scholarly journals Cleavage Inhibition of the Murine Coronavirus Spike Protein by a Furin-Like Enzyme Affects Cell-Cell but Not Virus-Cell Fusion

2004 ◽  
Vol 78 (11) ◽  
pp. 6048-6054 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Konrad Stadler ◽  
Gert-Jan Godeke ◽  
Berend Jan Bosch ◽  
Peter J. M. Rottier
Keyword(s):  
Cell Fusion ◽  
Spike Protein ◽  
Dependent Manner ◽  
Protein Cleavage ◽  
Concentration Dependent Manner ◽  
Cell Cell ◽  
Murine Coronavirus

ABSTRACT Cleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentration-dependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements.

scholarly journals Effect of mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell-cell fusion function

2021 ◽  
Author(s):  
Chelsea T. Barrett ◽  
Hadley E. Neal ◽  
Kearstin Edmonds ◽  
Carole L. Moncman ◽  
Rachel Thompson ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
Protein S ◽  
Proteolytic Processing ◽  
Spike Protein ◽  
Protein Cleavage ◽  
Fusion Event ◽  
Viral Binding ◽  
Mammalian Cell Lines ◽  
Cell Cell

AbstractThe SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S cell-cell fusion. Additionally, we examine S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this highly sought-after therapeutic target.

scholarly journals Acquisition of Cell–Cell Fusion Activity by Amino Acid Substitutions in Spike Protein Determines the Infectivity of a Coronavirus in Cultured Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (7) ◽  
pp. e6130 ◽  
Author(s):  
Yoshiyuki Yamada ◽  
Xiao Bo Liu ◽  
Shou Guo Fang ◽  
Felicia P. L. Tay ◽  
Ding Xiang Liu
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Cultured Cells ◽  
Spike Protein ◽  
Amino Acid Substitutions ◽  
Fusion Activity ◽  
Cell Cell

scholarly journals The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

2003 ◽  
Vol 77 (5) ◽  
pp. 3058-3066 ◽  
Author(s):  
Laurie J. Earp ◽  
Sue E. Delos ◽  
Robert C. Netter ◽  
Paul Bates ◽  
Judith M. White
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Low Ph ◽  
Target Cells ◽  
Dependent Manner ◽  
Neutral Ph ◽  
Subtype A ◽  
Avian Sarcoma ◽  
Virus Subtype ◽  
Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

scholarly journals Neurovirulent Murine Coronavirus JHM.SD Uses Cellular Zinc Metalloproteases for Virus Entry and Cell-Cell Fusion

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Judith M. Phillips ◽  
Tom Gallagher ◽  
Susan R. Weiss
Keyword(s):  
Cell Surface ◽  
Matrix Metalloproteinase ◽  
Serine Protease ◽  
Cell Fusion ◽  
Acid Ph ◽  
Endosomal Acidification ◽  
Zinc Metalloproteases ◽  
S Proteins ◽  
Cell Cell ◽  
Murine Coronavirus

ABSTRACT The coronavirus (CoV) S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the murine coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry, and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry, of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain, A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytium formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of matrix metalloproteinase (MMP)- and a disintegrin and metalloprotease (ADAM)-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to coronavirus fusion. IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), as well as a number of important animal pathogens. Because coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection. For example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76–84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and ADAM families, as potential targets for anticoronavirus therapy.

scholarly journals SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion

2021 ◽  
Author(s):  
Akatsuki Saito ◽  
Hesham Nasser ◽  
Keiya Uriu ◽  
Yusuke Kosugi ◽  
Takashi Irie ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Viral Genome ◽  
Neutralizing Antibody ◽  
Spike Protein ◽  
Human Society ◽  
Viral Infectivity ◽  
Viral Fusion ◽  
Antibody Resistance ◽  
Cell Cell

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and at least five variants of concerns (VOCs) have been considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2 lineage (delta variant), closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617 variants are highly fusogenic and form prominent syncytia. Bioinformatic analyses reveal that the P681R mutation in the spike protein is highly conserved in this lineage. Although the P681R mutation decreases viral infectivity, this mutation confers the neutralizing antibody resistance. Notably, we demonstrate that the P681R mutation facilitates the furin-mediated spike cleavage and enhances and accelerates cell-cell fusion. Our data suggest that the P681R mutation is a hallmark characterizing the virological phenotype of this newest VOC, which may associate with viral pathogenicity.

scholarly journals Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

2006 ◽  
Vol 80 (3) ◽  
pp. 1302-1310 ◽  
Author(s):  
Rene Broer ◽  
Bertrand Boson ◽  
Willy Spaan ◽  
François-Loïc Cosset ◽  
Jeroen Corver
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Transmembrane Domains ◽  
Spike Protein ◽  
Fusion Activity ◽  
Wild Type ◽  
Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

scholarly journals Different host cell proteases activate the SARS-coronavirus spike-protein for cell–cell and virus–cell fusion

Virology ◽  
2011 ◽  
Vol 413 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Graham Simmons ◽  
Stephanie Bertram ◽  
Ilona Glowacka ◽  
Imke Steffen ◽  
Chawaree Chaipan ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Spike Protein ◽  
Sars Coronavirus ◽  
Cell Cell

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

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.

scholarly journals N-(4-Hydroxyphenyl)retinamide suppresses SARS-CoV-2 spike protein-mediated cell-cell fusion and viral infection in vitro 

2021 ◽  
Author(s):  
Yasuhiro Hayashi ◽  
Kiyoto Tsuchiya ◽  
Mizuki Yamamoto ◽  
Yoko Nemoto-Sasaki ◽  
Kazunari Tanigawa ◽  
...  
Keyword(s):  
Viral Infection ◽  
Cell Fusion ◽  
Antitumour Activity ◽  
Cellular Membrane ◽  
Host Cells ◽  
Spike Protein ◽  
Sphingolipid Metabolism ◽  
Metabolism Enzymes ◽  
Cell Cell

Abstract The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), persists worldwide with limited therapeutic options. Since membrane fusion between SARS-CoV-2 and host cells is essential for the early step of the infection, the membrane compositions, including sphingolipids, in host cells are considered to affect the viral infection. However, the role of sphingolipids in the life cycle of SARS-CoV-2 remains unclear. Here, we assessed several inhibitors of sphingolipid metabolism enzymes against SARS-CoV-2 spike protein-mediated cell-cell fusion and viral infection in vitro. Among the compounds tested, only N-(4-hydroxyphenyl)retinamide (4-HPR, also known as fenretinide), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1) and well known for having antitumour activity, suppressed cell-cell fusion (50% effective concentration [EC50] = 4.1 µM) and viral infection ([EC50] = 4.4 µM), wherein the EC50 values are below its plasma concentration in previous clinical trials on tumours. DES1 catalyses the introduction of a double bond in dihydroceramide, and the inhibition efficiencies observed were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids and the decreased cellular membrane fluidity. These findings, together with the accumulated clinical data regarding the safety of 4-HPR, make it a likely candidate drug to treat COVID-19.

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

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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