Gene of the month: the 2019-nCoV/SARS-CoV-2 novel coronavirus spike protein

The year 2020 has seen a major and sustained outbreak of a novel betacoronavirus (severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2) infection that causes fever, severe respiratory illness and pneumonia, a disease called COVID-19. At the time of writing, the death toll was greater than 120 000 worldwide with more than 2 million documented infections. The genome of the CoV encodes a number of structural proteins that facilitate cellular entry and assembly of virions, of which the spike protein S appears to be critical for cellular entry. The spike protein guides the virus to attach to the host cell. The spike protein contains a receptor-binding domain (RBD), a fusion domain and a transmembrane domain. The RBD of spike protein S binds to Angiotensin Converting Enzyme 2 (ACE2) to initiate cellular entry. The spike protein of SARS-CoV-2 shows more than 90% amino acid similarity to the pangolin and bat CoVs and these also use ACE2 as a receptor. Binding of the spike protein to ACE2 exposes the cleavage sites to cellular proteases. Cleavage of the spike protein by transmembrane protease serine 2 and other cellular proteases initiates fusion and endocytosis. The spike protein contains an addition furin cleavage site that may allow it to be ‘preactivated’ and highly infectious after replication. The fundamental role of the spike protein in infectivity suggests that it is an important target for vaccine development, blocking therapy with antibodies and diagnostic antigen-based tests. This review briefly outlines the structure and function of the 2019 novel CoV/SARS-CoV-2 spike protein S.

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Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein

10.1101/2020.02.19.956581 ◽

2020 ◽

Cited By ~ 91

Author(s):

Alexandra C. Walls ◽

Young-Jun Park ◽

M. Alexandra Tortorici ◽

Abigail Wall ◽

Andrew T. McGuire ◽

...

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Target Cells ◽

Furin Cleavage ◽

Humoral Immune ◽

Binding Domains ◽

Furin Cleavage Site ◽

Recent Emergence ◽

Novel Coronavirus

SUMMARYThe recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

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Sequence Analysis Indicates that 2019-nCoV Virus Contains a Putative Furin Cleavage Site at the Boundary of S1 and S2 Domains of Spike Protein

10.31219/osf.io/nkcrf ◽

2020 ◽

Author(s):

Z. Galen WO

Keyword(s):

Amino Acid ◽

Cleavage Site ◽

Spike Protein ◽

Sequence Motif ◽

Furin Cleavage ◽

Host Cell Membrane ◽

Virus Family ◽

Prediction Program ◽

Furin Cleavage Site ◽

Novel Coronavirus

The infectious 2019-nCoV virus, which caused the current novel coronavirus pneumonia epidemic outbreak, possesses a unique 4-Amino Acid insert at the boundary of the two subdomains (S1 and S2) of Spike protein based on multiple protein sequence alignment with the large SARS and SARS-related virus family. Using Bat CoV_RaTG13 Spike protein as reference (sharing 97% aa identity) the 4-amino acid insert can be identified as PRRA (AA position 681-684). The effect of the 4-AA insertion is the presence of a furin signature sequence motif (PRRARSV) at the boundary of S1 and S2 domains of spike protein. This sequence motif consists the required Arg residue for P1 and P4 position of Furin site. In addition, it contains Arg at P3 site as well as Ser at P1’ site of furin motif. This sequence motif matches Aerolysin furin site in FurinDB and was predicted to be moderately strong (score 0.62) by ProP, a protease cleavage site prediction program. This finding suggests that the infectious 2019-nCoV virus, unlike SARS viruses, may be processed via cellular furin recognition and cleavage of the spike protein before host cell membrane fusion and entry. This putative furin site in spike protein of 2019-nCoV virus, if proven to be functional, suggests the potential of looking into agents inhibiting furin as therapeutic mean for the treatment of the novel coronavirus pneumonia.

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The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism

Viruses ◽

10.3390/v11100972 ◽

2019 ◽

Vol 11 (10) ◽

pp. 972 ◽

Cited By ~ 22

Author(s):

Jinlong Cheng ◽

Ye Zhao ◽

Gang Xu ◽

Keran Zhang ◽

Wenfeng Jia ◽

...

Keyword(s):

Neutralization Test ◽

Protein S ◽

Spike Protein ◽

Wild Type ◽

Effective Protection ◽

Furin Cleavage ◽

Infectious Bronchitis ◽

Furin Cleavage Site

Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2′ site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2′ site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2′ site (furin-S2′ site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2′ site as a determinant of neurotropism.

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The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

International Journal of Molecular Sciences ◽

10.3390/ijms22126490 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6490

Author(s):

Olga A. Postnikova ◽

Sheetal Uppal ◽

Weiliang Huang ◽

Maureen A. Kane ◽

Rafael Villasmil ◽

...

Keyword(s):

Amino Acid ◽

Insertion Sequence ◽

Experimental Studies ◽

Spike Protein ◽

Spike Glycoprotein ◽

Furin Cleavage ◽

New Host ◽

S Protein ◽

Furin Cleavage Site ◽

Ribosome Pausing

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

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Role of Structural and Non-Structural Proteins and Therapeutic Targets of SARS-CoV-2 for COVID-19

Cells ◽

10.3390/cells10040821 ◽

2021 ◽

Vol 10 (4) ◽

pp. 821

Author(s):

Rohitash Yadav ◽

Jitendra Kumar Chaudhary ◽

Neeraj Jain ◽

Pankaj Kumar Chaudhary ◽

Supriya Khanra ◽

...

Keyword(s):

Host Cell ◽

Protein S ◽

Structural Similarity ◽

Cell Receptor ◽

Molecular Assembly ◽

The Body ◽

Spike Protein ◽

Structural Proteins ◽

Structural Protein ◽

Novel Coronavirus

Coronavirus belongs to the family of Coronaviridae, comprising single-stranded, positive-sense RNA genome (+ ssRNA) of around 26 to 32 kilobases, and has been known to cause infection to a myriad of mammalian hosts, such as humans, cats, bats, civets, dogs, and camels with varied consequences in terms of death and debilitation. Strikingly, novel coronavirus (2019-nCoV), later renamed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and found to be the causative agent of coronavirus disease-19 (COVID-19), shows 88% of sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, 79% with SARS-CoV and 50% with MERS-CoV, respectively. Despite key amino acid residual variability, there is an incredible structural similarity between the receptor binding domain (RBD) of spike protein (S) of SARS-CoV-2 and SARS-CoV. During infection, spike protein of SARS-CoV-2 compared to SARS-CoV displays 10–20 times greater affinity for its cognate host cell receptor, angiotensin-converting enzyme 2 (ACE2), leading proteolytic cleavage of S protein by transmembrane protease serine 2 (TMPRSS2). Following cellular entry, the ORF-1a and ORF-1ab, located downstream to 5′ end of + ssRNA genome, undergo translation, thereby forming two large polyproteins, pp1a and pp1ab. These polyproteins, following protease-induced cleavage and molecular assembly, form functional viral RNA polymerase, also referred to as replicase. Thereafter, uninterrupted orchestrated replication-transcription molecular events lead to the synthesis of multiple nested sets of subgenomic mRNAs (sgRNAs), which are finally translated to several structural and accessory proteins participating in structure formation and various molecular functions of virus, respectively. These multiple structural proteins assemble and encapsulate genomic RNA (gRNA), resulting in numerous viral progenies, which eventually exit the host cell, and spread infection to rest of the body. In this review, we primarily focus on genomic organization, structural and non-structural protein components, and potential prospective molecular targets for development of therapeutic drugs, convalescent plasm therapy, and a myriad of potential vaccines to tackle SARS-CoV-2 infection.

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Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

Comparative Medicine ◽

10.30802/aalas-cm-21-000027 ◽

2021 ◽

Author(s):

Alison E Stout ◽

Qinghua Guo ◽

Jean K Millet ◽

Gary R Whittaker

Keyword(s):

Viral Pathogenesis ◽

Spike Protein ◽

Future Research ◽

Receptor Binding Domain ◽

Viral Reservoirs ◽

Furin Cleavage ◽

Proteolytic Activation ◽

Furin Cleavage Site ◽

Unique Aspect ◽

Genome Level

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the humanpopulation, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronavirusesRaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronavirusesremains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contributeto the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

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Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109905118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2109905118

Author(s):

Liping Zhang ◽

Matthew Mann ◽

Zulfeqhar A. Syed ◽

Hayley M. Reynolds ◽

E. Tian ◽

...

Keyword(s):

Cleavage Site ◽

Protein S ◽

Viral Infectivity ◽

The Novel ◽

Furin Cleavage ◽

Global Pandemic ◽

Furin Cleavage Site ◽

Respiratory Cells ◽

Syncytia Formation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

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SARS-CoV-2 convergent evolution as a guide to explore adaptive advantage

10.1101/2021.05.24.445534 ◽

2021 ◽

Author(s):

Jiri Zahradnik ◽

Jaroslav Nunvar ◽

Gideon Schreiber

Keyword(s):

Receptor Binding ◽

Convergent Evolution ◽

Great Majority ◽

Viral Population ◽

Binding Motif ◽

Furin Cleavage ◽

S Protein ◽

Adaptive Mutations ◽

Furin Cleavage Site ◽

Protein Receptor

Much can be learned from 1.2 million sequences of SARS-CoV-2 generated during the last 15 months. Out of the overwhelming number of mutations sampled so far, only few rose to prominence in the viral population. Many of these emerged recently and independently in multiple lineages. Such a textbook example of convergent evolution at the molecular level is not only curiosity but a guide to uncover the basis for adaptive advantage behind these events. Focusing on the extent of the convergent evolution in the spike (S) protein, our report confirms that the most concerning SARS-CoV-2 lineages carry the heaviest burden of convergent S-protein mutations, suggesting their fundamental adaptive advantage. The great majority (21/25) of S-protein sites under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. We further show that among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favored. While the probed mutation space in the S protein covered all amino-acids reachable by single nucleotide changes, substitutions requiring two nucleotide changes or epistatic mutations of multiple-residues have only recently started to emerge. Unfortunately, despite their convergent emergence and physical association, most of these adaptive mutations and their combinations remain understudied. We aim to promote research of current variants which are currently understudied but may become important in the future.

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

Journal of Virology ◽

10.1128/jvi.02744-05 ◽

2006 ◽

Vol 80 (14) ◽

pp. 6794-6800 ◽

Cited By ~ 76

Author(s):

Fang Li ◽

Marcelo Berardi ◽

Wenhui Li ◽

Michael Farzan ◽

Philip R. Dormitzer ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Receptor Binding ◽

Conformational Changes ◽

Viral Entry ◽

Protein S ◽

Spike Protein ◽

Fusion Peptides ◽

Conformational States ◽

Protease Cleavage

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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Mutation of a Conserved Hydrophobic Patch PreventsIncorporation of ZP3 into the Zona Pellucida SurroundingMouseEggs

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.8982-8991.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 8982-8991 ◽

Cited By ~ 27

Author(s):

Ming Zhao ◽

Lyn Gold ◽

Heidi Dorward ◽

Li-fang Liang ◽

Tanya Hoodbhoy ◽

...

Keyword(s):

Zona Pellucida ◽

Cleavage Site ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Proprotein Convertase ◽

Furin Cleavage ◽

Mouse Oocytes ◽

Hydrophobic Patch ◽

Furin Cleavage Site

ABSTRACT Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a “zona” domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.

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