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

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

10.21203/rs.3.rs-148750/v4 ◽

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.

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

10.21203/rs.3.rs-148750/v1 ◽

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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N- (4-Hydroxyphenyl) retinamide suppresses SARS-CoV-2 spike protein-mediated cell-cell fusion by a dihydroceramide Δ4-desaturase 1-independent mechanism

Journal of Virology ◽

10.1128/jvi.00807-21 ◽

2021 ◽

Author(s):

Yasuhiro Hayashi ◽

Kiyoto Tsuchiya ◽

Mizuki Yamamoto ◽

Yoko Nemoto-Sasaki ◽

Kazunari Tanigawa ◽

...

Keyword(s):

Cell Membrane ◽

Viral Infection ◽

Membrane Fusion ◽

Membrane Fluidity ◽

Cell Fusion ◽

Host Cells ◽

Spike Protein ◽

Sphingolipid Metabolism ◽

Independent Mechanism ◽

Cell Cell

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N -(4-hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion, and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeable ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells, compared to that in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. Importance Sphingolipids could play an important role in SARS-CoV-2 S-meditated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1 and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

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

10.21203/rs.3.rs-148750/v2 ◽

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009246 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1009246 ◽

Cited By ~ 1

Author(s):

Guido Papa ◽

Donna L. Mallery ◽

Anna Albecka ◽

Lawrence G. 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

Severe 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 may reduce but not abolish viral spread.

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