Conformational States of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Ectodomain

ABSTRACT The severe acute respiratory syndrome coronavirus enters cells through the activities of a spike protein (S) which has receptor-binding (S1) and membrane fusion (S2) regions. We have characterized four sequential states of a purified recombinant S ectodomain (S-e) comprising S1 and the ectodomain of S2. They are S-e monomers, uncleaved S-e trimers, cleaved S-e trimers, and dissociated S1 monomers and S2 trimer rosettes. Lowered pH induces an irreversible transition from flexible, L-shaped S-e monomers to clove-shaped trimers. Protease cleavage of the trimer occurs at the S1-S2 boundary; an ensuing S1 dissociation leads to a major rearrangement of the trimeric S2 and to formation of rosettes likely to represent clusters of elongated, postfusion trimers of S2 associated through their fusion peptides. The states and transitions of S suggest conformational changes that mediate viral entry into cells.

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Structural impact on SARS-CoV-2 spike protein by D614G substitution

Science ◽

10.1126/science.abf2303 ◽

2021 ◽

pp. eabf2303

Author(s):

Jun Zhang ◽

Yongfei Cai ◽

Tianshu Xiao ◽

Jianming Lu ◽

Hanqin Peng ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Receptor Binding ◽

Viral Entry ◽

Vaccine Development ◽

Spike Protein ◽

Receptor Binding Domain ◽

Structural Rearrangements ◽

Viral Spread ◽

Structural Impact

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. We report here cryo-EM structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity, and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

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Binding and entering: COVID finds a new home

PLoS Pathogens ◽

10.1371/journal.ppat.1009857 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009857

Author(s):

Michelle N. Vu ◽

Vineet D. Menachery

Keyword(s):

Middle East ◽

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Common Cold ◽

Binding Domain ◽

Middle East Respiratory Syndrome ◽

Spike Protein ◽

Receptor Binding Domain ◽

Protease Cleavage ◽

Pandemic Virus

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged as a virus with a pathogenicity closer to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and a transmissibility similar to common cold coronaviruses (CoVs). In this review, we briefly discuss the features of the receptor-binding domain (RBD) and protease cleavage of the SARS-CoV-2 spike protein that enable SARS-CoV-2 to be a pandemic virus.

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Nipah Virus Attachment Glycoprotein Stalk C-Terminal Region Links Receptor Binding to Fusion Triggering

Journal of Virology ◽

10.1128/jvi.02277-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1838-1850 ◽

Cited By ~ 28

Author(s):

Qian Liu ◽

Birgit Bradel-Tretheway ◽

Abrrey I. Monreal ◽

Jonel P. Saludes ◽

Xiaonan Lu ◽

...

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Receptor Binding ◽

Conformational Changes ◽

Viral Entry ◽

Nipah Virus ◽

Cell Receptor ◽

Terminal Region ◽

Content Type ◽

Cell Cell

ABSTRACTMembrane fusion is essential for paramyxovirus entry into target cells and for the cell-cell fusion (syncytia) that results from many paramyxoviral infections. The concerted efforts of two membrane-integral viral proteins, the attachment (HN, H, or G) and fusion (F) glycoproteins, mediate membrane fusion. The emergent Nipah virus (NiV) is a highly pathogenic and deadly zoonotic paramyxovirus. We recently reported that upon cell receptor ephrinB2 or ephrinB3 binding, at least two conformational changes occur in the NiV-G head, followed by one in the NiV-G stalk, that subsequently result in F triggering and F execution of membrane fusion. However, the domains and residues in NiV-G that trigger F and the specific events that link receptor binding to F triggering are unknown. In the present study, we identified a NiV-G stalk C-terminal region (amino acids 159 to 163) that is important for multiple G functions, including G tetramerization, conformational integrity, G-F interactions, receptor-induced conformational changes in G, and F triggering. On the basis of these results, we propose that this NiV-G region serves as an important structural and functional linker between the NiV-G head and the rest of the stalk and is critical in propagating the F-triggering signal via specific conformational changes that open a concealed F-triggering domain(s) in the G stalk. These findings broaden our understanding of the mechanism(s) of receptor-induced paramyxovirus F triggering during viral entry and cell-cell fusion.IMPORTANCEThe emergent deadly viruses Nipah virus (NiV) and Hendra virus belong to theHenipavirusgenus in theParamyxoviridaefamily. NiV infections target endothelial cells and neurons and, in humans, result in 40 to 75% mortality rates. The broad tropism of the henipaviruses and the unavailability of therapeutics threaten the health of humans and livestock. Viral entry into host cells is the first step of henipavirus infections, which ultimately cause syncytium formation. After attaching to the host cell receptor, henipaviruses enter the target cell via direct viral-cell membrane fusion mediated by two membrane glycoproteins: the attachment protein (G) and the fusion protein (F). In this study, we identified and characterized a region in the NiV-G stalk C-terminal domain that links receptor binding to fusion triggering via several important glycoprotein functions. These findings advance our understanding of the membrane fusion-triggering mechanism(s) of the henipaviruses and the paramyxoviruses.

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Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

Journal of Virology ◽

10.1128/jvi.00785-19 ◽

2019 ◽

Vol 93 (19) ◽

Cited By ~ 3

Author(s):

Miyuki Kawase ◽

Michiyo Kataoka ◽

Kazuya Shirato ◽

Shutoku Matsuyama

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Receptor Binding ◽

Conformational Changes ◽

Biochemical Analysis ◽

Spike Protein ◽

Viral Fusion ◽

Enveloped Viruses ◽

Viral Fusion Protein ◽

Protease Digestion

ABSTRACT A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses. IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

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Domains and Functions of Spike Protein in SARS-Cov-2 in the Context of Vaccine Design

Viruses ◽

10.3390/v13010109 ◽

2021 ◽

Vol 13 (1) ◽

pp. 109

Author(s):

Xuhua Xia

Keyword(s):

Membrane Fusion ◽

Receptor Binding ◽

Viral Entry ◽

Vaccine Development ◽

Cell Entry ◽

Spike Protein ◽

Structural Domains ◽

Theoretical Approaches ◽

A Cell ◽

And Function

The spike protein in SARS-CoV-2 (SARS-2-S) interacts with the human ACE2 receptor to gain entry into a cell to initiate infection. Both Pfizer/BioNTech’s BNT162b2 and Moderna’s mRNA-1273 vaccine candidates are based on stabilized mRNA encoding prefusion SARS-2-S that can be produced after the mRNA is delivered into the human cell and translated. SARS-2-S is cleaved into S1 and S2 subunits, with S1 serving the function of receptor-binding and S2 serving the function of membrane fusion. Here, I dissect in detail the various domains of SARS-2-S and their functions discovered through a variety of different experimental and theoretical approaches to build a foundation for a comprehensive mechanistic understanding of how SARS-2-S works to achieve its function of mediating cell entry and subsequent cell-to-cell transmission. The integration of structure and function of SARS-2-S in this review should enhance our understanding of the dynamic processes involving receptor binding, multiple cleavage events, membrane fusion, viral entry, as well as the emergence of new viral variants. I highlighted the relevance of structural domains and dynamics to vaccine development, and discussed reasons for the spike protein to be frequently featured in the conspiracy theory claiming that SARS-CoV-2 is artificially created.

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Functional Characterization of Heptad Repeat 1 and 2 Mutants of the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus

Journal of Virology ◽

10.1128/jvi.80.7.3225-3237.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3225-3237 ◽

Cited By ~ 13

Author(s):

Woan-Eng Chan ◽

Chin-Kai Chuang ◽

Shiou-Hwei Yeh ◽

Mau-Sun Chang ◽

Steve S.-L. Chen

Keyword(s):

Molecular Weight ◽

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Viral Entry ◽

Functional Characterization ◽

Surface Expression ◽

Spike Protein ◽

Heptad Repeat ◽

Cell Surface Expression ◽

Wild Type

ABSTRACT To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an α-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight SL form, while the wild type and all other mutants produced similar levels of both the SL form and the slower-migrating, higher-molecular-weight SH form. The wild-type SL form was processed to the SH form, whereas the SL form of the 913P mutant was inefficiently converted to the SH form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the SH form into the virions as effectively as did the wild-type SH form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.

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Structural impact on SARS-CoV-2 spike protein by D614G substitution

10.1101/2020.10.13.337980 ◽

2020 ◽

Cited By ~ 4

Author(s):

Jun Zhang ◽

Yongfei Cai ◽

Tianshu Xiao ◽

Jianming Lu ◽

Hanqin Peng ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Viral Entry ◽

Vaccine Development ◽

Spike Protein ◽

Receptor Binding Domain ◽

Structural Rearrangements ◽

S Protein ◽

Viral Spread ◽

Structural Impact

AbstractSubstitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

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Receptor-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein Contains Multiple Conformation-Dependent Epitopes that Induce Highly Potent Neutralizing Antibodies

The Journal of Immunology ◽

10.4049/jimmunol.174.8.4908 ◽

2005 ◽

Vol 174 (8) ◽

pp. 4908-4915 ◽

Cited By ~ 156

Author(s):

Yuxian He ◽

Hong Lu ◽

Pamela Siddiqui ◽

Yusen Zhou ◽

Shibo Jiang

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Multiple Conformation

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Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

eLife ◽

10.7554/elife.61552 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Qi Yang ◽

Thomas A Hughes ◽

Anju Kelkar ◽

Xinheng Yu ◽

Kai Cheng ◽

...

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Multiple Roles ◽

Spike Protein ◽

Receptor Binding Domain ◽

Mechanistic Studies ◽

Post Translational Modification ◽

Minor Contribution ◽

Chemical Inhibitors ◽

The Impact

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

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