Syncytia formation by SARS‐CoV‐2‐infected cells

Buchrieser, Julian; Dufloo, Jérémy; Hubert, Mathieu; Monel, Blandine; Planas, Delphine; Rajah, Maaran Michael; Planchais, Cyril; Porrot, Françoise; Guivel‐Benhassine, Florence; Werf, Sylvie van der; Casartelli, Nicoletta; Mouquet, Hugo; Bruel, Timothée; Schwartz, Olivier

doi:10.15252/embj.2020106267

Cited by 382 publications

(373 citation statements)

References 64 publications

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“…Studies on SARS-CoV-1 and recent papers and preprints on SARS-CoV-2 have shown a variety of phenotypes with different IFITMs on both viral entry and cellto-cell fusion mediated by the spike protein (Bozzo et al, 2020;Peacock et al, 2020;Shi et al, 2020). IFITM1 appears to block syncytium formation between infected and uninfected cells and this is overcome by TMPRSS2 expression, which is in keeping with our observations that in stably expressing cells small effects of IFITM1 on SARS-CoV-2 entry in A549-ACE2 cells can be abolished similarly (Buchrieser et al, 2020). Other data has implicated IFITM3, and demonstrated that it can be enhancing if its expression is restricted to the cell surface.…”

Section: Ifitm2supporting

confidence: 91%

The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2

Winstone

Brotos

Reid

et al. 2020

Preprint

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The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein (S). The presence of a polybasic cleavage site in SARS-CoV-2 S at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the S precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons, and more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2 and the degree of this restriction is governed by route of viral entry. Removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH- and cathepsin-dependent in late endosomes where, like SARS-CoV-1 S, it is more sensitive to IFITM2 restriction. Furthermore, we find that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 S may reduce viral replication through the activity of type I IFNs.IMPORTANCEThe furin cleavage site in the S protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2, and that the sensitivity is exacerbated by deletion of the furin cleavage site which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus may represent a potential therapeutic target for COVID-19 treatment development.

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Section: Ifitm2supporting

confidence: 91%

The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2

Winstone

Brotos

Reid

et al. 2020

Preprint

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“…Although a previous study by Fletcher et al 28 showed that a single Asn residue positioned at the end of the heptad had a minor impact on stability, we determined that the peptide pairs, P7A:P8A and P5A:P6A, with two Asn residues per peptide, had lower stability but still in the nanomolar range, and the Finally, high-affinity CC pairs were used to set up the assay for monitoring cell fusion. Genetic fusion to protein interaction domains can guide protein complex formation 46 , and strong affinities are needed for a robust output in comparison to relying solely on the reconstitution of split GFP 43 . We established an assay to monitor the SARS-CoV-2 spike-protein-mediated cell-cell fusion, which allowed for quantitation by reconstitution of a CC-split luciferase reporter or visualization of syncytia by the reconstituted CC-split GFP.…”

Section: Discussion/conclusionmentioning

confidence: 99%

“…We demonstrated the effect of inhibiting TMPRSS2 protease that is required to cleave S protein and expose the fusion protein of spike protein prior to fusion. Camostat mesylate 43 has been previously tested as an inhibitor in the active viral assay 43 (Fig 6A). Cell fusion was detectable without the presence of TMPRSS2 protease ( Fig S7A); however, a strong signal increase was observed upon the coexpression of the protease (Fig.…”

Section: Cc-mediated Reporters For Viral Fusion Protein-mediated Syncmentioning

confidence: 99%

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Plaper

Aupič

Dekleva

et al. 2020

Preprint

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Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

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“…SΔ20 might also increase virus replication, as similar mutants (SΔ18, SΔ19 and SΔ21) were recently reported to increase both infectivity and replication of vesicular stomatitis virus (VSV) and human immunodeficiency virus (HIV) pseudotyped with SARS-CoV-2 S protein in cultured cells [41–44]. It is also tempting to suggest a link between SARS-CoV-2 pathogenesis and the presence of SΔ20, since severe cases of the disease were recently linked to considerable lung damage and the occurrence of syncytia [45,46].…”

Section: Discussionmentioning

confidence: 99%

Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

Rocheleau

Laroche

et al. 2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a genome comprised of a ~30K nucleotides non-segmented, positive single-stranded RNA. Although its RNA-dependent RNA polymerase exhibits exonuclease proofreading activity, viral sequence diversity can be induced by replication errors and host factors. These variations can be observed in the population of viral sequences isolated from infected host cells and are not necessarily reflected in the genome of transmitted founder viruses. We profiled intra-sample genetic diversity of SARS-CoV-2 variants using 15,289 high-throughput sequencing datasets from infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing, and/or sequencing protocols. Despite high mutational background, we confidently identified intra-variable positions recurrent in the samples analyzed, including several positions at the end of the gene encoding the viral S protein. Notably, most of the samples possesses a C->A missense mutation resulting in the S protein lacking the last 20 amino acids (SΔ20). Here we demonstrate that SΔ20 exhibits increased cell-to-cell fusion and syncytia formations. Our findings are suggestive of the consistent emergence of high-frequency viral quasispecies that are not horizontally transmitted but involved in intra-host infection and spread.Author summaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease, COVID-19, has caused significant worldwide mortality and unprecedented economic burden. Here we studied the intra-host genetic diversity of SARS-CoV-2 genomes and identified a high-frequency and recurrent non-sense mutation yielding a truncated form of the viral spike protein, in both human COVID-19 samples and in cell culture experiments. Through the use of a functional assay, we observed that this truncated spike protein displays an elevated fusogenic potential and forms syncytia. Given the high frequency at which this mutation independently arises across various samples, it can be hypothesized that this deletion mutation provides a selective advantage to viral replication and may also have a role in pathogenesis in humans.

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Syncytia formation by SARS‐CoV‐2‐infected cells

Cited by 382 publications

References 64 publications

The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2

The polybasic cleavage site in the SARS-CoV-2 spike modulates viral sensitivity to Type I IFN and IFITM2

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Identification of a High-frequency Intra-host SARS-CoV-2 spike Variant with Enhanced Cytopathic and Fusogenic Effect

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