Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell–cell fusion but not interaction with M protein

Abstract The emergence of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, Omicron, is the most urgent concern in the global health in December 2021. Our statistical modelling estimates that Omicron is >3.0-fold and >5.6-fold more transmissible than Delta in South Africa and the UK, respectively. Intriguingly, cell culture experiments show that Omicron is less fusogenic than Delta and ancestral SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into the two subunits, which facilitates cell-cell fusion, Omicron S is faintly cleaved. Further, in hamster model, Omicron shows decreased lung infectivity and is less pathogenic compared to Delta and ancestral SARS-CoV-2. Our data suggest that the efficacy of SARS-CoV-2 S cleavage and viral fusogenicity are closely associated with viral pathogenicity, and Omicron evolved to exhibit increased transmissibility and attenuated pathogenicity.

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Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion

Cell & Bioscience ◽

10.1186/s13578-021-00626-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xuening Wang ◽

Chih-Hsiung Chen ◽

Saiaditya Badeti ◽

Jong Hyun Cho ◽

Alireza Naghizadeh ◽

...

Keyword(s):

Cell Fusion ◽

Cell Lines ◽

Cell Hybrid ◽

Host Cells ◽

Spike Protein ◽

Expression Vectors ◽

S Protein ◽

Er Retention ◽

Syncytia Formation ◽

Cell Cell

Abstract Background The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of the syncytia by SARS-CoV-2 are not fully understood. Results In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), as well as human ACE2 expression vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell–cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. Conclusions This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully. Δ19-S mRNA may represent a safer mRNA vaccine design in the future.

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New Consensus pattern in Spike CoV-2: potential implications in coagulation process and cell–cell fusion

Cell Death Discovery ◽

10.1038/s41420-020-00372-1 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Silvia Buonvino ◽

Sonia Melino

Keyword(s):

Cell Fate ◽

Cell Fusion ◽

Molecular Mechanisms ◽

Coagulation Factor ◽

Cell Interaction ◽

Factor X ◽

S Protein ◽

Coagulation Process ◽

Consensus Pattern ◽

Cell Cell

AbstractCoagulopathy and syncytial formation are relevant effects of the SARS-CoV-2 infection, but the underlying molecular mechanisms triggering these processes are not fully elucidated. Here, we identified a potential consensus pattern in the Spike S glycoprotein present within the cytoplasmic domain; this consensus pattern was detected in only 79 out of 561,000 proteins (UniProt bank). Interestingly, the pattern was present in both human and bat the coronaviruses S proteins, in many proteins involved in coagulation process, cell–cell interaction, protein aggregation and regulation of cell fate, such as von Willebrand factor, coagulation factor X, fibronectin and Notch, characterized by the presence of the cysteine-rich EGF-like domain. This finding may suggest functional similarities between the matched proteins and the CoV-2 S protein, implying a new possible involvement of the S protein in the molecular mechanism that leads to the coagulopathy and cell fusion in COVID-19 disease.

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A bivalent protein targeting glycans and HR1 domain in spike protein potently inhibited infection of SARS-CoV-2 and other human coronaviruses

Cell & Bioscience ◽

10.1186/s13578-021-00638-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yanxing Cai ◽

Wei Xu ◽

Jiayi Tang ◽

Najing Cao ◽

Qiaoshuai Lan ◽

...

Keyword(s):

Cell Fusion ◽

Mechanism Of Action ◽

Broad Spectrum ◽

Inhibitory Activity ◽

Inhibitory Peptide ◽

Newborn Mice ◽

S Protein ◽

Human Coronaviruses ◽

Potent Inhibitory Activity ◽

Cell Cell

Abstract Background Our previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. In this study, we aimed to design and construct a bivalent protein consisting of GRFT and EK1 components and evaluate its inhibitory activity and mechanism of action against infection by SARS-CoV-2 and its mutants, as well as other human coronaviruses (HCoVs). Methods The bivalent proteins were expressed in E. coli and purified with Ni-NTA column. HIV backbone-based pseudovirus (PsV) infection and HCoV S-mediated cell–cell fusion assays were performed to test their inhibitory activity. ELISA and Native-PAGE were conducted to illustrate the mechanism of action of these bivalent proteins. Five-day-old newborn mice were intranasally administrated with a selected bivalent protein before or after HCoV-OC43 challenge, and its protective effect was monitored for 14 days. Results Among the three bivalent proteins purified, GL25E exhibited the most potent inhibitory activity against infection of SARS-CoV-2 PsVs expressing wild-type and mutated S protein. GL25E was significantly more effective than GRFT and EK1 alone in inhibiting HCoV S-mediated cell–cell fusion, as well as infection by SARS-CoV-2 and other HCoVs, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. GL25E could inhibit authentic SASR-CoV-2, HCoV-OC43 and HCoV-229E infection in vitro and prevent newborn mice from authentic HCoV-OC43 infection in vivo. GL25E could bind to glycans in the S1 subunit and HR1 in the S2 subunit in S protein, showing a mechanism of action similar to that of GRFT and EK1 alone. Conclusions Since GL25E showed highly potent and broad-spectrum inhibitory activity against infection of SARS-CoV-2 and its mutants, as well as other HCoVs, it is a promising candidate for further development as a broad-spectrum anti-HCoV therapeutic and prophylactic to treat and prevent COVID-19 and other emerging HCoV diseases.

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Deletion of ER-retention Motif on SARS-CoV-2 Spike Protein Reduces Cell Hybrid During Cell-cell Fusion

10.21203/rs.3.rs-380389/v1 ◽

2021 ◽

Author(s):

Chih-Hsiung Chen ◽

Saiaditya Badeti ◽

Jong Hyun Cho ◽

Alireza Naghizadeh ◽

Xuening Wang ◽

...

Keyword(s):

Cell Fusion ◽

Cell Lines ◽

Cell Hybrid ◽

Host Cells ◽

Spike Protein ◽

In Vitro Assays ◽

S Protein ◽

Er Retention ◽

Syncytia Formation ◽

Cell Cell

Abstract The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of these syncytia by SARS-CoV-2 are not fully understood. In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were stably transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), or human ACE2 vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell-cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully.

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Altered Interaction of the Matrix Protein with the Cytoplasmic Tail of Hemagglutinin Modulates Measles Virus Growth by Affecting Virus Assembly and Cell-Cell Fusion

Journal of Virology ◽

10.1128/jvi.00248-07 ◽

2007 ◽

Vol 81 (13) ◽

pp. 6827-6836 ◽

Cited By ~ 52

Author(s):

Maino Tahara ◽

Makoto Takeda ◽

Yusuke Yanagi

Keyword(s):

Cell Fusion ◽

Measles Virus ◽

Matrix Protein ◽

Cytoplasmic Tail ◽

Vero Cells ◽

M Protein ◽

Virus Growth ◽

The Matrix ◽

H Protein ◽

Cell Cell

ABSTRACT Clinical isolates of measles virus (MV) use signaling lymphocyte activation molecule (SLAM) as a cellular receptor, whereas vaccine and laboratory strains may utilize the ubiquitously expressed CD46 as an additional receptor. MVs also infect, albeit inefficiently, SLAM− cells, via a SLAM- and CD46-independent pathway. Our previous study with recombinant chimeric viruses revealed that not only the receptor-binding hemagglutinin (H) but also the matrix (M) protein of the Edmonston vaccine strain can confer on an MV clinical isolate the ability to grow well in SLAM− Vero cells. Two substitutions (P64S and E89K) in the M protein which are present in many vaccine strains were found to be responsible for the efficient growth of recombinant virus in Vero cells. Here we show that the P64S and E89K substitutions allow a strong interaction of the M protein with the cytoplasmic tail of the H protein, thereby enhancing the assembly of infectious particles in Vero cells. These substitutions, however, are not necessarily advantageous for MVs, as they inhibit SLAM-dependent cell-cell fusion, thus reducing virus growth in SLAM+ B-lymphoblastoid B95a cells. When the cytoplasmic tail of the H protein is deleted, a virus with an M protein possessing the P64S and E89K substitutions no longer grows well in Vero cells yet causes cell-cell fusion and replicates efficiently in B95a cells. These results reveal a novel mechanism of adaptation and attenuation of MV in which the altered interaction of the M protein with the cytoplasmic tail of the H protein modulates MV growth in different cell types.

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Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity

Journal of Virology ◽

10.1128/jvi.00635-20 ◽

2020 ◽

Vol 94 (14) ◽

Cited By ~ 18

Author(s):

Yuanmei Zhu ◽

Danwei Yu ◽

Hongxia Yan ◽

Huihui Chong ◽

Yuxian He

Keyword(s):

Public Health ◽

Severe Acute Respiratory Syndrome ◽

Fusion Protein ◽

Membrane Fusion ◽

Cell Fusion ◽

Heptad Repeat ◽

Global Public Health ◽

S Protein ◽

Fusion Inhibitors ◽

Cell Cell

ABSTRACT The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.

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Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

10.1101/2020.08.13.243303 ◽

2020 ◽

Cited By ~ 4

Author(s):

Guido Papa ◽

Donna L. Mallery ◽

Anna Albecka ◽

Lawrence Welch ◽

Jérôme Cattin-Ortolá ◽

...

Keyword(s):

Cell Fusion ◽

Cell Receptor ◽

Furin Cleavage ◽

S Protein ◽

Viral Spread ◽

Host Cell Receptor ◽

Host Membrane ◽

Dependent Cell ◽

Syncytia Formation ◽

Cell Cell

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.

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Blockade of SARS-CoV-2 spike protein-mediated cell–cell fusion using COVID-19 convalescent plasma

Scientific Reports ◽

10.1038/s41598-021-84840-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ling Wang ◽

Juan Zhao ◽

Lam N. T. Nguyen ◽

James L. Adkins ◽

Madison Schank ◽

...

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Neutralizing Antibodies ◽

Molecular Mechanisms ◽

Virus Entry ◽

High Capacity ◽

Target Cells ◽

Global Public Health ◽

S Protein ◽

Cell Cell

AbstractThe recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell–cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell–cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

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Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

10.1101/2020.10.15.340604 ◽

2020 ◽

Author(s):

Samuel A. Theuerkauf ◽

Alexander Michels ◽

Vanessa Riechert ◽

Thorsten J. Maier ◽

Egbert Flory ◽

...

Keyword(s):

Cell Fusion ◽

Neutralizing Antibodies ◽

High Efficiency ◽

Dynamic Range ◽

Protein S ◽

High Dynamic Range ◽

Spike Protein ◽

S Protein ◽

Syncytia Formation ◽

Cell Cell

AbstractCell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split β-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.

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