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 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

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

2021 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Cell Fusion ◽  
Virus Entry ◽  
Proximal Region ◽  
Glycoprotein B ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Membrane Proximal Region ◽  
Domain V ◽  
Domain I ◽  
Cell Cell

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

scholarly journals Development of a Novel High-Throughput Surrogate Assay to Measure HIV Envelope/CCR5/CD4-Mediated Viral/Cell Fusion Using BacMam Baculovirus Technology

2003 ◽  
Vol 8 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Stephen Jenkinson ◽  
David C. Mc Coy ◽  
Sandy A. Kerner ◽  
Robert G. Ferris ◽  
Wendell K. Lawrence ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
High Throughput ◽  
Viral Envelope ◽  
Host Cells ◽  
Luciferase Reporter ◽  
Viral Fusion ◽  
Fusion Event ◽  
Surrogate Assay ◽  
Cell Cell

The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.

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 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.

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 mM) and viral infection ([EC50] = 4.4 mM), 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.

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