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• W4206142017 abstract "Article4 November 2020free access Source DataTransparent process Syncytia formation by SARS-CoV-2-infected cells Julian Buchrieser Julian Buchrieser orcid.org/0000-0003-4790-7577 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Jérémy Dufloo Jérémy Dufloo orcid.org/0000-0002-4963-1378 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Bio Sorbonne Paris Cité (BioSPC), Université de Paris, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Mathieu Hubert Mathieu Hubert Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Blandine Monel Blandine Monel Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Delphine Planas Delphine Planas Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Vaccine Research Institute, Créteil, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Maaran Michael Rajah Maaran Michael Rajah Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Bio Sorbonne Paris Cité (BioSPC), Université de Paris, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Cyril Planchais Cyril Planchais Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, INSERM U1222, Paris, France Search for more papers by this author Françoise Porrot Françoise Porrot Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Florence Guivel-Benhassine Florence Guivel-Benhassine Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Sylvie Van der Werf Sylvie Van der Werf Molecular Genetics of RNA Viruses, Department of Virology, Institut Pasteur, CNRS UMR 3569, Université de Paris, Paris, France National Reference Center for Respiratory Viruses, Institut Pasteur, Paris, France Search for more papers by this author Nicoletta Casartelli Nicoletta Casartelli Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Hugo Mouquet Hugo Mouquet orcid.org/0000-0002-4230-610X Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, INSERM U1222, Paris, France Search for more papers by this author Timothée Bruel Timothée Bruel orcid.org/0000-0002-3952-4261 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Olivier Schwartz Corresponding Author Olivier Schwartz [email protected] orcid.org/0000-0002-0729-1475 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Vaccine Research Institute, Créteil, France Search for more papers by this author Julian Buchrieser Julian Buchrieser orcid.org/0000-0003-4790-7577 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Jérémy Dufloo Jérémy Dufloo orcid.org/0000-0002-4963-1378 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Bio Sorbonne Paris Cité (BioSPC), Université de Paris, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Mathieu Hubert Mathieu Hubert Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Blandine Monel Blandine Monel Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Delphine Planas Delphine Planas Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Vaccine Research Institute, Créteil, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Maaran Michael Rajah Maaran Michael Rajah Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Bio Sorbonne Paris Cité (BioSPC), Université de Paris, Paris, FranceThese authors contributed equally to this work as first co-authors Search for more papers by this author Cyril Planchais Cyril Planchais Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, INSERM U1222, Paris, France Search for more papers by this author Françoise Porrot Françoise Porrot Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Florence Guivel-Benhassine Florence Guivel-Benhassine Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Sylvie Van der Werf Sylvie Van der Werf Molecular Genetics of RNA Viruses, Department of Virology, Institut Pasteur, CNRS UMR 3569, Université de Paris, Paris, France National Reference Center for Respiratory Viruses, Institut Pasteur, Paris, France Search for more papers by this author Nicoletta Casartelli Nicoletta Casartelli Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Hugo Mouquet Hugo Mouquet orcid.org/0000-0002-4230-610X Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, INSERM U1222, Paris, France Search for more papers by this author Timothée Bruel Timothée Bruel orcid.org/0000-0002-3952-4261 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Search for more papers by this author Olivier Schwartz Corresponding Author Olivier Schwartz [email protected] orcid.org/0000-0002-0729-1475 Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France CNRS-UMR3569, Paris, France Vaccine Research Institute, Créteil, France Search for more papers by this author Author Information Julian Buchrieser1,2, Jérémy Dufloo1,2,3, Mathieu Hubert1,2, Blandine Monel1,2, Delphine Planas1,2,4, Maaran Michael Rajah1,2,3, Cyril Planchais5, Françoise Porrot1,2, Florence Guivel-Benhassine1,2, Sylvie Van der Werf6,7, Nicoletta Casartelli1,2, Hugo Mouquet5, Timothée Bruel1,2 and Olivier Schwartz *,1,2,4 1Virus and Immunity Unit, Department of Virology, Institut Pasteur, Paris, France 2CNRS-UMR3569, Paris, France 3Bio Sorbonne Paris Cité (BioSPC), Université de Paris, Paris, France 4Vaccine Research Institute, Créteil, France 5Laboratory of Humoral Immunology, Department of Immunology, Institut Pasteur, INSERM U1222, Paris, France 6Molecular Genetics of RNA Viruses, Department of Virology, Institut Pasteur, CNRS UMR 3569, Université de Paris, Paris, France 7National Reference Center for Respiratory Viruses, Institut Pasteur, Paris, France *Corresponding author. Tel: +33 145688353; E-mail: [email protected] The EMBO Journal (2020)39:e106267https://doi.org/10.15252/embj.2020106267 Correction(s) for this article Syncytia formation by SARS-CoV-2-infected cells01 February 2021 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2-infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation. Synopsis Cells infected with SARS-CoV-2 fuse with neighboring cells to form syncytia. This process is accelerated by the TMPRSS2 protease and restricted by interferon-induced transmembrane proteins (IFITMs). SARS-CoV-2-infected cells can fuse with neighboring cells to form syncytia. IFITM proteins, particularly IFITM1, restrict syncytia formation. TMPRSS2 protease accelerates syncytia formation and reverts the inhibitory effects of the IFITMs. Introduction COVID-19 consists of a spectrum of syndromes from a mild, flu-like illness to severe pneumonia. Disease severity is linked to lung epithelial destruction, resulting from both immune-mediated damages and viral cytopathic effects. SARS-CoV-2 infection of respiratory epithelial cells likely activates monocytes, macrophages, and dendritic cells, resulting in secretion of proinflammatory cytokines (Huang et al, 2020; Ong et al., 2020; Zhou et al, 2020). Excessive systemic cytokine production may lead to thrombosis, hypotension, acute respiratory distress syndrome (ARDS), and fatal multi-organ failure. The innate type-I and type-III interferon (IFN) response, which normally controls viral replication is also reduced in severe cases (Blanco-Melo et al, 2020; preprint: Hadjadj et al, 2020; Park & Iwasaki, 2020). However, prolonged IFN-production aggravates disease by impairing lung epithelial regeneration (Broggi et al, 2020; Major et al, 2020). In the lung, SARS-CoV-2 infects ciliated cells in the airway, alveolar type 2 pneumocytes, and epithelial progenitors among others (Bost et al, 2020; Hou et al, 2020; Subbarao & Mahanty, 2020). SARS-CoV-2 and other coronaviruses are cytopathic (Freundt et al, 2010; preprint: Gorshkov et al, 2020; Ogando et al, 2020; Ren et al, 2020; Tang et al, 2020). The death of infected cells is also a trigger of immune activation. SARS-CoV-2 entry into cell is initiated by interactions between the spike glycoprotein (S) and its receptor, angiotensin-converting enzyme 2 (ACE2), followed by S cleavage and priming by the cellular protease TMPRSS2 or other surface and endosomal proteases (Letko et al, 2020; Matsuyama et al, 2020; Hoffmann et al, 2020b). The structure of S in complex with ACE2 has been elucidated (Lan et al, 2020; Walls et al, 2020; Wang et al, 2020). S consists of three S1-S2 dimers, displaying conformational changes upon virus entry leading to fusion. Besides fusion mediated by virions, S proteins present at the plasma membrane can trigger receptor-dependent syncytia formation. These syncytia have been observed in cell cultures and in tissues from individuals infected with SARS-CoV-1, MERS-CoV, or SARS-CoV-2 (Franks et al, 2003; Matsuyama et al, 2010; Chan et al, 2013; Qian et al, 2013; preprint: Giacca et al, 2020; Hoffmann et al, 2020a; Tian et al, 2020; Xu et al, 2020), but they were not precisely characterized. It has been proposed that they may originate from direct infection of target cells or from the indirect immune-mediated fusion of myeloid cells. Fused pneumocytes expressing SARS-CoV-2 RNA and S proteins were observed in post-mortem lung tissues of 20 out of 41 COVID-19-infected patients, indicating that productive infection leads to syncytia formation, at least in critical cases (preprint: Giacca et al, 2020). SARS-CoV-2 replication is in part controlled by the innate host response, through mechanisms that are currently being unveiled. Interferon-stimulated genes (ISGs) inhibit discrete steps of the viral life cycle. At the entry level, the interferon (IFN)-Induced Transmembrane proteins (IFITM1, IFITM2, or IFITM3) block many viruses by inhibiting virus–cell fusion at hemifusion or pore formation stages (Shi et al, 2017). IFITMs act by modifying the rigidity and/or curvature of the membranes in which they reside (Abdel Motal et al, 1993; Compton Alex et al, 2014; Shi et al, 2017; Zani & Yount, 2018). Due to different sorting motifs, IFITM1 is mostly found at the plasma membrane, whereas IFITM2/3 accumulates in the endo-lysosomal compartment after transiting through the surface. IFITMs inhibit SARS-CoV, 229E, and MERS-CoV entry, but promote infection by HCoV-OC43, a coronavirus that causes the common cold (Huang et al, 2011; Bertram et al, 2013; Warren et al, 2014; Wrensch et al, 2014; Zhao et al, 2014; Zhao et al, 2018). IFITMs, as well as other ISGs, including LY6E and Cholesterol 25-hydrolase (CH25H), impair SARS-CoV-2 replication by blocking the fusion of virions (Pfaender et al, 2020; preprint: Zang et al, 2020; Zhao et al, 2020). Most of the experiments regarding these ISGs have been performed with single-cycle viral pseudotypes. Little is known about the impact of IFITMs on SARS-CoV-2-induced syncytia formation. Here, we characterized the mechanisms of SARS-CoV-2-induced cell–cell fusion and examined how syncytia formation is impacted by IFITMs and TMPRSS2. Results Syncytia formation by SARS-CoV-2-infected cells We first examined whether SARS-CoV-2-infected cells may form syncytia. To this aim, we derived U2OS cells stably expressing ACE2. We selected this cell line because its flat shape facilitates imaging. We generated U2OS-ACE2 cells carrying a GFP–Split complementation system (Buchrieser et al, 2019), in which two cells separately produce half of the reporter protein, producing GFP only upon fusion (Fig 1A). These U2OS-ACE2-derived cells, that we termed “S-Fuse” cells, were exposed to various doses of SARS-CoV-2. Video-microscopy analysis showed that syncytia appeared rapidly, starting at 6 h post-infection and grew in size, as bystander cells are incorporated in fused cells (Fig 1B and C and Movie EV1). Most of the syncytia end up dying, as assessed by the acquisition of propidium iodide (PI) (Fig 1B and C and Movie EV1). The extent of fusion was then quantified by measuring the GFP+ area with a high-content imager. The total area of syncytia within each well correlated with the viral inoculum, indicating that the assay provides a quantitative assessment of viral infection (Fig 2A). S was expressed by the syncytia, but also by single infected cells that have not yet fused, as assessed by immunofluorescence (Fig 1D). Flow cytometry on unpermeabilized infected cells further showed that S was present at the surface (Fig 1E). Figure 1. SARS-CoV-2 induced syncytia formation A. GFP-Split U2OS-ACE2 were co-cultured at a 1:1 ratio and infected with SARS-CoV-2. Syncytia formation and cell death was monitored by video microscopy or at endpoint using confocal microscopy and high-content imaging. B. Still images of GFP (syncytia) and propidium iodide (PI) (cell death) at different time points. Scale bar: 100 µm. C. Quantification of U2OS-ACE2 fusion and death by time-lapse microscopy. Results are mean ± SD from three fields per condition. D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm. E. Surface S staining of infected U2OS-ACE2 cells analyzed by flow cytometry. Results are representative of at least three independent experiments. Download figure Download PowerPoint Figure 2. Impact of TMPRSS2 and IFITMs on syncytia formation by U2OS-ACE2-infected cells Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20 h. Image quantification method is described in Fig EV2. A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and nuclei count normalized to NI. B. IFITM1, but not IFITM2 and 3, inhibits SARS-CoV-2-induced syncytia formation. Right panels: Area of GFP+ cells C. The fusion of IFITM1+ cells with U2OS-ACE2 syncytia is drastically reduced. Right panel: Area of IFITM+ GFP+ cells D. IFITM1 decreases the number of infected cells. Right panel: Fraction of cells positive for S, normalized to control cells (transduced with pQCXIP-empty vector). Data information: Left panels: one representative experiment is shown. Scale bars: 100 µm. Right panels: Data are mean ± SD of 3–9 independent experiments. Statistical analysis: B–C: One-way ANOVA, D: Two-way ANOVA. ns: non-significant, *P < 0. 05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Impact of IFITMs and TMPRSS2 on syncytia formation We then asked whether TMPRSS2 and IFITM1, IFITM2, and IFITM3 impact syncytia formation. We generated S-Fuse cells stably producing each of the four proteins. Their expression was verified by flow cytometry or Western blotting (Fig EV1). We then compared their sensitivity to SARS-CoV-2 infection. The presence of TMPRSS2 increased the appearance of fused cells by 5- to 10-fold (Fig 2A). On the contrary, IFITM1 significantly inhibited syncytia formation (Fig 2B and Movie EV1). Therefore, TMPRSS2 and IFITM1 exert opposite effects on syncytia formation. IFITM2 and 3 were poorly active (Fig 2B), probably because they mostly accumulate within the endosomal compartment, which limits their ability to alter fusion events occurring at the plasma membrane. Since only 40% of each cell population expressed high levels of IFITM, as assessed by flow cytometry (Fig EV1) and immunofluorescence (Fig 2C), we asked whether IFITM+ cells were present in the syncytia. A co-staining with anti-IFITM antibodies indicated that syncytia did not incorporate IFITM1+ cells present in the culture, whereas this was not the case for IFITM2 and 3 (Figs 2C and EV3). Moreover, the inhibitory effect of IFITM1 on syncytia was associated a decreased number of S+ cells (Fig 2D). Click here to expand this figure. Figure EV1. ACE2 and IFITM expression in U2OS-ACE2 cell derivatives A. ACE2 expression assessed by Western blot in U2OS-ACE2 GFP-split cells expressing or not IFITM1. Cell lysates were analyzed by for ACE2 and actin as a loading control. B–E. Analysis of ACE2 and IFITMs levels by flow cytometry B. Gating strategy. Cells were gated by size and granularity. Positive and negative gates were then set on control U2OS cells lacking the protein of interest. C. ACE2 levels on parental U2OS (control) and various IFITM derivatives. D. IFITM1 levels on parental U2OS (control) and U2OS-ACE2 IFITM1 cells. E. IFITM2 and IFITM3 levels on parental U2OS (control) and U2OS-ACE2 IFITM2 and U2OS-ACE2 IFITM3 cells. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Image quantification methodology A. Quantification of fusion and viability. To measure the extent of cell–cell fusion, the GFP area was automatically delimited, measured, and then divided by the total cell area. For cell viability, nuclei were automatically counted, and the total number of nuclei per well was normalized to that of non-infected control cells. B. Quantification of syncytia expressing IFITMs. For IFITM-GFP overlap quantification, the IFITM+ area was first selected on the 647 nm channel (image 2) and the GFP-positive area was quantified within the IFITM+ area (image 3). Image 4 shows the overlap area in gray for simpler visualization. C. Quantification of infected cells expressing the S protein. The cells were stained with anti-S antibodies, and the nuclei were detected with Hoechst. The S+ area was delimited (each selected object is pseudo-colored). Nuclei present within the S+ area were scored and divided by the total number of nuclei, to calculate the number of infected cells per well. Data information: Scale bars: 100 µm. The same field was used in this example for A and C, Hoechst images are therefore identical in A and C. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Expression of IFITM1, IFITM2, and IFITM3 in SARS-CoV-2-infected syncytiaSARS-CoV-2-induced syncytia in U2OS cells are IFITM1 dim or negative, but IFITM2/3 positive. Left: Non-infected U2OS-ACE2 cells. Middle and right: Cells infected with SARS-CoV-2 at the indicated MOI. The GFP area is delimited in white. With IFITM1, the fused GFP area does not overlap with the red IFITM1 area. For IFITM2 and IFITM3, an overlap with the GFP is visible. Scale bars: 100 µm. Quantifications are presented Fig 2C. Download figure Download PowerPoint We next assessed whether other cell types form syncytia upon SARS-CoV-2 infection. To this aim, we used 293T cells transiently transfected with ACE2 and also generated stable A549-ACE2 cells. The two cell lines readily formed syncytia upon infection (Fig EV4A and B). In order to rule out the possibility that syncytia formation is solely dependent on ACE2 over-expression, we investigated the naturally permissible Vero cells with the GFP-split system. We did not detect fused infected Vero cells (Fig EV5A); thus, we used as donors U20S-ACE2-infected cells that we co-cultivated with uninfected Vero cells. Numerous heterocellular syncytia were formed in a short period of time (8 h) (Fig EV5D). The ability of Vero cells to fuse was again confirmed when donor 293T cells were transfected with S and co-cultivated with Vero E6 acceptor cells (Fig EV5C). Additionally, Vero cells are also capable of forming syncytia upon transfection of only the S protein (Fig EV5B). Of note, Caco2 cells did not fuse upon SARS-CoV-2 infection (Fig EV4C). Taken together, our data strongly suggest that the ability to form syncytia upon SARS-CoV-2 infection is dependent on cell type as well as on the surface levels of S and ACE2. Fusion is detected in Vero cells with endogenous levels of ACE2. Click here to expand this figure. Figure EV4. Characterization of SARS-CoV-2-induced syncytia in different cell lines A. 293T-GFP1-10 and -GFP11 (1:1 ratio) were transfected with ACE2 and infected with SARS-CoV-2 at the indicated MOIs. Cell fusion was visualized by the GFP+ area 18 h post-infection. One representative image per condition is shown, and GFP area is delimited in white. B. A549-ACE2 cells were infected with SARS-CoV-2 at the indicated MOIs for 24 h and stained with anti-S antibodies and Hoechst. One representative image per condition is shown, and syncytia are manually delimited in white. C. Caco2 cells were infected with SARS-CoV-2 at the indicated MOIs for 24 h and stained for ZO1 tight junction marker, Hoechst and S. No clear syncytia formation was observed at any of the MOIs tested. Data information: Scale bar: 50 µm. Data are representative of three independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Characterization of syncytia formation in Vero cells A. Vero-GFP1-10 and -GFP11 (1:1 ratio) were infected with SARS-CoV-2 for 24 h and stained with anti-S antibodies and Hoechst. Cell fusion was visualized by the GFP+ area. No syncytia were observed in infected Vero cells. B. Vero-GFP1-10 and -GFP11 (1:1 ratio) were transfected with S expression plasmid and cell fusion was visualized by the GFP+ area at 18 h post-transfection. S expression induced extensive syncytia formation. C. S-expressing 293T GFP11 (Donor cells) were co-cultured overnight with Vero GFP1-10 (Acceptor cells). Cell fusion was quantified by measuring the GFP+ area. S-expressing 293Ts readily fuse with Vero cells. D. SARS-CoV-2-infected U2OS ACE2+ GFP1-10 (Donor cells) were co-cultured for 8 h with Vero GFP11 (Acceptor cells). Cell fusion was quantified by measuring the GFP+ area. Infected U2OS fuse with Vero cells. Data information: Scale bar: 50 µm. Representative images of three independent experiments. Download figure Download PowerPoint Syncytia formation by S-expressing cells We further characterized the mechanisms of fusion and its regulation by IFITMs and TMPRSS2. We asked whether S alone was sufficient to trigger fusion by transfecting an expression plasmid in 293-T cells harboring the GFP-Split system (Fig 3A). Many large and multinucleated GFP+ cells were detected, when ACE2 was co-expressed (Fig 3B). S-mediated cell fusion was significantly decreased when cells were co-transfected with flag-tagged IFITM1, 2, or 3 plasmids, compared with a control plasmid (Fig 3B and C). IFITM1 was slightly more inhibitory than IFITM2 and 3 in this system. The transient expression of TMPRSS2 enhanced fusion by 2.5-fold. Interestingly, when the serine protease was present, the inhibitory effect of IFITMs was no longer visible (Fig 3B and C). The anti-fusogenic effect of IFITM1 varied with the amount of transfected IFITM1 plasmid, but TMPRSS2 counteracted IFITM1 at all doses (Fig EV6A–C). We next measured the kinetics of S-mediated cell fusion by live video-microscopy, monitoring in real-time the GFP+ area. TMPRSS2 accelerated the appearance of GFP+ cells, indicating that it increases the speed of cell–cell fusion (Movie EV2 and Fig 3D). At 12 h post-transfection, the syncytia area was already fourfold larger than in the control condition (Fig 3D). A similar kinetic analysis indicated that IFITM1 strongly inhibited fusion, whereas IFITM2 and IFITM3 were less efficient (Fig 3E). In the presence of TMPRSS2, the rapid fusion kinetics were similar with or without IFITMs (Fig 3E). Figure 3. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells A. 293T-GFP1-10 and -GFP11 cells (1:1 ratio) were co-transfected with S, ACE2, TMPRSS2, IFITM, or control plasmids. Cell fusion was quantified by measuring the GFP+ area by high-content imaging after 18 h. (B and C) or analyzed over time by video microscopy (D and E). B. Representative images of cell–cell fusion. Scale bar: 100 µm. C. Quantification of GFP+ areas. Results are mean ± SD from five independent experiments. D. TMPRSS2 accelerates fusion. Cells were monitored by video microscopy, and the GFP area was quantified over time. Left panel: one representative experiment. Results are mean ± SD from three fields per condition. Right panel: Mean ± SD from seven independent experiments (at 12 h post-transfection). E. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells. One representative out of three independent experiment is shown. Data information: Statistical analysis: B, C: One-way ANOVA, ns: non-significant, ***P < 0.001, ****P < 0.0001. D: Wilcoxon matched-pairs signed-rank test, **P < 0.01. Download figure Download PowerPoint Click here to expand this figure. Figure EV6. Dose-response analysis of IFITM1 activity and impact of TMPRSS2 on IFITM1, 2, and 3 levels measured by Western blot A. 293T-GFP1-10 and -GFP11 (1:1 ratio) were co-transfected with S, ACE2, TMPRSS2, and increasing amounts of IFITM1 plasmids. Cell fusion was quantified by measuring the GFP+ area by high-content imaging at 18 h post-transfection B. Representative images of cell–cell fusion, with the indicated amounts of transfected plasmids. Scale bar: 100 µm. C. Quantification of GFP+ area. Results are mean ± SD from three independent experiments. Statistical analysis: One-way ANOVA, ns: non-significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. D. 293T cells were co-transfected with TMPRSS2 and IFITM1, IFITM2, IFITM3, or control plasmids, and analyzed 18 h post-transfection by Western blot. TMPRSS2 does not cleave or reduce the levels of IFITMs. Download figure Download PowerPoint We next studied whether IFITMs and TMPRSS2 impact cell–cell fusion by acting on S-expressing cells (“donor cells”), on ACE2-expressing cells (“acceptor cells”) or on both. To this end, we used a co-culture system of 293T-GFP1-10 donor cells with 293T-GFP11 acceptor cells. IFITMs and TMPRSS2 were transfected into either donor or acceptor cells (Fig 4A). In the absence of TMPRSS2, IFITMs were poorly efficient when present in donor cells, but inhibited fusion in acceptor cells. As already observed, IFITM1 was more active than IFITM2 and IFITM3. When TMPRSS2 was present in donor cells, IFITMs lost the weak effect they displayed when they were also in donor cells but retained their ability to inhibit fusion when expressed in acceptor cells (Fig 4A). Finally, when TMPRSS2 was expressed in acceptor cells, inhibition of fusion by IFITM was abolished regardless of their side of expression (Fig 4A). Therefore, IFITMs and TMPRSS" @default.
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• W4206142017 title "Syncytia formation by SARS‐CoV‐2‐infected cells" @default.
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