Developing a Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein Model in a Viral Membrane

ABSTRACTThis technical study describes all-atom modeling and simulation of a fully-glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB:6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently-determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully-glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19), so researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.

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SARS-CoV-2 full length spike protein for COVID-19 vaccine development and diagnostic testing

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2020.12.546 ◽

2021 ◽

Vol 147 (2) ◽

pp. AB152

Author(s):

Crystal Richardson ◽

Mayuresh Abhyankar ◽

Jillian Bracaglia ◽

Sayeh Agah ◽

Zachary Schuhmacher ◽

...

Keyword(s):

Vaccine Development ◽

Diagnostic Testing ◽

Full Length ◽

Spike Protein

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Refinement of the Transmembrane Domain in Constructing the Membrane-Embedded SARS-COV-2 Spike Protein Model

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1430 ◽

2021 ◽

Vol 120 (3) ◽

pp. 211a

Author(s):

Tianle Chen ◽

Karan Kapoor ◽

Emad Tajkhorshid

Keyword(s):

Transmembrane Domain ◽

Spike Protein ◽

Protein Model

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Modified Vaccinia Ankara Based SARS-CoV-2 Vaccine Expressing Full-Length Spike Induces Strong Neutralizing Antibody Response

10.1101/2020.06.27.175166 ◽

2020 ◽

Cited By ~ 1

Author(s):

Nanda Kishore Routhu ◽

Sailaja Gangadhara ◽

Narayanaiah Cheedarla ◽

Ayalnesh Shiferaw ◽

Sheikh Abdul Rahman ◽

...

Keyword(s):

Antibody Response ◽

Room Temperature ◽

Vaccine Candidate ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Full Length ◽

Spike Protein ◽

Strongly Correlated ◽

Igg Antibodies ◽

Potential Vaccine

AbstractThere is a great need for the development of vaccines for preventing SARS-CoV-2 infection and mitigating the COVID-19 pandemic. Here, we developed two modified vaccinia Ankara (MVA) based vaccines which express either a membrane anchored full-length spike protein (MVA/S) stabilized in a prefusion state or the S1 region of the spike (MVA/S1) which forms trimers and is secreted. Both immunogens contained the receptor-binding domain (RBD) which is a known target of antibody-mediated neutralization. Following immunizations with MVA/S or MVA/S1, both spike protein recombinants induced strong IgG antibodies to purified full-length SARS-CoV-2 spike protein. The MVA/S induced a robust antibody response to purified RBD, S1 and S2 whereas MVA/S1 induced an antibody response to the S1 region outside of the RBD region. Both vaccines induced an antibody response in the lung and that was associated with induction of bronchus-associated lymphoid tissue. MVA/S but not MVA/S1 vaccinated mice generated robust neutralizing antibody responses against SARS-CoV-2 that strongly correlated with RBD antibody binding titers. Mechanistically, S1 binding to ACE-2 was strong but reduced following prolonged pre-incubation at room temperature suggesting confirmation changes in RBD with time. These results demonstrate MVA/S is a potential vaccine candidate against SARS-CoV-2 infection.

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Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

10.1101/2020.09.08.286732 ◽

2020 ◽

Author(s):

Matthew Stuible ◽

Christian Gervais ◽

Simon Lord-Dufour ◽

Sylvie Perret ◽

Denis L’Abbe ◽

...

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Affinity Purification ◽

Transient Gene Expression ◽

Full Length ◽

Spike Protein ◽

Hek293 Cells ◽

High Yield ◽

Diagnostic Tools ◽

Purification Method

ABSTRACTRecombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/rcTA cells gave substantially better yields than the other methods. Different forms of the spike were expressed, including the wild-type SARS-CoV-2 sequence and a mutated/stabilized form (to favor expression of the full-length spike in prefusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

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Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

Biomedicines ◽

10.3390/biomedicines10010049 ◽

2021 ◽

Vol 10 (1) ◽

pp. 49

Author(s):

Maria Ennemoser ◽

Julia Rieger ◽

Eva Muttenthaler ◽

Tanja Gerlza ◽

Kurt Zatloukal ◽

...

Keyword(s):

Heparan Sulfate ◽

Viral Transmission ◽

Full Length ◽

Spike Protein ◽

Target Cells ◽

Pentosan Polysulfate ◽

Cell Infection ◽

Virus Surface ◽

Kd Values

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600–680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50–500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

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Structure, Dynamics, Receptor Binding, and Antibody Binding of Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane

10.1101/2020.10.18.343715 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yeol Kyo Choi ◽

Yiwei Cao ◽

Martin Frank ◽

Hyeonuk Woo ◽

Sang-Jun Park ◽

...

Keyword(s):

Receptor Binding ◽

Vaccine Development ◽

Accessible Surface Area ◽

Full Length ◽

S Protein ◽

Viral Membrane ◽

Accessible Surface ◽

Dynamics Simulations ◽

The Impact ◽

Insight Into

ABSTRACTThe spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2), and is considered the major target for drug and vaccine development. We previously built fully-glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of receptor binding domain (RBD) and different templates for the stalk region. In this work, multiple μs-long all-atom molecular dynamics simulations were performed to provide deeper insight into the structure and dynamics of S protein, and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of RBD, and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody-glycan clash analysis instead of a commonly-used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibody. Overall, our observations offer structural and dynamic insight into SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.

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Phage display of functional, full-length human and viral membrane proteins

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2008.07.051 ◽

2008 ◽

Vol 18 (22) ◽

pp. 5937-5940 ◽

Cited By ~ 14

Author(s):

Sudipta Majumdar ◽

Agnes Hajduczki ◽

Aaron S. Mendez ◽

Gregory A. Weiss

Keyword(s):

Membrane Proteins ◽

Phage Display ◽

Full Length ◽

Viral Membrane

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Full-Length Computational Model of the SARS-CoV-2 Spike Protein and Its Implications for a Viral Membrane Fusion Mechanism

Viruses ◽

10.3390/v13061126 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1126

Author(s):

Wataru Nishima ◽

Marta Kulik

Keyword(s):

Membrane Fusion ◽

Neutralizing Antibody ◽

Infection Process ◽

Proteolytic Processing ◽

Full Length ◽

Spike Protein ◽

Fusion Model ◽

Protein Uptake ◽

Host Membrane ◽

Short Time

The SARS-CoV-2 virus has now become one of the greatest causes of infectious death and morbidity since the 1918 flu pandemic. Substantial and unprecedented progress has been made in the elucidation of the viral infection process in a short time; however, our understanding of the structure–function dynamics of the spike protein during the membrane fusion process and viral uptake remains incomplete. Employing computational approaches, we use full-length structural models of the SARS-CoV-2 spike protein integrating Cryo-EM images and biophysical properties, which fill the gaps in our understanding. We propose a membrane fusion model incorporating structural transitions associated with the proteolytic processing of the spike protein, which initiates and regulates a series of events to facilitate membrane fusion and viral genome uptake. The membrane fusion mechanism highlights the notable role of the S1 subunit and eventual mature spike protein uptake through the host membrane. Our comprehensive view accounts for distinct neutralizing antibody binding effects targeting the spike protein and the enhanced infectivity of the SARS-CoV-2 variant.

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