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Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine

  • Yasunori Watanabe
    Yasunori Watanabe
    School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.
    Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.
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  • , 
  • Luiza Mendonça
    Luiza Mendonça
    Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, U.K.
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  • Elizabeth R. Allen
    Elizabeth R. Allen
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
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  • , 
  • Andrew Howe
    Andrew Howe
    Electron Bio-imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, U.K.
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  • , 
  • Mercede Lee
    Mercede Lee
    The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
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  • , 
  • Joel D. Allen
    Joel D. Allen
    School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.
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    Orcidhttp://orcid.org/0000-0003-2547-968X
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  • Himanshi Chawla
    Himanshi Chawla
    School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.
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  • David Pulido
    David Pulido
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
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  • Francesca Donnellan
    Francesca Donnellan
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
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  • Hannah Davies
    Hannah Davies
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
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  • Marta Ulaszewska
    Marta Ulaszewska
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
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  • Sandra Belij-Rammerstorfer
    Sandra Belij-Rammerstorfer
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    NIHR Oxford Biomedical Research Centre, Oxford, U.K.
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  • Susan Morris
    Susan Morris
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
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  • Anna-Sophia Krebs
    Anna-Sophia Krebs
    Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, U.K.
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  • Wanwisa Dejnirattisai
    Wanwisa Dejnirattisai
    The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
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  • , 
  • Juthathip Mongkolsapaya
    Juthathip Mongkolsapaya
    The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
    Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
    Chinese Academy of Medical Science (CAMS) Oxford Institute (COI), University of Oxford, Oxford, U.K.
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  • Piyada Supasa
    Piyada Supasa
    The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
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  • , 
  • Gavin R. Screaton
    Gavin R. Screaton
    The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
    Division of Medical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, U.K.
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  • , 
  • Catherine M. Green
    Catherine M. Green
    The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
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  • , 
  • Teresa Lambe*
    Teresa Lambe
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    NIHR Oxford Biomedical Research Centre, Oxford, U.K.
    *Email: [email protected] (T.L.).
    More by Teresa Lambe
  • , 
  • Peijun Zhang*
    Peijun Zhang
    Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, U.K.
    Electron Bio-imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, U.K.
    *Email: [email protected] (P.Z).
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    Orcidhttp://orcid.org/0000-0003-1803-691X
  • , 
  • Sarah C. Gilbert*
    Sarah C. Gilbert
    The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    NIHR Oxford Biomedical Research Centre, Oxford, U.K.
    *Email: [email protected] (S.C.G.).
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  • , and 
  • Max Crispin*
    Max Crispin
    School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.
    *Email: [email protected] (M.C.).
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    Orcidhttp://orcid.org/0000-0002-1072-2694
Cite this: ACS Cent. Sci. 2021, 7, 4, 594–602
Publication Date (Web):April 2, 2021
https://doi.org/10.1021/acscentsci.1c00080

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirm the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

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Synopsis

The structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine are described. Also, native-like post-translational processing and assembly are demonstrated.

Introduction

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Vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are an essential countermeasure to stem the COVID-19 pandemic. Vaccine development efforts aim to produce both a strong T cell response and a neutralizing immune response against the virus, the main target being the spike (S) proteins that protrude from the viral envelope. (1) The S protein is responsible for mediating host-cell entry, with the S1 and S2 subunits facilitating angiotensin-converting enzyme 2 (ACE2) receptor binding and membrane fusion, respectively. (2−4) The SARS-CoV-2 S gene encodes the extensively glycosylated trimeric class I viral fusion protein with 22 N-linked glycans per protomer. (5)
While the coronavirus S glycoprotein is the principal target for SARS-CoV-2 vaccine design, leading vaccine candidates and recently licensed vaccines utilize a variety of constructs and strategies. (6) For example, both Moderna’s mRNA-1273 and Pfizer’s BNT162b2 (7) encode full length S with two mutations to stabilize the prefusion conformation, (8) and Sinovac’s CoronaVac inactivated virus vaccine presents the wild-type S on the viral surface, (9) although the majority of spikes are in the postfusion conformation. (10) One key aim for SARS-CoV-2 vaccine development is to elicit a robust immune response against the spike, and more specifically the receptor-binding domain (RBD), where many neutralizing epitopes are located. (10−15) To this end, many vaccine candidates include (two or more) stabilizing mutations in the S protein, such that the protein maintains the prefusion conformation and avoids shedding of S1. (3)
The replication-deficient chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19/AZD1222 (hereafter referred to as ChAdOx1 nCoV-19), encodes the full-length wild-type SARS-CoV-2 spike protein. ChAdOx1 nCoV-19 has previously been shown to elicit not only strong neutralizing antibody responses but also robust spike-specific T-cell responses. (16−19) Although adenovirus-vectored vaccines are a promising way to deliver viral glycoprotein antigens, the processing and presentation of the SARS-CoV-2 spike are yet to be characterized. Understanding the molecular features of the expressed viral antigen is important for the interpretation of the immune response to this vaccine. Here, we determine the native-like functionality, prefusion trimeric structure, and glycosylation of SARS-CoV-2 S protein expressed from the ChAdOx1 nCoV-19 vaccine.

Results and Discussion

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Expression of Prefusion Conformation SARS-CoV-2 S Glycoprotein on Cell Surfaces upon ChAdOx1 nCoV-19 Infection

ChAdOx1 nCoV-19 encodes a wild-type S sequence, including the transmembrane domains, which is trafficked to the cell surface with a tissue plasminogen activator (tPA) signal sequence (Figure 1A). Using flow cytometry, we first detected the presence of the S glycoprotein at the cell surface of ChAdOx1 nCoV-19 infected HeLa S3 cells (Figure 1B). Sera from mice vaccinated with ChAdOx1 nCoV-19 was used to detect the expression level of S at the cell surface, revealing ∼ 60–70% of all cells expressing S (range of duplicate averages across three experimental repeats) (Supplementary (Sup.) Figure 1). A ChAdOx1 vaccine encoding an irrelevant filovirus antigen (EBOV) was used as a negative control, (20) which showed a low level of binding to the anti-ChAdOx1 nCoV-19 vaccine serum (∼0.5–2% cells across all experiments). This observation accounts for antibodies raised against the vector itself rather that the vaccine antigen. Subsequently, we sought to examine the properties of the cell surface expressed S protein using recombinant ACE2 and a panel of human mAbs which bind to specific regions of S (Figure 1B). Binding of infected cells to recombinant ACE2 confirms the correct folding of S and native presentation and functionality of the RBD (Figure 1B). This observation is further supported by the binding of the human mAbs, in particular Ab45 which recognizes RBD, Ab71 which recognizes the trimeric spike, and Ab111 which recognizes the NTD. Ab44 which recognizes S2 also demonstrates considerable binding. These data confirm significant presence of the prefusion trimer at the cell surface. In the absence of a postfusion specific anti-S2 antibody we were unable to quantify if some postfusion spike is present at the cell surface.

Figure 1

Figure 1. ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino acid sequons (NXS/T, where X ≠ P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice), or a panel of human mAbs (Ab44, Ab45, Ab71, and Ab111, which recognize S2, RBD, trimeric S, and NTD, respectively) and compared to noninfected controls, analyzed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with antispike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19; middle (red), ACE2; and right (shades of green) human mAbs. In dark gray cells infected with an irrelevant ChAdOx1 vaccine and in light gray noninfected cells are shown as a control. Experimental replicates were performed two times, and representative data are shown.

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While this observation that the majority of cells infected with ChAdOx1 nCoV-19 present native-like spikes on the cell surface (Figure 1B), it is interesting to note that a population may shed the S1 subunit. Whether this is a beneficial or detrimental feature with respect to the elicitation of immune responses during vaccination is unknown. Shedding of S1 subunits from viruses occurs during native infection, (21−24) and the ChAdOx1 nCoV-19-derived S proteins mimic this native feature of the viral spike. Nevertheless it has been shown that vaccination with ChAdOx nCoV-19 elicits robust antibody and T cell responses. (16−19)

Structural Analysis of Membrane-Associated ChAdOx1 nCoV-19 Derived SARS-CoV-2 S Protein

After the surface expression of the S protein driven by ChAdOx1 nCoV-19 vector infection was confirmed by cytometry, we sought to probe the structure of S proteins on native cell surfaces using cryo-electron tomography and subtomogram averaging (cryoET STA). We imaged U2OS and HeLa cells infected with ChAdOx1 nCoV-19. These cells were chosen for the thin cell peripheries which make them accessible for cryoET analysis. The tomograms revealed that the surface of the cells is densely covered with protruding densities consistent with the size and shape of the prefusion conformation of SARS-CoV-2 S protein (Figure 2A and 2B, movie 1). These densities are absent in control uninfected cells (Sup. Figure 2). To determine whether these spikes represent the SARS-CoV-2 prefusion spike, we performed subtomogram averaging of 11391 spikes from cell surfaces using emClarity. (25) The averaged density map, with 3-fold symmetry applied, is at 11.6 Å resolution (at 0.5 FSC cutoff) (Figure 2C), resolving the overall spike structure, which overlaps very well with prefusion spike atomic models in the literature (4,26−29) (Figure 2D and 2E). We subsequently preformed cryo-immunolabeling using ChAdOx1 nCoV-19 vaccinated mice sera which confirmed the presentation of abundant S protein on the cell surface, but not on control cells infected with ChAdOx-GFP or uninfected cells (Sup. Figure 3).

Figure 2

Figure 2. CryoET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm. (B) Detailed view of the boxed area marked in (A). White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C–E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cutoff (C), shown from side view (D) and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) (29) is fitted for reference.

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In order to investigate the presence of postfusion S protein conformations, we employed template searching for pre- and postfusion spikes on cells infected with ChAdOx nCoV-19 (Sup. Figure 4). This analysis revealed a presence of abundant prefusion spikes on the cell surface, while little postfusion spikes are detected. Given the lack of a postfusion conformation specific anti-S2 antibody, we were not able to probe, by cryo-immunolabeling, postfusion spikes on the surface of cells.
The presentation of prefusion S proteins was achieved through encoding SARS-CoV-2 S protein in the ChAdOx1 backbone without the incorporation of stabilizing mutations. Given that most neutralizing antibodies target epitopes displayed on the prefusion spike, the cryoET analysis revealing the expression of trimeric SARS-CoV-2 spike in the prefusion conformation strongly supports ChAdOx nCoV-19 as an effective vaccine strategy for the generation of a neutralizing immune response.

Site-Specific Glycan Analysis of the ChAdOx1 nCoV-19 Derived SARS-CoV-2 S Protein Reveals Native-Like Glycan Maturation

The SARS-CoV-2 S gene encodes 22 N-linked glycosylation sequons which span both the S1 and S2 subunits (Figure 1A). These host-derived glycans mask immunogenic protein epitopes from the humoral immune system, a common strategy utilized by viruses to evade the immune system. (30) It is therefore of considerable importance that spike proteins produced upon vaccination successfully recapitulates the glycosylation observed on the virus, since antibodies may be elicited against protein epitopes that are occluded during natural infection. We sought to resolve the site-specific glycosylation of SARS-CoV-2 S proteins produced by cells following infection with ChAdOx1 nCoV-19. To this end, human embryonic kidney 293 F (HEK293F) cells were incubated with ChAdOx1 nCoV-19. Western blot analysis of the protein pellets from the cell lysates using both S1- and S2-specific antibodies confirmed the presence and maturation of the S protein into the S1 and S2 subunits following furin cleavage. Since the whole cell was lysed, intracellular material of uncleaved S0 protein was also detected (Figure 3A). These gel bands were excised and analyzed by liquid chromatography–mass spectrometry (LC-MS) separately.

Figure 3

Figure 3. Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1 = Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2 = Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3 = 2P-stabilized SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (gray), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarize the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (gray) N-linked glycan populations. Representative glycans are modeled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB), (3) with one RBD in the “up” conformation. The modeled glycans are colored according to oligomannose/hybrid-type glycan content with glycan sites labeled in green (80–100%), orange (30–79%), pink (0–29%), or gray (not detected).

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To determine the site-specific glycosylation and glycan occupancy of the spike protein, we employed trypsin, chymotrypsin, α-lytic protease to generate glycopeptide samples. In order to increase the signal intensity of the glycopeptides, these samples were treated sequentially with Endoglycosidase H to cleave oligomannose- and hybrid-type glycans between the core GlcNAc residues, leaving a single GlcNAc attached to the Asn residue (+203 Da). Subsequent PNGase F treatment removed the remaining complex-type glycans which deamidates the Asn to Asp resulting in a + 3 Da shift as the reaction was conducted in 18O water. (31−33) This homogenization of the glycans into oligomannose/hybrid-type and complex-type glycan categories was performed in order to increase coverage across the S protein. The mass of peptides with unoccupied glycan sites exhibits no mass shift. The resulting peptide/glycopeptide pool from both the S1/S2 (cleaved) and S0 (uncleaved) proteins were subjected to LC-MS analysis (Figure 3B).
The glycans presented on this S0 immature form of the protein were predominantly underprocessed oligomannose/hybrid-type glycans (85%) (Sup. Table 1), which reflects the intracellular origin of the protein, yet to be trafficked through to the cell membrane. This is consistent with furin protease being located in the trans-Golgi apparatus. In contrast, the cleaved S1/S2 protein, likely to be presented on the surface of cells, possesses significantly lower levels of these under-processed glycans (56%) and elevated levels of complex-type glycans (38%). Furthermore, high levels of glycan occupancy were observed across the protein. This is especially important since glycans are known to shield more immunogenic protein epitopes; thus, the expression of SARS-CoV-2 S proteins using the ChAdOx1 vaccine platform results in presentation of epitopes similar to those seen during natural infection. (5,30,34) We note that the overall levels of oligomannose/hybrid-type glycans on the S1/S2 protein were elevated compared to previous analyses; however, we attribute this to the lack of coverage of the glycosylation sites toward the C-terminus which are predominantly complex-type on recombinant proteins and viruses. (5,27,35)
Using the cryo-EM structure of the trimeric SARS-CoV-2 S protein, we mapped the glycosylation status of the S1/S2 protein (Figure 3C). A mixture of oligomannose/hybrid and complex-type sites were observed, with glycan sites such as N234, which are known to have stabilizing effects on the RBD, preserving the predominantly oligomannose state reported in both recombinant proteins and viruses. (5,27,35,36) Similarly, the glycan at N165 which also stabilizes the RBD “up” conformation was determined to be complex-type on the S protein arising from infection of cells with ChAdOx1 nCoV19. Since glycans are sensitive reporters of local protein architecture, it is encouraging that such glycans, known to have structural roles, conserve their processing state which provides additional evidence of native-like prefusion protein structure. Furthermore, there were no detectable traces of O-linked glycans on the ChAdOx1-derived S protein which may be due to their low abundances. This has been reflected in the low abundances of O-linked glycans from previous analysis on recombinant proteins, (5,35) and lack of densities on EM maps. (26) A site-specific comparison between previously analyzed stabilized recombinant S protein analyses and the ChAdOx1 nCoV-19-derived S protein is provided (Sup. Figure 5).
Adenoviruses have broad cell tropism due to the widespread distribution of their cellular receptors such as coxsackie and adenovirus receptor (CAR) and CD46. (37,38) Replication-deficient adenovirus vectored vaccines, such as ChAdOx nCoV-19, are administered intramuscularly and predominantly induce antigen expression in muscle cells, fibroblasts, and professional antigen presenting cells (APCs, including dendritic cells) present in germinal centers of the lymph nodes. These APCs are primed by direct adenovirus infection or by cross-priming from nonimmune cells. (38)
During vaccination, SARS-CoV-2 S protein processing can be dependent on both the receptors present on and the enzymes expressed by the S-producing cells. (39) Presence of the cell entry receptor ACE2 and host-cell proteases including furin and TMPRSS2 can drive conformational rearrangements resulting in postfusion conformations of S. Tissue distribution of ACE2 is primarily in the nasal mucosa and GI tracts, and TMPRSS2 is predominantly expressed in lungs and GI tracts, where cells are susceptible to SARS-CoV-2 infection. In contrast, the muscle, fibroblast, and immune cell types targeted by intramuscularly administered ChAdOx1 nCoV-19 have not been shown to express high levels of ACE2 or TMPRSS2. (40−42) The cell lines chosen for this study of the higher order structure and conformation vaccine-derived S (HeLa S3 and U20S) similarly do not express these enzymes in high abundance and are therefore a suitable surrogate for this in vitro study. Although HEK293F cells are not a natural host cell type in which the S protein would be expressed upon administration of ChAdOx nCoV-19, due to the conservation of the enzymatic processing of the early stages of the mammalian N-linked glycosylation pathway, (43,44) they represent a suitable model system to study the nature of the carbohydrate modifications of the S protein. It may be useful in future to examine vaccine derived antigen processing in primary cells or even biopsy material, especially if alternative routes, such as intranasal administration, are considered.
Overall, this study provides holistic biophysical scrutiny of the spike protein that is produced upon vaccination with ChAdOx1 nCoV-19. Importantly, we reveal the native-like mimicry of SARS-CoV-2 S protein’s receptor binding functionality, prefusion structure, and processing of glycan modifications. While there is some evidence of S1 subunit shedding, as observed on the actual SARS-CoV-2 virus, the impact of the catabolism of the trimeric spike on immunogenicity and vaccine efficacy is yet to be determined. The data presented here will assist comparison across SARS-CoV-2 vaccination strategies and aid the development of next-generation immunogens.

Materials and Methods

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Production of ChAdOx1 nCoV-19

ChAdOx1 nCoV-19 was produced as described previously. (17) The spike protein (S) of SARS-Cov-2 (Genbank accession number YP_009724390.1) was codon optimized for expression in human cell lines and synthesized by GeneArt Gene Synthesis (Thermo Fisher Scientific). The sequence encoding amino acids 2–1273 were cloned into a shuttle plasmid following InFusion cloning (Clontech). The shuttle plasmid encodes a modified human cytomegalovirus major immediate early promoter (IE CMV) with tetracycline operator (TetO) sites, a polyadenylation signal from bovine growth hormone (BGH), and a tPA signal sequence upstream of the inserted gene.

Production of ChAdOx1 nCoV-19 Derived SARS-CoV-2 S

A liter of human embryonic kidney 293 Freestyle (HEK293F) cells, at 1 × 106 cells/ml density, were infected with ChAdOx1 nCoV-19 at an MOI of ∼ 1 viral particles per cell. The cells were incubated at 37 °C for 48 h and pelleted in a centrifuge. The pellets were washed twice with phosphate buffered saline before addition of 5 mL of lysis buffer (20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.5% IGEPAL, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EDTA) and sonication. The mixture was centrifuged at 4000 rpm for 30 min, and the supernatant was collected. A 5 mL aliquot of lysis buffer and 3.5 mL of chloroform were added, and the tube was vortexed. H2O (9 mL) was added, and after vortexing, the tube was centrifuged at 4000 rpm for 30 min. The upper liquid layer was removed, and 9 mL of methanol were added. After vortexing, the tube was centrifuged again. The methanol was subsequently removed, and the pellet was allowed to air-dry in the dark. Pellets were dissolved in 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.

Expression and Purification of Recombinant 2P-Stabilized SARS-CoV-2 S

Recombinant 2P-stabilized SARS-CoV-2 S protein was expressed as previously described. (3,5) In brief, the expression vector containing the 2P-stabilized S protein was used to transiently transfect FreeStyle293F cells. The protein was purified from the supernatant using nickel-affinity chromatography before size-exclusion chromatography.

Western Blot Analysis

Protein pellets from the HEK293F cell lysates were loaded onto an SDS-PAGE gel and then transferred to a PVDF membrane. Polyclonal rabbit anti-S1 antibody (Sinobiological, Cat: 40592-T62) and anti-S2 antibody (Abcam, Cat: ab272504) were used as primary antibodies.

FACS Analysis for Cell Surface Spike Expression

Hela S3 cells at 4 × 105 cell/mL were infected with 10 MOI ChAdOx1 nCoV-19 and incubated at 37 °C for 40–48 h. Cells were harvested and pelleted by centrifugation at 500g for 5 min. Resuspended cells were split into three pools for the alternative staining and were incubated with either pooled prime-boost sera derived from outbred CD1 mice immunized intramuscularly with 108 infectious units of ChAdOx1 nCoV-19, (18) prepared at 1:50 in PBS-BSA-0.5% or human mAbs prepared at 1 μg/mL or recombinant ACE2 expressed with a human Fc tag prepared at 2 μg/mL (see below). Cells were incubated for 2 h at rt. Cells were washed 3× with PBS-BSA-0.5% and then incubated for 1 h at rt with secondary antibodies conjugated with Alexafluor488 diluted at 1:1000 in PBS-BSA 0.5% (for ChAdOx1 nCov-19 serum staining Goat Anti mouse AlexaFluor 488 (Life Tech A11029), for ACE2 staining, Goat Anti Human AlexaFluor 488 (Life Tech)). Cells were washed twice with PBS-BSA-0.5% before resuspending in PBS and analyzed by flow cytometry using a Fortessa X20 FACS analyzer. Samples were considered positive for spike expression if they had a fluorescence intensity above a threshold value determined by the maximum intensity of the noninfected control cells (Figure 1D). Experiments were performed twice in duplicate, and representative data are shown. Data were analyzed using FlowJo v9 (TreeStar).

ACE2 Expression

The ectodomain of the human angiotensin-converting enzyme 2 (ACE2) soluble construct encoding residues 18–740 (from NCBI Reference Sequence: NP_001358344.1.), where the transmembrane and cytoplasmic domains were removed, was expressed using human embryonic kidney 293 F cells (HEK293F). A N-terminal monoFc followed by TEV cleavage was included as well as a C-terminal four amino acid C-tag (EPEA) for affinity purification. ACE2 ectodomain was transiently expressed in Expi293 (Thermo Fisher Scientific) and protein purified from culture supernatants by C-tag affinity purification followed by gel filtration in Tris-buffered saline (TBS) pH 7.4 buffer.

Isolation of Human Monoclonal Antibodies from Peripheral B Cells by Spike-Specific Single B Cells Sorting

To isolate Spike-specific B cells, PBMCs were labeled with recombinant trimeric spike-twin-Strep and stained with antibody cocktail consisting of CD3-FITC, CD14-FITC, CD56-FITC, CD16-FITC, IgM-FITC, IgA-FITC, IgD-FITC, IgG-BV786, CD19-BUV395, and Strep-MAB-DY549 (iba) to probe the Strep tag of spike. CD19+, IgG+, CD3–, CD14–, CD56–, CD16–, IgM–, IgA–, IgD–, Spike+ cells were then single cell sorted into 96-well PCR plates containing RNase inhibitor (N2611; Promega) and stored at – 80 °C.
Genes encoding Ig VH, Ig Vκ and Vλ from positive wells were amplified by RT-PCR (210210; QIAGEN) and nested PCR (203205; Qiagen) using ‘cocktails’ of primers specific for human IgG. PCR products of genes encoding heavy and light chains were joined with the expression vector for human IgG1 or Ig κ-chain or λ-chain (gifts from H. Wardemann) by Gibson assembly. Plasmids encoding heavy and light chains were then cotransfected into the 293T cell line by the polyethylenimine method (408727; Sigma), and antibodies were harvested for further study.

CryoET Sample Preparation and Imaging

EM grids (G300F1, R2/2 Quantifoil holey carbon, gold) were glow-discharged and treated with bovine fibronectin (20 μg/mL) for 30 min. Grids were washed with PBS and UV-treated for 1 h. U2OS or HeLa cells (1.6 × 105) resuspended in 2 mL of DMEM 10% FBS, Pen/Strep were seeded on top of the grids in 6-well plate wells. Cells and grids were incubated for 24 h at 37 °C/5% CO2 to allow cell attachment to grid carbon. ChAdOx1 nCoV-19 was added to the cells at MOI 1, and cells were returned to 37 °C/5% CO2 for 48 h. Grids were plunge-frozen in liquid ethane at – 183 °C using the Leica GP2 plunger, after receiving 1 μL of 5× concentrated 10 nm Au fiducial solution (EMS) in the back-side and being blotted from the back. Grids were stored at liquid nitrogen until time of imaging.
Tilt series acquisition was carried out with an FEI Titan Krios G2 (Thermo Fisher Scientific) electron microscope operated at 300 kV and equipped with a Gatan BioQuantum energy filter and post-GIF K3 detector (Gatan, Pleasanton, CA) housed at electron Bioimaging Center (eBIC/Diamond Light Source, UK). Tilt series were acquired using SerialEM (45) at a pixel size of 2.13 Å. Zero-loss imaging was used for all tilt series with a 20 eV slit width. Defocus values ranged from – 2 μm to – 7 μm.
A grouped dose-symmetric scheme was used for all tilt series, (46) with a range of ± 60 degrees at 3 degree increments in groups of 3 and total dose of 120–135 e–/Å2. At each tilt, 5 movie frames were recorded using Correlated Double Sampling (CDS) in super-resolution mode and saved in lzw compressed tif format with no gain normalization. Movies were subsequently gain normalized during motion correction and Fourier cropped back to physical pixel size with MotionCor2. (47)
Tilt series were manually aligned using eTomo, (48) and tilt series and alignment files were imported to emClarity. (25) 11391 subtomograms were selected after template matching using EMDB-21452 (4) as the template, low-pass filtered to 40 Å. Subtomograms were separated in 2 completely independent half-data sets and iteratively aligned and averaged resulting in a final 11.6 Å map at 0.5 FSC threshold. Model fitting and visualization were performed in Chimera. (49)

Glycopeptide Analysis by Liquid Chromatography–Mass Spectrometry

Approximately 90 gel bands containing the furin cleaved (S1/S2) and uncleaved (S0) SARS-CoV-2 S protein were excised in order to gather sufficient material. The gel bands were destained in 50% 100 mM ammonium bicarbonate, 50% acetonitrile overnight. In-gel reduction and alkylation were performed according to the protocol by Shevchenko et al. (50) The proteins were digested using trypsin, chymotrypsin, and α-lytic protease, which cleave at the amino acids R/K, F/Y/W, and T/A/S/V, respectively. After extraction, the peptide/glycopeptides were first treated with endoglycosidase H to deplete oligomannose-type glycans and leave a single GlcNAc residue at the corresponding site. Subsequently, the reaction mixture was completely dried and resuspended in a mixture containing PNGase F using only H218O (Sigma-Aldrich) throughout. This reaction cleaves the remaining complex-type glycans but leaves the GlcNAc residues remaining after Endo H digestion intact. The use of H218O enables complex-type glycan sites to be differentiated from unoccupied glycan sites since the hydrolysis of the glycosidic bond by PNGase F leaves an 18O isotope on the resulting aspartic acid residue. The resulting peptides are purified using C18 Zip-tip (MerckMillipore) cleanup following the manufacturer’s protocol. Eluted glycopeptides were dried and resuspended in 0.1% formic acid prior to analysis by liquid chromatography–mass spectrometry. An Easy-nLC 1200 (Thermo Fisher Scientific) system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) using higher energy collision-induced dissociation fragmentation was used. Peptides were separated using an EasySpray PepMap RSLC C18 column (75 cm × 75 μm) with a 275 min linear gradient consisting of 0–32% acetonitrile in 0.1% formic acid over 240 min, followed by 35 min of 80% acetonitrile in 0.1% formic acid. The flow rate was set to 200 nL/min. The spray voltage was set to 2.7 kV, and the temperature of the heated capillary was set to 40 °C. The ion transfer tube temperature was set to 275 °C. The scan range was 400–1600 m/z. The HCD collision energy was set to 27%. Precursor and fragment detection were performed at a resolution 100 000 and 30 000 for MS1 and MS2, respectively. The AGC target for MS1 and MS2 was 4e5 and 5e4, respectively, and the injection time for MS1 and MS2 was 50 and 54 ms, respectively.
Glycopeptide fragmentation data were extracted from the raw file using Byonic and Byologic software (Version 3.5; Protein Metrics Inc.). The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b- and y-fragment ions were observed. Two modifications were searched for + 203 Da corresponding to a single GlcNAc residue, still attached to an asparagine residue of an N-linked glycan site, following the cleavage by Endo H, and + 3 Da corresponding to the 18O deamidation product of a complex glycan. The relative quantities of each glycan type at each site as well as the unoccupied proportion were determined by comparison of the extracted ion chromatographic areas.

Glycosylated Model Construction

Structural models of N-linked glycan presentation on SARS-CoV-2 S were created using electron microscopy structures (PDB ID 6VSB) along with complex-, hybrid-, and oligomannose-type N-linked glycans (PDB ID 4BYH, 4B7I, and 2WAH). A representative glycoform presented at each site was modeled on to the N-linked carbohydrate attachment sites in Coot. (51)

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00080.

  • Supplementary Figures 1–5 and Table 1; FACS gating strategy, tomographic slices, cryo-EM images, site-specific glycosylation comparisons between recombinant proteins, and values for glycoforms observed on S proteins derived from ChAdOx1 nCoV-19 (PDF)

  • Movie 1: Cryo-electron tomography of U2OS cells infected with ChAdOx1 nCoV-19 (MP4)

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Author Information

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  • Corresponding Authors
    • Teresa Lambe - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.;  NIHR Oxford Biomedical Research Centre, Oxford, U.K.;  Email: [email protected]
    • Peijun Zhang - Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, U.K.;  Electron Bio-imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, U.K.;  Orcidhttp://orcid.org/0000-0003-1803-691X;  Email: [email protected]
    • Sarah C. Gilbert - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.;  NIHR Oxford Biomedical Research Centre, Oxford, U.K.;  Email: [email protected]
    • Max Crispin - School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.;  Orcidhttp://orcid.org/0000-0002-1072-2694;  Email: [email protected]
  • Authors
    • Yasunori Watanabe - School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.;  Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.
    • Luiza Mendonça - Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, U.K.
    • Elizabeth R. Allen - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    • Andrew Howe - Electron Bio-imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, OX11 0DE, U.K.
    • Mercede Lee - The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
    • Joel D. Allen - School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.;  Orcidhttp://orcid.org/0000-0003-2547-968X
    • Himanshi Chawla - School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, U.K.
    • David Pulido - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    • Francesca Donnellan - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    • Hannah Davies - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    • Marta Ulaszewska - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    • Sandra Belij-Rammerstorfer - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.;  NIHR Oxford Biomedical Research Centre, Oxford, U.K.
    • Susan Morris - The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, U.K.
    • Anna-Sophia Krebs - Division of Structural Biology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, U.K.
    • Wanwisa Dejnirattisai - The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
    • Juthathip Mongkolsapaya - The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.;  Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand;  Chinese Academy of Medical Science (CAMS) Oxford Institute (COI), University of Oxford, Oxford, U.K.
    • Piyada Supasa - The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
    • Gavin R. Screaton - The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.;  Division of Medical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, U.K.
    • Catherine M. Green - The Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K.
  • Notes
    The authors declare the following competing financial interest(s): Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19. S.C.G. is co-founder of Vaccitech (collaborators in the early development of this vaccine candidate) and named as an inventor on a patent covering use of ChAdOx1-vectored vaccines and a patent application covering this SARS-CoV-2 vaccine. T.L. is named as an inventor on a patent application covering this SARS-CoV-2 vaccine and consultant to Vaccitech. Since initial submission of the manuscript, Y.W. has accepted a position at AstraZeneca; the design, experimental execution, and the write-up of this study were completed prior to this development.

    Data and materials availability: The cryoEM density maps for ChAdOx nCoV-2019 was deposited in the EMDB under accession code EMD-12530. Mass spectrometry raw files have been deposited in the MassIVE proteomics database (MSV000086998).

Acknowledgments

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Early work on producing ChAdOx1 nCoV-19 was supported by Funding from the Department of Health and Social Care (DHSC) managed by the Engineering and Physical Sciences Research Council (EPSRC) for the Future Vaccine Manufacturing Research Hub Grant Reference: EP/R013756/1. This work was supported by the International AIDS Vaccine Initiative (IAVI) through Grant INV-008352/OPP1153692 and the IAVI Neutralizing Antibody Center through the Collaboration for AIDS Vaccine Discovery Grant OPP1196345/INV-008813, both funded by the Bill and Melinda Gates Foundation. This work was also supported by the National Institute for Allergy and Infectious Diseases through the Scripps Consortium for HIV Vaccine Development (CHAVD) (AI144462), the University of Southampton Coronavirus Response Fund, the Bright Future Trust, the National Institute for Allergy and Infectious Diseases Grant AI150481, and the UK Wellcome Trust Investigator Award 206422/Z/17/Z. We acknowledge Diamond for access and support of the CryoEM facilities at the UK national electron bioimaging centre (eBIC, Proposal NR18477, NR21005, and NT21004), funded by the Wellcome Trust, MRC and BBSRC. The computational aspects of this research were supported by the Wellcome Trust Core Award Grant Number 203141/Z/16/Z and the NIHR Oxford BRC. C.G. is supported by Wellcome Award 090532/Z/09/Z. National Institute for Health Research Biomedical Research Centre Funding Scheme, the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (Grant Number: 2018-I2M-2-002). G.R.S. is supported as a Wellcome Trust Senior Investigator (Grant 095541/A/11/Z).

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    The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET
    Liu, Chuang; Mendonca, Luiza; Yang, Yang; Gao, Yuanzhu; Shen, Chenguang; Liu, Jiwei; Ni, Tao; Ju, Bin; Liu, Congcong; Tang, Xian; Wei, Jinli; Ma, Xiaomin; Zhu, Yanan; Liu, Weilong; Xu, Shuman; Liu, Yingxia; Yuan, Jing; Wu, Jing; Liu, Zheng; Zhang, Zheng; Liu, Lei; Wang, Peiyi; Zhang, Peijun
    Structure (Oxford, United Kingdom) (2020), 28 (11), 1218-1224.e4CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    The ongoing global pandemic of coronavirus disease 2019 (COVID-19) resulted from the outbreak of SARS-CoV-2 in Dec. 2019. Currently, multiple efforts are being made to rapidly develop vaccines and treatments to fight COVID-19. Current vaccine candidates use inactivated SARS-CoV-2 viruses; therefore, it is important to understand the architecture of inactivated SARS-CoV-2. We have genetically and structurally characterized β-propiolactone-inactivated viruses from a propagated and purified clin. strain of SARS-CoV-2. We obsd. that the virus particles are roughly spherical or moderately pleiomorphic. Although a small fraction of prefusion spikes are found, most spikes appear nail shaped, thus resembling a postfusion state, where the S1 protein of the spike has disassocd. from S2. Cryoelectron tomog. and subtomogram averaging of these spikes yielded a d. map that closely matches the overall structure of the SARS-CoV postfusion spike and its corresponding glycosylation site. Our findings have major implications for SARS-CoV-2 vaccine design, esp. those using inactivated viruses.
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  11. 11
    Zost, S. J.; Gilchuk, P.; Case, J. B.; Binshtein, E.; Chen, R. E.; Nkolola, J. P.; Schäfer, A.; Reidy, J. X.; Trivette, A.; Nargi, R. S. Potently Neutralizing and Protective Human Antibodies against SARS-CoV-2. Nature 2020, 584 (7821), 443– 449,  DOI: 10.1038/s41586-020-2548-6
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    11
    Potently neutralizing and protective human antibodies against SARS-CoV-2
    Zost, Seth J.; Gilchuk, Pavlo; Case, James Brett; Binshtein, Elad; Chen, Rita E.; Nkolola, Joseph P.; Schafer, Alexandra; Reidy, Joseph X.; Trivette, Andrew; Nargi, Rachel S.; Sutton, Rachel E.; Suryadevara, Naveenchandra; Martinez, David R.; Williamson, Lauren E.; Chen, Elaine C.; Jones, Taylor; Day, Samuel; Myers, Luke; Hassan, Ahmed O.; Kafai, Natasha M.; Winkler, Emma S.; Fox, Julie M.; Shrihari, Swathi; Mueller, Benjamin K.; Meiler, Jens; Chandrashekar, Abishek; Mercado, Noe B.; Steinhardt, James J.; Ren, Kuishu; Loo, Yueh-Ming; Kallewaard, Nicole L.; McCune, Broc T.; Keeler, Shamus P.; Holtzman, Michael J.; Barouch, Dan H.; Gralinski, Lisa E.; Baric, Ralph S.; Thackray, Larissa B.; Diamond, Michael S.; Carnahan, Robert H.; Crowe Jr, James E.
    Nature (London, United Kingdom) (2020), 584 (7821), 443-449CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health and the medical countermeasures available so far are limited. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-2. We analyze a large panel of human monoclonal antibodies that target the spike (S) glycoprotein, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural, and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In 2 mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130, or a combination of both of these antibodies protected mice from wt. loss and reduced the viral burden and levels of inflammation in the lungs. In addn., passive transfer of either of 2 of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.
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  12. 12
    Robbiani, D. F.; Gaebler, C.; Muecksch, F.; Lorenzi, J. C. C.; Wang, Z.; Cho, A.; Agudelo, M.; Barnes, C. O.; Gazumyan, A.; Finkin, S. Convergent Antibody Responses to SARS-CoV-2 in Convalescent Individuals. Nature 2020, 584 (7821), 437– 442,  DOI: 10.1038/s41586-020-2456-9
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    12
    Convergent antibody responses to SARS-CoV-2 in convalescent individuals
    Robbiani, Davide F.; Gaebler, Christian; Muecksch, Frauke; Lorenzi, Julio C. C.; Wang, Zijun; Cho, Alice; Agudelo, Marianna; Barnes, Christopher O.; Gazumyan, Anna; Finkin, Shlomo; Hagglof, Thomas; Oliveira, Thiago Y.; Viant, Charlotte; Hurley, Arlene; Hoffmann, Hans-Heinrich; Millard, Katrina G.; Kost, Rhonda G.; Cipolla, Melissa; Gordon, Kristie; Bianchini, Filippo; Chen, Spencer T.; Ramos, Victor; Patel, Roshni; Dizon, Juan; Shimeliovich, Irina; Mendoza, Pilar; Hartweger, Harald; Nogueira, Lilian; Pack, Maggi; Horowitz, Jill; Schmidt, Fabian; Weisblum, Yiska; Michailidis, Eleftherios; Ashbrook, Alison W.; Waltari, Eric; Pak, John E.; Huey-Tubman, Kathryn E.; Koranda, Nicholas; Hoffman, Pauline R.; West Jr, Anthony P.; Rice, Charles M.; Hatziioannou, Theodora; Bjorkman, Pamela J.; Bieniasz, Paul D.; Caskey, Marina; Nussenzweig, Michel C.
    Nature (London, United Kingdom) (2020), 584 (7821), 437-442CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. We report on 149 COVID-19-convalescent individuals. Plasma samples collected an av. of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titers; titers were <50 in 33% of samples, <1000 in 79% of samples, and only 1% of samples had titers >5000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titers, antibodies to 3 distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concns. (IC50 values) as low as 2 ng/mL. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
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  13. 13
    Du, S.; Cao, Y.; Zhu, Q.; Yu, P.; Qi, F.; Wang, G.; Du, X.; Bao, L.; Deng, W.; Zhu, H. Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy. Cell 2020, 183, 1013,  DOI: 10.1016/j.cell.2020.09.035
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    13
    Structurally resolved SARS-CoV-2 antibody shows high efficacy in severely infected hamsters and provides a potent cocktail pairing strategy
    Du, Shuo; Cao, Yunlong; Zhu, Qinyu; Yu, Pin; Qi, Feifei; Wang, Guopeng; Du, Xiaoxia; Bao, Linlin; Deng, Wei; Zhu, Hua; Liu, Jiangning; Nie, Jianhui; Zheng, Yinghui; Liang, Haoyu; Liu, Ruixue; Gong, Shuran; Xu, Hua; Yisimayi, Ayijiang; Lv, Qi; Wang, Bo; He, Runsheng; Han, Yunlin; Zhao, Wenjie; Bai, Yali; Qu, Yajin; Gao, Xiang; Ji, Chenggong; Wang, Qisheng; Gao, Ning; Huang, Weijin; Wang, Youchun; Xie, X. Sunney; Su, Xiao-dong; Xiao, Junyu; Qin, Chuan
    Cell (Cambridge, MA, United States) (2020), 183 (4), 1013-1023.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is crit. for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.
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  14. 14
    Barnes, C. O.; Jette, C. A.; Abernathy, M. E.; Dam, K.-M. A.; Esswein, S. R.; Gristick, H. B.; Malyutin, A. G.; Sharaf, N. G.; Huey-Tubman, K. E.; Lee, Y. E. SARS-CoV-2 Neutralizing Antibody Structures Inform Therapeutic Strategies. Nature 2020, 1– 9,  DOI: 10.1038/s41586-020-2852-1
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    Brouwer, P. J. M.; Caniels, T. G.; van der Straten, K.; Snitselaar, J. L.; Aldon, Y.; Bangaru, S.; Torres, J. L.; Okba, N. M. A.; Claireaux, M.; Kerster, G. Potent Neutralizing Antibodies from COVID-19 Patients Define Multiple Targets of Vulnerability. Science 2020, eabc5902  DOI: 10.1126/science.abc5902
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    Folegatti, P. M.; Ewer, K. J.; Aley, P. K.; Angus, B.; Becker, S.; Belij-Rammerstorfer, S.; Bellamy, D.; Bibi, S.; Bittaye, M.; Clutterbuck, E. A.; Safety and Immunogenicity of the ChAdOx1 NCoV-19 Vaccine against SARS-CoV-2: A Preliminary Report of a Phase 1/2, Single-Blind, Randomised Controlled Trial. Lancet 2020, 0 (0). DOI: 10.1016/S0140-6736(20)31604-4 .
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    van Doremalen, N.; Lambe, T.; Spencer, A.; Belij-Rammerstorfer, S.; Purushotham, J. N.; Port, J. R.; Avanzato, V. A.; Bushmaker, T.; Flaxman, A.; Ulaszewska, M. ChAdOx1 NCoV-19 Vaccine Prevents SARS-CoV-2 Pneumonia in Rhesus Macaques. Nature 2020, 1– 5,  DOI: 10.1038/s41586-020-2608-y
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    Graham, S. P.; McLean, R. K.; Spencer, A. J.; Belij-Rammerstorfer, S.; Wright, D.; Ulaszewska, M.; Edwards, J. C.; Hayes, J. W. P.; Martini, V.; Thakur, N. Evaluation of the Immunogenicity of Prime-Boost Vaccination with the Replication-Deficient Viral Vectored COVID-19 Vaccine Candidate ChAdOx1 NCoV-19. npj Vaccines 2020, 5 (1), 1– 6,  DOI: 10.1038/s41541-020-00221-3
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    Ramasamy, M. N.; Minassian, A. M.; Ewer, K. J.; Flaxman, A. L.; Folegatti, P. M.; Owens, D. R.; Voysey, M.; Aley, P. K.; Angus, B.; Babbage, G.; Safety and Immunogenicity of ChAdOx1 NCoV-19 Vaccine Administered in a Prime-Boost Regimen in Young and Old Adults (COV002): A Single-Blind, Randomised, Controlled, Phase 2/3 Trial. Lancet 2020, 0 (0). DOI: 10.1016/s0140-6736(20)32466-1 .
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    Sebastian, S.; Flaxman, A.; Cha, K. M.; Ulaszewska, M.; Gilbride, C.; Sharpe, H.; Wright, E.; Spencer, A. J.; Dowall, S.; Hewson, R.; A Multi-Filovirus Vaccine Candidate: Co-Expression of Ebola, Sudan, and Marburg Antigens in a Single Vector. Vaccines 2020, 8 (2). DOI: 10.3390/vaccines8020241 .
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    Zhang, L.; Jackson, C. B.; Mou, H.; Ojha, A.; Peng, H.; Quinlan, B. D.; Rangarajan, E. S.; Pan, A.; Vanderheiden, A.; Suthar, M. S. SARS-CoV-2 Spike-Protein D614G Mutation Increases Virion Spike Density and Infectivity. Nat. Commun. 2020, 11 (1), 1– 9,  DOI: 10.1038/s41467-020-19808-4
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    Pallesen, J.; Wang, N.; Corbett, K. S.; Wrapp, D.; Kirchdoerfer, R. N.; Turner, H. L.; Cottrell, C. A.; Becker, M. M.; Wang, L.; Shi, W. Immunogenicity and Structures of a Rationally Designed Prefusion MERS-CoV Spike Antigen. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (35), E7348– E7357,  DOI: 10.1073/pnas.1707304114
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    22
    Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen
    Pallesen, Jesper; Wang, Nianshuang; Corbett, Kizzmekia S.; Wrapp, Daniel; Kirchdoerfer, Robert N.; Turner, Hannah L.; Cottrell, Christopher A.; Becker, Michelle M.; Wang, Lingshu; Shi, Wei; Kong, Wing-Pui; Andres, Erica L.; Kettenbach, Arminja N.; Denison, Mark R.; Chappell, James D.; Graham, Barney S.; Ward, Andrew B.; McLellan, Jason S.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), E7348-E7357CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also detd. high-resoln. structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.
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    Walls, A. C.; Tortorici, M. A.; Snijder, J.; Xiong, X.; Bosch, B. J.; Rey, F. A.; Veesler, D. Tectonic Conformational Changes of a Coronavirus Spike Glycoprotein Promote Membrane Fusion. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (42), 11157– 11162,  DOI: 10.1073/pnas.1708727114
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    23
    Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion
    Walls, Alexandra C.; Tortorici, M. Alejandra; Snijder, Joost; Xiong, Xiaoli; Bosch, Berend-Jan; Rey, Felix A.; Veesler, David
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (42), 11157-11162CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by 2 global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here, we report the cryo-electron microscopy (cryo-EM) structure of a coronavirus S glycoprotein in the post-fusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the pre-fusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochem. characterized the mol. events assocd. with refolding of the metastable pre-fusion S glycoprotein to the post-fusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The obsd. similarity between post-fusion coronavirus S and paramyxovirus F structures demonstrated that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.
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    Berger, I.; Schaffitzel, C. The SARS-CoV-2 Spike Protein: Balancing Stability and Infectivity. Cell Research; Springer Nature: November 2, 2020; pp 1059– 1060. DOI: 10.1038/s41422-020-00430-4 .
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    Himes, B. A.; Zhang, P. EmClarity: Software for High-Resolution Cryo-Electron Tomography and Subtomogram Averaging. Nat. Methods 2018, 15 (11), 955– 961,  DOI: 10.1038/s41592-018-0167-z
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    25
    emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging
    Himes, Benjamin A.; Zhang, Peijun
    Nature Methods (2018), 15 (11), 955-961CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    Macromol. complexes are intrinsically flexible and often challenging to purify for structure detn. by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomog. (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resoln., yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity (https://github.com/bHimes/emClarity/wiki), a GPU-accelerated image-processing package featuring an iterative tomog. tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component anal. classification method. We demonstrate that our approach offers substantial improvement in the resoln. of maps and in the sepn. of different functional states of macromol. complexes compared with current state-of-the-art software.
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    Turoňová, B.; Sikora, M.; Schürmann, C.; Hagen, W.; Welsch, S.; Blanc, F.; von Bülow, S.; Gecht, M.; Bagola, K.; Hörner, C.; In Situ Structural Analysis of SARS-CoV-2 Spike Reveals Flexibility Mediated by Three Hinges. bioRxiv 2020, DOI: 10.1101/2020.06.26.173476 .
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    Yao, H.; Song, Y.; Chen, Y.; Wu, N.; Xu, J.; Sun, C.; Zhang, J.; Weng, T.; Zhang, Z.; Wu, Z. Molecular Architecture of the SARS-CoV-2 Virus. Cell 2020, 183, 730,  DOI: 10.1016/j.cell.2020.09.018
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    Molecular Architecture of the SARS-CoV-2 Virus
    Yao, Hangping; Song, Yutong; Chen, Yong; Wu, Nanping; Xu, Jialu; Sun, Chujie; Zhang, Jiaxing; Weng, Tianhao; Zhang, Zheyuan; Wu, Zhigang; Cheng, Linfang; Shi, Danrong; Lu, Xiangyun; Lei, Jianlin; Crispin, Max; Shi, Yigong; Li, Lanjuan; Li, Sai
    Cell (Cambridge, MA, United States) (2020), 183 (3), 730-738.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the mol. assembly of the authentic SARS-CoV-2 virus using cryoelectron tomog. (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were detd. to av. resolns. of 8.7-11 Å. Compns. of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diam. lumen.
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    Ke, Z.; Oton, J.; Qu, K.; Cortese, M.; Zila, V.; McKeane, L.; Nakane, T.; Zivanov, J.; Neufeldt, C. J.; Cerikan, B. Structures and Distributions of SARS-CoV-2 Spike Proteins on Intact Virions. Nature 2020, 1– 5,  DOI: 10.1038/s41586-020-2665-2
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    Toelzer, C.; Gupta, K.; Yadav, S. K. N.; Borucu, U.; Davidson, A. D.; Kavanagh Williamson, M.; Shoemark, D. K.; Garzoni, F.; Staufer, O.; Milligan, R. Free Fatty Acid Binding Pocket in the Locked Structure of SARS-CoV-2 Spike Protein. Science 2020, 370 (6517), 725– 730,  DOI: 10.1126/science.abd3255
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    29
    Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein
    Toelzer, Christine; Gupta, Kapil; Yadav, Sathish K. N.; Borucu, Ufuk; Davidson, Andrew D.; Kavanagh Williamson, Maia; Shoemark, Deborah K.; Garzoni, Frederic; Staufer, Oskar; Milligan, Rachel; Capin, Julien; Mulholland, Adrian J.; Spatz, Joachim; Fitzgerald, Daniel; Berger, Imre; Schaffitzel, Christiane
    Science (Washington, DC, United States) (2020), 370 (6517), 725-730CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathol. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor-binding domains tightly bind the essential free fatty acid linoleic acid (LA) in 3 composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.
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    Watanabe, Y.; Bowden, T. A.; Wilson, I. A.; Crispin, M. Exploitation of Glycosylation in Enveloped Virus Pathobiology. Biochim. Biophys. Acta, Gen. Subj. 2019, 1863 (10), 1480– 1497,  DOI: 10.1016/j.bbagen.2019.05.012
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    30
    Exploitation of glycosylation in enveloped virus pathobiology
    Watanabe, Yasunori; Bowden, Thomas A.; Wilson, Ian A.; Crispin, Max
    Biochimica et Biophysica Acta, General Subjects (2019), 1863 (10), 1480-1497CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
    A review. Glycosylation is a ubiquitous post-translational modification responsible for a multitude of crucial biol. roles. As obligate parasites, viruses exploit host-cell machinery to glycosylate their own proteins during replication. Viral envelope proteins from a variety of human pathogens including HIV-1, influenza virus, Lassa virus, SARS, Zika virus, dengue virus, and Ebola virus have evolved to be extensively glycosylated. These host-cell derived glycans facilitate diverse structural and functional roles during the viral life-cycle, ranging from immune evasion by glycan shielding to enhancement of immune cell infection. In this review, we highlight the imperative and auxiliary roles glycans play, and how specific oligosaccharide structures facilitate these functions during viral pathogenesis. We discuss the growing efforts to exploit viral glycobiol. in the development of anti-viral vaccines and therapies.
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  31. 31
    Cao, L.; Pauthner, M.; Andrabi, R.; Rantalainen, K.; Berndsen, Z.; Diedrich, J. K.; Menis, S.; Sok, D.; Bastidas, R.; Park, S.-K. R. Differential Processing of HIV Envelope Glycans on the Virus and Soluble Recombinant Trimer. Nat. Commun. 2018, 9 (1), 3693,  DOI: 10.1038/s41467-018-06121-4
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    Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer
    Cao Liwei; Diedrich Jolene K; Park Sung-Kyu Robin; Delahunty Claire M; He Lin; Yates John R 3rd; Paulson James C; Cao Liwei; Pauthner Matthias; Andrabi Raiees; Menis Sergey; Sok Devin; Bastidas Raiza; Guenaga Javier; Wyatt Richard T; Schief William R; Burton Dennis R; Paulson James C; Cao Liwei; Pauthner Matthias; Andrabi Raiees; Rantalainen Kimmo; Berndsen Zachary; Menis Sergey; Sok Devin; Bastidas Raiza; Guenaga Javier; Wyatt Richard T; Schief William R; Ward Andrew B; Burton Dennis R; Paulson James C; Rantalainen Kimmo; Berndsen Zachary; Ward Andrew B; Burton Dennis R
    Nature communications (2018), 9 (1), 3693 ISSN:.
    As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.
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    Cao, L.; Diedrich, J. K.; Kulp, D. W.; Pauthner, M.; He, L.; Park, S.-K. R.; Sok, D.; Su, C. Y.; Delahunty, C. M.; Menis, S. Global Site-Specific N-Glycosylation Analysis of HIV Envelope Glycoprotein. Nat. Commun. 2017, 8, 14954,  DOI: 10.1038/ncomms14954
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    Global site-specific N-glycosylation analysis of HIV envelope glycoprotein
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    Nature Communications (2017), 8 (), 14954CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. BNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quant. anal. of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.
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    Struwe, W. B.; Chertova, E.; Allen, J. D.; Seabright, G. E.; Watanabe, Y.; Harvey, D. J.; Medina-Ramirez, M.; Roser, J. D.; Smith, R.; Westcott, D. Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a Soluble Trimeric Immunogen. Cell Rep. 2018, 24 (8), 1958– 1966, e5  DOI: 10.1016/j.celrep.2018.07.080
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    Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a Soluble Trimeric Immunogen
    Struwe, Weston B.; Chertova, Elena; Allen, Joel D.; Seabright, Gemma E.; Watanabe, Yasunori; Harvey, David J.; Medina-Ramirez, Max; Roser, James D.; Smith, Rodman; Westcott, David; Keele, Brandon F.; Bess, Julian W. Jr.; Sanders, Rogier W.; Lifson, Jeffrey D.; Moore, John P.; Crispin, Max
    Cell Reports (2018), 24 (8), 1958-1966.e5CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Many broadly neutralizing antibodies (bnAbs) against HIV-1 recognize and/or penetrate the glycan shield on native, virion-assocd. envelope glycoprotein (Env) spikes. The same bnAbs also bind to recombinant, sol. trimeric immunogens based on the SOSIP design. While SOSIP trimers are close structural and antigenic mimics of virion Env, the extent to which their glycan structures resemble ones on infectious viruses is undefined. Here, we compare the overall glycosylation of gp120 and gp41 subunits from BG505 (clade A) virions produced in a lymphoid cell line with those from recombinant BG505 SOSIP trimers, including CHO-derived clin. grade material. We also performed detailed site-specific analyses of gp120. Glycans relevant to key bnAb epitopes are generally similar on the recombinant SOSIP and virion-derived Env proteins, although the latter do contain hotspots of elevated glycan processing. Knowledge of native vs. recombinant Env glycosylation will guide vaccine design and manufg. programs.
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    Watanabe, Y.; Berndsen, Z. T.; Raghwani, J.; Seabright, G. E.; Allen, J. D.; Pybus, O. G.; McLellan, J. S.; Wilson, I. A.; Bowden, T. A.; Ward, A. B. Vulnerabilities in Coronavirus Glycan Shields despite Extensive Glycosylation. Nat. Commun. 2020, 11 (1), 2688,  DOI: 10.1038/s41467-020-16567-0
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    Vulnerabilities in coronavirus glycan shields despite extensive glycosylation
    Watanabe, Yasunori; Berndsen, Zachary T.; Raghwani, Jayna; Seabright, Gemma E.; Allen, Joel D.; Pybus, Oliver G.; McLellan, Jason S.; Wilson, Ian A.; Bowden, Thomas A.; Ward, Andrew B.; Crispin, Max
    Nature Communications (2020), 11 (1), 2688CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan d. on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan d. of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organization of glycosylation across class I viral fusion proteins influence not only individual glycan compns. but also the immunol. pressure across the protein surface.
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    Zhao, P.; Praissman, J. L.; Grant, O. C.; Chen, B.; Brief, I.; Cai, Y.; Xiao, T.; Rosenbalm, K. E.; Aoki, K.; Kellman, B. P. Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor. Cell Host Microbe 2020, 28, 1– 16,  DOI: 10.1016/j.chom.2020.08.004
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    Brun, J.; Vasiljevic, S.; Gangadharan, B.; Hensen, M.; Chandran, A. V.; Hill, M. L.; Kiappes, J. L.; Dwek, R. A.; Alonzi, D. S.; Struwe, W. B.; Analysis of SARS-CoV-2 Spike Glycosylation Reveals Shedding of a Vaccine Candidate. bioRxiv . bioRxiv November 16, 2020; DOI: 10.1101/2020.11.16.384594 .
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    Essalmani, R.; Jain, J.; Susan-Resiga, D.; Andréo, U.; Evagelidis, A.; Derbali, R. M.; Huynh, D. N.; Dallaire, F.; Laporte, M.; Delpal, A.; Furin Cleaves SARS-CoV-2 Spike-Glycoprotein at S1/S2 and S2’ for Viral Fusion/Entry: Indirect Role of TMPRSS2. bioRxiv 2020, DOI: 10.1101/2020.12.18.423106 .
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    Uhlen, M.; Fagerberg, L.; Hallstrom, B. M.; Lindskog, C.; Oksvold, P.; Mardinoglu, A.; Sivertsson, A.; Kampf, C.; Sjostedt, E.; Asplund, A. Tissue-Based Map of the Human Proteome. Science 2015, 347 (6220), 1260419– 1260419,  DOI: 10.1126/science.1260419
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    Proteomics. Tissue-based map of the human proteome
    Uhlen Mathias; Fagerberg Linn; Oksvold Per; Sivertsson ¡ÑÜAsa; Lundberg Emma; Odeberg Jacob; Alm Tove; Nilsson Peter; Schwenk Jochen M; von Feilitzen Kalle; Forsberg Mattias; Persson Lukas; Johansson Fredric; Zwahlen Martin; Hallstrom Bjorn M; Lindskog Cecilia; Kampf Caroline; Asplund Anna; Olsson IngMarie; Djureinovic Dijana; Edqvist Per-Henrik; Ponten Fredrik; Mardinoglu Adil; Sjostedt Evelina; Edlund Karolina; Navani Sanjay; Szigyarto Cristina Al-Khalili; Takanen Jenny Ottosson; Hober Sophia; Berling Holger; Tegel Hanna; Rockberg Johan; Hamsten Marica; Mulder Jan; von Heijne Gunnar; Nielsen Jens
    Science (New York, N.Y.) (2015), 347 (6220), 1260419 ISSN:.
    Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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    Hamming, I.; Timens, W.; Bulthuis, M. L. C.; Lely, A. T.; Navis, G. J.; van Goor, H. Tissue Distribution of ACE2 Protein, the Functional Receptor for SARS Coronavirus. A First Step in Understanding SARS Pathogenesis. J. Pathol. 2004, 203 (2), 631– 637,  DOI: 10.1002/path.1570
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    Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis
    Hamming, I.; Timens, W.; Bulthuis, M. L. C.; Lely, A. T.; Navis, G. J.; van Goor, H.
    Journal of Pathology (2004), 203 (2), 631-637CODEN: JPTLAS; ISSN:0022-3417. (John Wiley & Sons Ltd.)
    Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the etiol. agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for SARS-CoV. Although ACE2 mRNA is known to be present in virtually all organs, its protein expression is largely unknown. Since identifying the possible route of infection has major implications for understanding the pathogenesis and future treatment strategies for SARS, the present study investigated the localization of ACE2 protein in various human organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). The most remarkable finding was the surface expression of ACE2 protein on lung alveolar epithelial cells and enterocytes of the small intestine. Furthermore, ACE2 was present in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied. In conclusion, ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, which might provide possible routes of entry for the SARS-CoV. This epithelial expression, together with the presence of ACE2 in vascular endothelium, also provides a first step in understanding the pathogenesis of the main SARS disease manifestations.
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    Sungnak, W.; Huang, N.; Bécavin, C.; Berg, M.; Queen, R.; Litvinukova, M.; Talavera-López, C.; Maatz, H.; Reichart, D.; Sampaziotis, F. SARS-CoV-2 Entry Factors Are Highly Expressed in Nasal Epithelial Cells Together with Innate Immune Genes. Nat. Med. 2020, 26 (5), 681– 687,  DOI: 10.1038/s41591-020-0868-6
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    Sungnak, Waradon; Huang, Ni; Becavin, Christophe; Berg, Marijn; Queen, Rachel; Litvinukova, Monika; Talavera-Lopez, Carlos; Maatz, Henrike; Reichart, Daniel; Sampaziotis, Fotios; Worlock, Kaylee B.; Yoshida, Masahiro; Barnes, Josephine L.; HCA Lung Biological Network
    Nature Medicine (New York, NY, United States) (2020), 26 (5), 681-687CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)
    We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-assocd. genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clin. samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.
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    Kornfeld, R.; Kornfeld, S. Assembly of Asparagine-Linked Oligosaccharides. Annu. Rev. Biochem. 1985, 54 (1), 631– 664,  DOI: 10.1146/annurev.bi.54.070185.003215
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    Assembly of asparagine-linked oligosaccharides
    Kornfeld R; Kornfeld S
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    There is no expanded citation for this reference.
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    Mastronarde, D. N. Automated Electron Microscope Tomography Using Robust Prediction of Specimen Movements. J. Struct. Biol. 2005, 152 (1), 36– 51,  DOI: 10.1016/j.jsb.2005.07.007
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    Automated electron microscope tomography using robust prediction of specimen movements
    Mastronarde David N
    Journal of structural biology (2005), 152 (1), 36-51 ISSN:1047-8477.
    A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.
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    Hagen, W. J. H.; Wan, W.; Briggs, J. A. G. Implementation of a Cryo-Electron Tomography Tilt-Scheme Optimized for High Resolution Subtomogram Averaging. J. Struct. Biol. 2017, 197 (2), 191– 198,  DOI: 10.1016/j.jsb.2016.06.007
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    Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging
    Hagen Wim J H; Wan William; Briggs John A G
    Journal of structural biology (2017), 197 (2), 191-198 ISSN:.
    Cryo-electron tomography (cryoET) allows 3D structural information to be obtained from cells and other biological samples in their close-to-native state. In combination with subtomogram averaging, detailed structures of repeating features can be resolved. CryoET data is collected as a series of images of the sample from different tilt angles; this is performed by physically rotating the sample in the microscope between each image. The angles at which the images are collected, and the order in which they are collected, together are called the tilt-scheme. Here we describe a "dose-symmetric tilt-scheme" that begins at low tilt and then alternates between increasingly positive and negative tilts. This tilt-scheme maximizes the amount of high-resolution information maintained in the tomogram for subsequent subtomogram averaging, and may also be advantageous for other applications. We describe implementation of the tilt-scheme in combination with further data-collection refinements including setting thresholds on acceptable drift and improving focus accuracy. Requirements for microscope set-up are introduced, and a macro is provided which automates the application of the tilt-scheme within SerialEM.
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    Zheng, S. Q.; Palovcak, E.; Armache, J. P.; Verba, K. A.; Cheng, Y.; Agard, D. A. MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy. Nat. Methods 2017, 14 (4), 331– 332,  DOI: 10.1038/nmeth.4193
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    Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul; Verba, Kliment A.; Cheng, Yifan; Agard, David A.
    Nature Methods (2017), 14 (4), 331-332CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    A review on anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Here we describe MotionCor2, a software tool for anisotropic correction of beam-induced motion. Overall, MotionCor2 is extremely robust and sufficiently accurate at correcting local motions so that the very time-consuming and computationally intensive particle polishing in RELION can be skipped, importantly, it also works on a wide range of data sets, including cryo tomog. tilt series.
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    Kremer, J. R.; Mastronarde, D. N.; McIntosh, J. R. Computer Visualization of Three-Dimensional Image Data Using IMOD. J. Struct. Biol. 1996, 116 (1), 71– 76,  DOI: 10.1006/jsbi.1996.0013
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    Journal of structural biology (1996), 116 (1), 71-6 ISSN:1047-8477.
    We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK28zmtFyqtg%253D%253D&md5=b6ab779bdec60f8644e254124c4dbbe7
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    Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera - A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25 (13), 1605– 1612,  DOI: 10.1002/jcc.20084
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    Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Couch, Gregory S.; Greenblatt, Daniel M.; Meng, Elaine C.; Ferrin, Thomas E.
    Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
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    Shevchenko, A.; Tomas, H.; Havli, J.; Olsen, J. V.; Mann, M. In-Gel Digestion for Mass Spectrometric Characterization of Proteins and Proteomes. Nat. Protoc. 2006, 1 (6), 2856– 2860,  DOI: 10.1038/nprot.2006.468
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    Structural analysis of glycoproteins: building N-linked glycans with Coot
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Cited By

This article is cited by 4 publications.

  1. Suman Maity, Atanu Acharya. Many Roles of Carbohydrates: A Computational Spotlight on the Coronavirus S Protein Binding. ACS Applied Bio Materials 2023, Article ASAP.
  2. Serge Perez, Olga Makshakova. Multifaceted Computational Modeling in Glycoscience. Chemical Reviews 2022, 122 (20) , 15914-15970. https://doi.org/10.1021/acs.chemrev.2c00060
  3. Victor Yin, Szu-Hsueh Lai, Tom G. Caniels, Philip J. M. Brouwer, Mitch Brinkkemper, Yoann Aldon, Hejun Liu, Meng Yuan, Ian A. Wilson, Rogier W. Sanders, Marit J. van Gils, Albert J. R. Heck. Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses. ACS Central Science 2021, 7 (11) , 1863-1873. https://doi.org/10.1021/acscentsci.1c00804
  4. Joel D. Allen, Himanshi Chawla, Firdaus Samsudin, Lorena Zuzic, Aishwary Tukaram Shivgan, Yasunori Watanabe, Wan-ting He, Sean Callaghan, Ge Song, Peter Yong, Philip J. M. Brouwer, Yutong Song, Yongfei Cai, Helen M. E. Duyvesteyn, Tomas Malinauskas, Joeri Kint, Paco Pino, Maria J. Wurm, Martin Frank, Bing Chen, David I. Stuart, Rogier W. Sanders, Raiees Andrabi, Dennis R. Burton, Sai Li, Peter J. Bond, Max Crispin. Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation. Biochemistry 2021, 60 (27) , 2153-2169. https://doi.org/10.1021/acs.biochem.1c00279
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  • Abstract

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    Figure 1

    Figure 1. ChAdOx1 nCoV-19 produces membrane associated SARS-CoV-2 S glycoprotein in native conformations able to bind its host receptor, ACE2. (A) Schematic representation of the vaccine encoded SARS-CoV-2 S protein, showing the position of N-linked glycosylation amino acid sequons (NXS/T, where X ≠ P) as branches. Protein domains are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM), with the additional tPA secretion signal at the N-terminus. (B) HeLa S3 cells were infected with ChAdOx1 nCoV-19 and incubated with recombinant ACE2, anti-ChAdOx1 nCoV-19 (derived from vaccinated mice), or a panel of human mAbs (Ab44, Ab45, Ab71, and Ab111, which recognize S2, RBD, trimeric S, and NTD, respectively) and compared to noninfected controls, analyzed by flow cytometry. (Left). Relative frequency of cells and AlexaFluor 488 fluorescence associated with antispike detection is plotted. Left, (blue) anti-ChAdOx1 nCoV-19; middle (red), ACE2; and right (shades of green) human mAbs. In dark gray cells infected with an irrelevant ChAdOx1 vaccine and in light gray noninfected cells are shown as a control. Experimental replicates were performed two times, and representative data are shown.

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    Figure 2

    Figure 2. CryoET and subtomogram average of ChAdOx1 nCoV-19 derived spike. (A) Tomographic slice of U2OS cell transduced with ChAdOx1 nCoV-19. The slice is 6.4 Å thick; PM = plasma membrane, scale bar = 100 nm. (B) Detailed view of the boxed area marked in (A). White arrowheads indicate spike proteins on the cell surface; scale bar = 50 nm. (C–E) Subtomogram average of ChAdOx1 nCoV-19 spikes at 11.6 Å resolution as indicated by Fourier-Shell correlation at 0.5 cutoff (C), shown from side view (D) and top view (E). SARS-CoV-2 atomic model (PDB 6ZB5) (29) is fitted for reference.

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    Figure 3

    Figure 3. Site-specific glycan processing of SARS-CoV-2 S upon infection with ChAdOx1 nCoV-19. (A) Western blot analysis of SARS-CoV-2 spike proteins, using anti-S1 and anti-S1+S2 antibodies. Lane 1 = Protein pellet from 293F cell lysates infected with ChAdOx1 nCoV-19. Lane 2 = Reduced protein pellet from 293F infected with ChAdOx1 nCoV-19. Lane 3 = 2P-stabilized SARS-CoV-2 S protein. The white boxes correspond to gel bands that were excised for mass spectrometric analysis. (B) Site-specific N-linked glycosylation of SARS-CoV-2 S0 and S1/S2 glycoproteins. The bar graphs represent the relative quantities of digested glycopeptides possessing the identifiers of oligomannose/hybrid-type glycans (green), complex-type glycans (pink), unoccupied PNGs (gray), or not determined (N.D.) at each N-linked glycan sequon on the S protein, listed from N to C terminus. (C) Glycosylated model of the cleaved (S1/S2) SARS-CoV-2 spike. The pie charts summarize the mass spectrometric analysis of the oligomannose/hybrid (green), complex (pink), or unoccupied (gray) N-linked glycan populations. Representative glycans are modeled onto the prefusion structure of trimeric SARS-CoV-2 S glycoprotein (PDB ID: 6VSB), (3) with one RBD in the “up” conformation. The modeled glycans are colored according to oligomannose/hybrid-type glycan content with glycan sites labeled in green (80–100%), orange (30–79%), pink (0–29%), or gray (not detected).

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      Convergent antibody responses to SARS-CoV-2 in convalescent individuals
      Robbiani, Davide F.; Gaebler, Christian; Muecksch, Frauke; Lorenzi, Julio C. C.; Wang, Zijun; Cho, Alice; Agudelo, Marianna; Barnes, Christopher O.; Gazumyan, Anna; Finkin, Shlomo; Hagglof, Thomas; Oliveira, Thiago Y.; Viant, Charlotte; Hurley, Arlene; Hoffmann, Hans-Heinrich; Millard, Katrina G.; Kost, Rhonda G.; Cipolla, Melissa; Gordon, Kristie; Bianchini, Filippo; Chen, Spencer T.; Ramos, Victor; Patel, Roshni; Dizon, Juan; Shimeliovich, Irina; Mendoza, Pilar; Hartweger, Harald; Nogueira, Lilian; Pack, Maggi; Horowitz, Jill; Schmidt, Fabian; Weisblum, Yiska; Michailidis, Eleftherios; Ashbrook, Alison W.; Waltari, Eric; Pak, John E.; Huey-Tubman, Kathryn E.; Koranda, Nicholas; Hoffman, Pauline R.; West Jr, Anthony P.; Rice, Charles M.; Hatziioannou, Theodora; Bjorkman, Pamela J.; Bieniasz, Paul D.; Caskey, Marina; Nussenzweig, Michel C.
      Nature (London, United Kingdom) (2020), 584 (7821), 437-442CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. We report on 149 COVID-19-convalescent individuals. Plasma samples collected an av. of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titers; titers were <50 in 33% of samples, <1000 in 79% of samples, and only 1% of samples had titers >5000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titers, antibodies to 3 distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concns. (IC50 values) as low as 2 ng/mL. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
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    13. 13
      Du, S.; Cao, Y.; Zhu, Q.; Yu, P.; Qi, F.; Wang, G.; Du, X.; Bao, L.; Deng, W.; Zhu, H. Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy. Cell 2020, 183, 1013,  DOI: 10.1016/j.cell.2020.09.035
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      13
      Structurally resolved SARS-CoV-2 antibody shows high efficacy in severely infected hamsters and provides a potent cocktail pairing strategy
      Du, Shuo; Cao, Yunlong; Zhu, Qinyu; Yu, Pin; Qi, Feifei; Wang, Guopeng; Du, Xiaoxia; Bao, Linlin; Deng, Wei; Zhu, Hua; Liu, Jiangning; Nie, Jianhui; Zheng, Yinghui; Liang, Haoyu; Liu, Ruixue; Gong, Shuran; Xu, Hua; Yisimayi, Ayijiang; Lv, Qi; Wang, Bo; He, Runsheng; Han, Yunlin; Zhao, Wenjie; Bai, Yali; Qu, Yajin; Gao, Xiang; Ji, Chenggong; Wang, Qisheng; Gao, Ning; Huang, Weijin; Wang, Youchun; Xie, X. Sunney; Su, Xiao-dong; Xiao, Junyu; Qin, Chuan
      Cell (Cambridge, MA, United States) (2020), 183 (4), 1013-1023.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is crit. for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.
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    14. 14
      Barnes, C. O.; Jette, C. A.; Abernathy, M. E.; Dam, K.-M. A.; Esswein, S. R.; Gristick, H. B.; Malyutin, A. G.; Sharaf, N. G.; Huey-Tubman, K. E.; Lee, Y. E. SARS-CoV-2 Neutralizing Antibody Structures Inform Therapeutic Strategies. Nature 2020, 1– 9,  DOI: 10.1038/s41586-020-2852-1
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      Brouwer, P. J. M.; Caniels, T. G.; van der Straten, K.; Snitselaar, J. L.; Aldon, Y.; Bangaru, S.; Torres, J. L.; Okba, N. M. A.; Claireaux, M.; Kerster, G. Potent Neutralizing Antibodies from COVID-19 Patients Define Multiple Targets of Vulnerability. Science 2020, eabc5902  DOI: 10.1126/science.abc5902
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      Folegatti, P. M.; Ewer, K. J.; Aley, P. K.; Angus, B.; Becker, S.; Belij-Rammerstorfer, S.; Bellamy, D.; Bibi, S.; Bittaye, M.; Clutterbuck, E. A.; Safety and Immunogenicity of the ChAdOx1 NCoV-19 Vaccine against SARS-CoV-2: A Preliminary Report of a Phase 1/2, Single-Blind, Randomised Controlled Trial. Lancet 2020, 0 (0). DOI: 10.1016/S0140-6736(20)31604-4 .
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      van Doremalen, N.; Lambe, T.; Spencer, A.; Belij-Rammerstorfer, S.; Purushotham, J. N.; Port, J. R.; Avanzato, V. A.; Bushmaker, T.; Flaxman, A.; Ulaszewska, M. ChAdOx1 NCoV-19 Vaccine Prevents SARS-CoV-2 Pneumonia in Rhesus Macaques. Nature 2020, 1– 5,  DOI: 10.1038/s41586-020-2608-y
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      Graham, S. P.; McLean, R. K.; Spencer, A. J.; Belij-Rammerstorfer, S.; Wright, D.; Ulaszewska, M.; Edwards, J. C.; Hayes, J. W. P.; Martini, V.; Thakur, N. Evaluation of the Immunogenicity of Prime-Boost Vaccination with the Replication-Deficient Viral Vectored COVID-19 Vaccine Candidate ChAdOx1 NCoV-19. npj Vaccines 2020, 5 (1), 1– 6,  DOI: 10.1038/s41541-020-00221-3
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      Ramasamy, M. N.; Minassian, A. M.; Ewer, K. J.; Flaxman, A. L.; Folegatti, P. M.; Owens, D. R.; Voysey, M.; Aley, P. K.; Angus, B.; Babbage, G.; Safety and Immunogenicity of ChAdOx1 NCoV-19 Vaccine Administered in a Prime-Boost Regimen in Young and Old Adults (COV002): A Single-Blind, Randomised, Controlled, Phase 2/3 Trial. Lancet 2020, 0 (0). DOI: 10.1016/s0140-6736(20)32466-1 .
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      Sebastian, S.; Flaxman, A.; Cha, K. M.; Ulaszewska, M.; Gilbride, C.; Sharpe, H.; Wright, E.; Spencer, A. J.; Dowall, S.; Hewson, R.; A Multi-Filovirus Vaccine Candidate: Co-Expression of Ebola, Sudan, and Marburg Antigens in a Single Vector. Vaccines 2020, 8 (2). DOI: 10.3390/vaccines8020241 .
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      Zhang, L.; Jackson, C. B.; Mou, H.; Ojha, A.; Peng, H.; Quinlan, B. D.; Rangarajan, E. S.; Pan, A.; Vanderheiden, A.; Suthar, M. S. SARS-CoV-2 Spike-Protein D614G Mutation Increases Virion Spike Density and Infectivity. Nat. Commun. 2020, 11 (1), 1– 9,  DOI: 10.1038/s41467-020-19808-4
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      Pallesen, J.; Wang, N.; Corbett, K. S.; Wrapp, D.; Kirchdoerfer, R. N.; Turner, H. L.; Cottrell, C. A.; Becker, M. M.; Wang, L.; Shi, W. Immunogenicity and Structures of a Rationally Designed Prefusion MERS-CoV Spike Antigen. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (35), E7348– E7357,  DOI: 10.1073/pnas.1707304114
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      22
      Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen
      Pallesen, Jesper; Wang, Nianshuang; Corbett, Kizzmekia S.; Wrapp, Daniel; Kirchdoerfer, Robert N.; Turner, Hannah L.; Cottrell, Christopher A.; Becker, Michelle M.; Wang, Lingshu; Shi, Wei; Kong, Wing-Pui; Andres, Erica L.; Kettenbach, Arminja N.; Denison, Mark R.; Chappell, James D.; Graham, Barney S.; Ward, Andrew B.; McLellan, Jason S.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), E7348-E7357CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also detd. high-resoln. structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.
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    23. 23
      Walls, A. C.; Tortorici, M. A.; Snijder, J.; Xiong, X.; Bosch, B. J.; Rey, F. A.; Veesler, D. Tectonic Conformational Changes of a Coronavirus Spike Glycoprotein Promote Membrane Fusion. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (42), 11157– 11162,  DOI: 10.1073/pnas.1708727114
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      23
      Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion
      Walls, Alexandra C.; Tortorici, M. Alejandra; Snijder, Joost; Xiong, Xiaoli; Bosch, Berend-Jan; Rey, Felix A.; Veesler, David
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (42), 11157-11162CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by 2 global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here, we report the cryo-electron microscopy (cryo-EM) structure of a coronavirus S glycoprotein in the post-fusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the pre-fusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochem. characterized the mol. events assocd. with refolding of the metastable pre-fusion S glycoprotein to the post-fusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The obsd. similarity between post-fusion coronavirus S and paramyxovirus F structures demonstrated that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.
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    24. 24
      Berger, I.; Schaffitzel, C. The SARS-CoV-2 Spike Protein: Balancing Stability and Infectivity. Cell Research; Springer Nature: November 2, 2020; pp 1059– 1060. DOI: 10.1038/s41422-020-00430-4 .
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      Himes, B. A.; Zhang, P. EmClarity: Software for High-Resolution Cryo-Electron Tomography and Subtomogram Averaging. Nat. Methods 2018, 15 (11), 955– 961,  DOI: 10.1038/s41592-018-0167-z
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      25
      emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging
      Himes, Benjamin A.; Zhang, Peijun
      Nature Methods (2018), 15 (11), 955-961CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Macromol. complexes are intrinsically flexible and often challenging to purify for structure detn. by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomog. (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resoln., yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity (https://github.com/bHimes/emClarity/wiki), a GPU-accelerated image-processing package featuring an iterative tomog. tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component anal. classification method. We demonstrate that our approach offers substantial improvement in the resoln. of maps and in the sepn. of different functional states of macromol. complexes compared with current state-of-the-art software.
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    26. 26
      Turoňová, B.; Sikora, M.; Schürmann, C.; Hagen, W.; Welsch, S.; Blanc, F.; von Bülow, S.; Gecht, M.; Bagola, K.; Hörner, C.; In Situ Structural Analysis of SARS-CoV-2 Spike Reveals Flexibility Mediated by Three Hinges. bioRxiv 2020, DOI: 10.1101/2020.06.26.173476 .
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      Yao, H.; Song, Y.; Chen, Y.; Wu, N.; Xu, J.; Sun, C.; Zhang, J.; Weng, T.; Zhang, Z.; Wu, Z. Molecular Architecture of the SARS-CoV-2 Virus. Cell 2020, 183, 730,  DOI: 10.1016/j.cell.2020.09.018
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      27
      Molecular Architecture of the SARS-CoV-2 Virus
      Yao, Hangping; Song, Yutong; Chen, Yong; Wu, Nanping; Xu, Jialu; Sun, Chujie; Zhang, Jiaxing; Weng, Tianhao; Zhang, Zheyuan; Wu, Zhigang; Cheng, Linfang; Shi, Danrong; Lu, Xiangyun; Lei, Jianlin; Crispin, Max; Shi, Yigong; Li, Lanjuan; Li, Sai
      Cell (Cambridge, MA, United States) (2020), 183 (3), 730-738.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the mol. assembly of the authentic SARS-CoV-2 virus using cryoelectron tomog. (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were detd. to av. resolns. of 8.7-11 Å. Compns. of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diam. lumen.
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    28. 28
      Ke, Z.; Oton, J.; Qu, K.; Cortese, M.; Zila, V.; McKeane, L.; Nakane, T.; Zivanov, J.; Neufeldt, C. J.; Cerikan, B. Structures and Distributions of SARS-CoV-2 Spike Proteins on Intact Virions. Nature 2020, 1– 5,  DOI: 10.1038/s41586-020-2665-2
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      Toelzer, C.; Gupta, K.; Yadav, S. K. N.; Borucu, U.; Davidson, A. D.; Kavanagh Williamson, M.; Shoemark, D. K.; Garzoni, F.; Staufer, O.; Milligan, R. Free Fatty Acid Binding Pocket in the Locked Structure of SARS-CoV-2 Spike Protein. Science 2020, 370 (6517), 725– 730,  DOI: 10.1126/science.abd3255
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      29
      Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein
      Toelzer, Christine; Gupta, Kapil; Yadav, Sathish K. N.; Borucu, Ufuk; Davidson, Andrew D.; Kavanagh Williamson, Maia; Shoemark, Deborah K.; Garzoni, Frederic; Staufer, Oskar; Milligan, Rachel; Capin, Julien; Mulholland, Adrian J.; Spatz, Joachim; Fitzgerald, Daniel; Berger, Imre; Schaffitzel, Christiane
      Science (Washington, DC, United States) (2020), 370 (6517), 725-730CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathol. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor-binding domains tightly bind the essential free fatty acid linoleic acid (LA) in 3 composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.
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    30. 30
      Watanabe, Y.; Bowden, T. A.; Wilson, I. A.; Crispin, M. Exploitation of Glycosylation in Enveloped Virus Pathobiology. Biochim. Biophys. Acta, Gen. Subj. 2019, 1863 (10), 1480– 1497,  DOI: 10.1016/j.bbagen.2019.05.012
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      30
      Exploitation of glycosylation in enveloped virus pathobiology
      Watanabe, Yasunori; Bowden, Thomas A.; Wilson, Ian A.; Crispin, Max
      Biochimica et Biophysica Acta, General Subjects (2019), 1863 (10), 1480-1497CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
      A review. Glycosylation is a ubiquitous post-translational modification responsible for a multitude of crucial biol. roles. As obligate parasites, viruses exploit host-cell machinery to glycosylate their own proteins during replication. Viral envelope proteins from a variety of human pathogens including HIV-1, influenza virus, Lassa virus, SARS, Zika virus, dengue virus, and Ebola virus have evolved to be extensively glycosylated. These host-cell derived glycans facilitate diverse structural and functional roles during the viral life-cycle, ranging from immune evasion by glycan shielding to enhancement of immune cell infection. In this review, we highlight the imperative and auxiliary roles glycans play, and how specific oligosaccharide structures facilitate these functions during viral pathogenesis. We discuss the growing efforts to exploit viral glycobiol. in the development of anti-viral vaccines and therapies.
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    31. 31
      Cao, L.; Pauthner, M.; Andrabi, R.; Rantalainen, K.; Berndsen, Z.; Diedrich, J. K.; Menis, S.; Sok, D.; Bastidas, R.; Park, S.-K. R. Differential Processing of HIV Envelope Glycans on the Virus and Soluble Recombinant Trimer. Nat. Commun. 2018, 9 (1), 3693,  DOI: 10.1038/s41467-018-06121-4
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      31
      Differential processing of HIV envelope glycans on the virus and soluble recombinant trimer
      Cao Liwei; Diedrich Jolene K; Park Sung-Kyu Robin; Delahunty Claire M; He Lin; Yates John R 3rd; Paulson James C; Cao Liwei; Pauthner Matthias; Andrabi Raiees; Menis Sergey; Sok Devin; Bastidas Raiza; Guenaga Javier; Wyatt Richard T; Schief William R; Burton Dennis R; Paulson James C; Cao Liwei; Pauthner Matthias; Andrabi Raiees; Rantalainen Kimmo; Berndsen Zachary; Menis Sergey; Sok Devin; Bastidas Raiza; Guenaga Javier; Wyatt Richard T; Schief William R; Ward Andrew B; Burton Dennis R; Paulson James C; Rantalainen Kimmo; Berndsen Zachary; Ward Andrew B; Burton Dennis R
      Nature communications (2018), 9 (1), 3693 ISSN:.
      As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.
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    32. 32
      Cao, L.; Diedrich, J. K.; Kulp, D. W.; Pauthner, M.; He, L.; Park, S.-K. R.; Sok, D.; Su, C. Y.; Delahunty, C. M.; Menis, S. Global Site-Specific N-Glycosylation Analysis of HIV Envelope Glycoprotein. Nat. Commun. 2017, 8, 14954,  DOI: 10.1038/ncomms14954
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      32
      Global site-specific N-glycosylation analysis of HIV envelope glycoprotein
      Cao, Liwei; Diedrich, Jolene K.; Kulp, Daniel W.; Pauthner, Matthias; He, Lin; Park, Sung-Kyu Robin; Sok, Devin; Su, Ching Yao; Delahunty, Claire M.; Menis, Sergey; Andrabi, Raiees; Guenaga, Javier; Georgeson, Erik; Kubitz, Michael; Adachi, Yumiko; Burton, Dennis R.; Schief, William R.; Yates, John R., III; Paulson, James C.
      Nature Communications (2017), 8 (), 14954CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. BNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields >95% sequence coverage for Env, provides semi-quant. anal. of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of high-mannose/hybrid and complex-type glycans.
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    33. 33
      Struwe, W. B.; Chertova, E.; Allen, J. D.; Seabright, G. E.; Watanabe, Y.; Harvey, D. J.; Medina-Ramirez, M.; Roser, J. D.; Smith, R.; Westcott, D. Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a Soluble Trimeric Immunogen. Cell Rep. 2018, 24 (8), 1958– 1966, e5  DOI: 10.1016/j.celrep.2018.07.080
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      33
      Site-Specific Glycosylation of Virion-Derived HIV-1 Env Is Mimicked by a Soluble Trimeric Immunogen
      Struwe, Weston B.; Chertova, Elena; Allen, Joel D.; Seabright, Gemma E.; Watanabe, Yasunori; Harvey, David J.; Medina-Ramirez, Max; Roser, James D.; Smith, Rodman; Westcott, David; Keele, Brandon F.; Bess, Julian W. Jr.; Sanders, Rogier W.; Lifson, Jeffrey D.; Moore, John P.; Crispin, Max
      Cell Reports (2018), 24 (8), 1958-1966.e5CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Many broadly neutralizing antibodies (bnAbs) against HIV-1 recognize and/or penetrate the glycan shield on native, virion-assocd. envelope glycoprotein (Env) spikes. The same bnAbs also bind to recombinant, sol. trimeric immunogens based on the SOSIP design. While SOSIP trimers are close structural and antigenic mimics of virion Env, the extent to which their glycan structures resemble ones on infectious viruses is undefined. Here, we compare the overall glycosylation of gp120 and gp41 subunits from BG505 (clade A) virions produced in a lymphoid cell line with those from recombinant BG505 SOSIP trimers, including CHO-derived clin. grade material. We also performed detailed site-specific analyses of gp120. Glycans relevant to key bnAb epitopes are generally similar on the recombinant SOSIP and virion-derived Env proteins, although the latter do contain hotspots of elevated glycan processing. Knowledge of native vs. recombinant Env glycosylation will guide vaccine design and manufg. programs.
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      Watanabe, Y.; Berndsen, Z. T.; Raghwani, J.; Seabright, G. E.; Allen, J. D.; Pybus, O. G.; McLellan, J. S.; Wilson, I. A.; Bowden, T. A.; Ward, A. B. Vulnerabilities in Coronavirus Glycan Shields despite Extensive Glycosylation. Nat. Commun. 2020, 11 (1), 2688,  DOI: 10.1038/s41467-020-16567-0
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      Abstr.: Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan d. on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan d. of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organization of glycosylation across class I viral fusion proteins influence not only individual glycan compns. but also the immunol. pressure across the protein surface.
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      Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.
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      Journal of Pathology (2004), 203 (2), 631-637CODEN: JPTLAS; ISSN:0022-3417. (John Wiley & Sons Ltd.)
      Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the etiol. agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for SARS-CoV. Although ACE2 mRNA is known to be present in virtually all organs, its protein expression is largely unknown. Since identifying the possible route of infection has major implications for understanding the pathogenesis and future treatment strategies for SARS, the present study investigated the localization of ACE2 protein in various human organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). The most remarkable finding was the surface expression of ACE2 protein on lung alveolar epithelial cells and enterocytes of the small intestine. Furthermore, ACE2 was present in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied. In conclusion, ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, which might provide possible routes of entry for the SARS-CoV. This epithelial expression, together with the presence of ACE2 in vascular endothelium, also provides a first step in understanding the pathogenesis of the main SARS disease manifestations.
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      Sungnak, W.; Huang, N.; Bécavin, C.; Berg, M.; Queen, R.; Litvinukova, M.; Talavera-López, C.; Maatz, H.; Reichart, D.; Sampaziotis, F. SARS-CoV-2 Entry Factors Are Highly Expressed in Nasal Epithelial Cells Together with Innate Immune Genes. Nat. Med. 2020, 26 (5), 681– 687,  DOI: 10.1038/s41591-020-0868-6
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjurY%253D&md5=cc313df9c190333b98af43951a2146fa
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL2M3nvVSjsA%253D%253D&md5=e4e0de7e37f61bbe1c7bb100927460ce
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      A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2MrjtVSrtw%253D%253D&md5=16b6502753c0f1f63ff559a784bce8f0
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      Hagen, W. J. H.; Wan, W.; Briggs, J. A. G. Implementation of a Cryo-Electron Tomography Tilt-Scheme Optimized for High Resolution Subtomogram Averaging. J. Struct. Biol. 2017, 197 (2), 191– 198,  DOI: 10.1016/j.jsb.2016.06.007
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      Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging
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      Cryo-electron tomography (cryoET) allows 3D structural information to be obtained from cells and other biological samples in their close-to-native state. In combination with subtomogram averaging, detailed structures of repeating features can be resolved. CryoET data is collected as a series of images of the sample from different tilt angles; this is performed by physically rotating the sample in the microscope between each image. The angles at which the images are collected, and the order in which they are collected, together are called the tilt-scheme. Here we describe a "dose-symmetric tilt-scheme" that begins at low tilt and then alternates between increasingly positive and negative tilts. This tilt-scheme maximizes the amount of high-resolution information maintained in the tomogram for subsequent subtomogram averaging, and may also be advantageous for other applications. We describe implementation of the tilt-scheme in combination with further data-collection refinements including setting thresholds on acceptable drift and improving focus accuracy. Requirements for microscope set-up are introduced, and a macro is provided which automates the application of the tilt-scheme within SerialEM.
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      Zheng, S. Q.; Palovcak, E.; Armache, J. P.; Verba, K. A.; Cheng, Y.; Agard, D. A. MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy. Nat. Methods 2017, 14 (4), 331– 332,  DOI: 10.1038/nmeth.4193
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      Nature Methods (2017), 14 (4), 331-332CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      A review on anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Here we describe MotionCor2, a software tool for anisotropic correction of beam-induced motion. Overall, MotionCor2 is extremely robust and sufficiently accurate at correcting local motions so that the very time-consuming and computationally intensive particle polishing in RELION can be skipped, importantly, it also works on a wide range of data sets, including cryo tomog. tilt series.
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      We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
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      Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera - A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25 (13), 1605– 1612,  DOI: 10.1002/jcc.20084
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      Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
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      Shevchenko, A.; Tomas, H.; Havli, J.; Olsen, J. V.; Mann, M. In-Gel Digestion for Mass Spectrometric Characterization of Proteins and Proteomes. Nat. Protoc. 2006, 1 (6), 2856– 2860,  DOI: 10.1038/nprot.2006.468
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      In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-yr-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of anal. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS anal. to identify proteins at sensitivities better than a few femtomoles of protein starting material.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFGjt7nM&md5=7c82516bf8197dff95fe73a371af2f0c
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      Emsley, P.; Crispin, M. Structural Analysis of Glycoproteins: Building N-Linked Glycans with Coot. Acta Crystallogr. Sect. D Struct. Biol. 2018, 74 (4), 256– 263,  DOI: 10.1107/S2059798318005119
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      Structural analysis of glycoproteins: building N-linked glycans with Coot
      Emsley, Paul; Crispin, Max
      Acta Crystallographica, Section D: Structural Biology (2018), 74 (4), 256-263CODEN: ACSDAD; ISSN:2059-7983. (International Union of Crystallography)
      Coot is a graphics application that is used to build or manipulate macromol. models; its particular forte is manipulation of the model at the residue level. The model-building tools of Coot have been combined and extended to assist or automate the building of N-linked glycans. The model is built by the addn. of monosaccharides, placed by variation of internal coordinates. The subsequent model is refined by real-space refinement, which is stabilized with modified and addnl. restraints. It is hoped that these enhanced building tools will help to reduce building errors of N-linked glycans and improve our knowledge of the structures of glycoproteins.
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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00080.

    • Supplementary Figures 1–5 and Table 1; FACS gating strategy, tomographic slices, cryo-EM images, site-specific glycosylation comparisons between recombinant proteins, and values for glycoforms observed on S proteins derived from ChAdOx1 nCoV-19 (PDF)

    • Movie 1: Cryo-electron tomography of U2OS cells infected with ChAdOx1 nCoV-19 (MP4)


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