S. Srivastava ◽

Alison M. Berezuk ◽

...

Keyword(s):

Electron Microscopy ◽

Viral Fitness ◽

Spike Protein ◽

Wild Type ◽

Antibody Neutralization ◽

Vaccination Rates ◽

Modest Increase ◽

Terminal Domain ◽

Cryo Electron Microscopy ◽

Neutralizing Epitopes

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

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Cryo-electron Microscopy Structure of the Swine Acute Diarrhea Syndrome Coronavirus Spike Glycoprotein Provides Insights into Evolution of Unique Coronavirus Spike Proteins

Journal of Virology ◽

10.1128/jvi.01301-20 ◽

2020 ◽

Vol 94 (22) ◽

Author(s):

Hongxin Guan ◽

Youwang Wang ◽

Vanja Perčulija ◽

Abdullah F. U. H. Saeed ◽

Yichang Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Immune Evasion ◽

Acute Diarrhea ◽

Structural Features ◽

The Other ◽

Spike Protein ◽

Interspecies Transmission ◽

Cryo Electron Microscopy ◽

Diarrhea Syndrome ◽

Spike Proteins

ABSTRACT Coronaviruses (CoV) have caused a number of major epidemics in humans and animals, including the current pandemic of coronavirus disease 2019 (COVID-19), which has brought a renewed focus on the evolution and interspecies transmission of coronaviruses. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was recently identified in piglets in southern China, is an alphacoronavirus that originates from the same genus of horseshoe bats as severe acute respiratory syndrome CoV (SARS-CoV) and that was reported to be capable of infecting cells from a broad range of species, suggesting a considerable potential for interspecies transmission. Given the importance of the coronavirus spike (S) glycoprotein in host range determination and viral entry, we report a cryo-electron microscopy (cryo-EM) structure of the SADS-CoV S trimer in the prefusion conformation at a 3.55-Å resolution. Our structure reveals that the SADS-CoV S trimer assumes an intrasubunit quaternary packing mode in which the S1 subunit N-terminal domain (S1-NTD) and the S1 subunit C-terminal domain (S1-CTD) of the same protomer pack together by facing each other in the lying-down state. SADS-CoV S has several distinctive structural features that may facilitate immune escape, such as a relatively compact architecture of the S trimer and epitope masking by glycan shielding. Comparison of SADS-CoV S with the spike proteins of the other coronavirus genera suggested that the structural features of SADS-CoV S are evolutionarily related to those of the spike proteins of the other genera rather than to the spike protein of a typical alphacoronavirus. These data provide new insights into the evolutionary relationship between spike glycoproteins of SADS-CoV and those of other coronaviruses and extend our understanding of their structural and functional diversity. IMPORTANCE In this article, we report the atomic-resolution prefusion structure of the spike protein from swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV is a pathogenic alphacoronavirus that was responsible for a large-scale outbreak of fatal disease in pigs and that was reported to be capable of interspecies transmission. We describe the overall structure of the SADS-CoV spike protein and conducted a detailed analysis of its main structural elements. Our results and analyses are consistent with those of previous phylogenetic studies and suggest that the SADS-CoV spike protein is evolutionarily related to the spike proteins of betacoronaviruses, with a strong similarity in S1-NTDs and a marked divergence in S1-CTDs. Moreover, we discuss the possible immune evasion strategies used by the SADS-CoV spike protein. Our study provides insights into the structure and immune evasion strategies of the SADS-CoV spike protein and broadens the understanding of the evolutionary relationships between coronavirus spike proteins of different genera.

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Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies

PLoS Biology ◽

10.1371/journal.pbio.3001237 ◽

2021 ◽

Vol 19 (4) ◽

pp. e3001237

Author(s):

Xing Zhu ◽

Dhiraj Mannar ◽

Shanti S. Srivastava ◽

Alison M. Berezuk ◽

Jean-Philippe Demers ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Structural Changes ◽

Neutralizing Antibody ◽

Antibody Fragments ◽

Spike Protein ◽

Receptor Binding Domain ◽

Structural Explanation ◽

Binding Interface ◽

Cryo Electron Microscopy

The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

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