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

ABSTRACT Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

Download Full-text

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

10.1101/2021.06.17.448820 ◽

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.

Download Full-text

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

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103255747 ◽

2003 ◽

Vol 8 (4) ◽

pp. 463-470 ◽

Cited By ~ 23

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.78.11.6048-6054.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 6048-6054 ◽

Cited By ~ 100

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.80.3.1302-1310.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1302-1310 ◽

Cited By ~ 38

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text