On the consequences of cell fusion in COVID-19

This commentary proposes how the coronavirus SARS-CoV-2 can contribute to clinical manifestations of COVID-19s by producing syncytia.

A hallmark of severe COVID-19 is the abundance of syncytia, the products of fusion between two or more cells, in the lungs of the patients (Buchrieser et al., 2020;Bussani et al., 2020;Braga et al., 2021). These syncytia were attributed to the ability of spike, a protein encoded by SARS-CoV-2, to fuse cells to each other, and prompted a search for already approved drugs that prevent this fusion. In a recent report (Braga et al., 2021), some of such were found to inhibit TMEM16F, a protein that has two activities: a calcium-activated ion channel that regulates chloride secretion, and a scramblase, a lipid transporter that relocates phosphatidylserine (PS) to the cell surface (Scudieri et al., 2015) in a process known as PS externalization.
PS externalization is required for cell fusion in many systems (Brukman et al., 2019;Zhang et al., 2020), which has explained why inhibiting a scramblase prevented the formation of spikeinduced syncytia. However, the report has concluded that although PS externalization "is required for plasma membrane fusion, chloride secretion might have relevance in COVID-19 pathogenesis" (Braga et al., 2021). This assumption, that the scramblase activity merely helped to identify the ion channel activity as a potential therapeutic target, reflects a common opinion that syncytia made in the body by infectious viruses are inconsequential.
To evaluate this assumption let us consider how syncytia and cell fusion that creates them might be involved in COVID-19.

Cell fusion as a cause of conditions associated with the activation of the blood coagulation cascade
COVID-19 is associated with widespread thrombosis (Hanff et al., 2020). Discovering syncytia in COVID-19 patients led to a suggestion that "the fusogenic properties of the MERS-CoV-and SARS-CoV-2-infected cells might be linked to the pathogenesis of thrombosis" (Bussani et al., 2020).
What could this link be?
I would like to suggest as a candidate the scramblase activity induced by spike-mediated fusion because PS externalization resulting from this activity not only enables cell fusion but also controls the rate limiting steps of the blood coagulation cascade (Zwaal, Comfurius and Bevers, 1998;Grover and Mackman, 2018;Smith and Morrissey, 2019) (Fig. 1). In agreement with this role, a deficiency of TMEM16F, the scramblase induced by spike, is responsible for Scott syndrome, a bleeding disorder (Suzuki et al., 2010).Viruses and the coagulation cascade. Viral infections activate the coagulation cascade primarily by triggering the assembly of extrinsic tenase, a complex between Tissue Factor (TF) and Factor VIIa, on the outer surface of the cellular membrane (Mackman et al., 2020) (Fig. 1). Full activation of the tenase requires PS externalization (Antoniak and Mackman, 2014), which, in synergy with TF binding, increases Factor VIIa activity by eight orders of magnitude (Bom and Bertina, 1990;Smith, Travers and Morrissey, 2015).
Since FVIIa and its precursor FVII are always present in the blood, the limiting steps for fully activating FVIIa are PS externalization and the presence of TF capable of binding and activating FVIIa. Accordingly, TF has been scrutinized as a potential target for COVID-19 therapy (Cañas et al., 2021). TF is regulated by controlling its expression, by tissue factor pathway inhibitor (TFPI), and by priming TF through a process known as de-encryption. The primary candidate for the de-encrypter is externalized PS [Grover 2018]. How PS externaliza-tion is induced in TF-expressing cells is unclear (Smith and Morrissey, 2019).
The hypothesis. The remarkable synergy of TF and externalized PS in activating FVIIa and the report that spike-induced syncytia externalize PS (Braga et al., 2021) together suggest a hypothesis that these syncytia can be a platform for assembling fully active extrinsic tenase capable of triggering blood coagulation (Fig. 2).
Whether this activation would result in clinically significant thrombosis would depend on whether syncytia express TF, which is likely because endothelial cells express TF during viral infections, and because the lungs are abundant in other cell types that express this protein (Grover and Mackman, 2018), whether syncytia come in contact with blood, whether they are large or abundant enough to assemble sufficient number of tenase complexes, which can be tested using available clotting assays, and the state of the coagulation system in a patient. Since even one thrombus can cause problems, the contribution of syncytia to COVID-19 could be clinically relevant.
Fusion at a distance. SARS-CoV-2 spike, as other viral fusogens, can fuse cells by two mechanisms. In the first, known as fusion from within, an infected cell begins to produce viral components, including spike, which is transported to the plasma membrane and can fuse it to the membrane of an adjacent cell if that cell has a spike receptor. Another mechanism, fusion from without, is executed by viral particles or lipid vesicles studded with spike that serve as a bridge between two cells (Gallaher and Bratt, 1972;Theuerkauf et al., 2021) (Fig. 2). In this case whether the virus is infectious or inactivated is irrelevant, as its ability to produce spike in the cell is not required.
Since SARS-Cov-2 infection induces the release of extracellular vesicles (also called microvesicles) in patients with moderate or severe COVID-19 (Rosell et al., 2021), and that SARS-Cov-2 spike can fuse cells if delivered on membrane vesicles (Theuerkauf et al., 2021), the site of thrombosis caused by syncytia can differ from that of infection.

Thrombosis by death.
Syncytia might also contribute to thrombosis by mechanism unrelated to the induction of a scramblase. A syncytium upon its demise would uncover a patch of the trombogenic basement membrane. Since even a single 20 micron fiber of collagen is sufficient to trigger platelet-dependent clotting (Zhu, Tomaiuolo and

Figure 1. An outline of the blood coagulation cascade
Blood coagulation cascade is a network of proteases, their precursors, cofactors, cells, enzymes, feedbacks, and feedforwards whose complexity and still unresolved questions make this outline by necessity rudimentary, with the primary goal to illustrate where the proteins that require binding to externalized PS (phosphatidylserine) for activation are in the network.
Most proteins involved in coagulation are called factors and are labeled by Roman numbers, such as Factor X or FX (hence enzymes that process FX are tenases). For simplicity, here the letter F is omitted. Activated factors are labeled with an a, as in FXa. Orange arrows represent proteolytic activity, grey arrow show a transition between forms. Blue horizontal lines represent cellular membrane with the cell surface facing down. Accordingly, the pinheads of externalized PS also face down. Note that most PS is actively relocated to face the cytoplasm unless the cell dies or the distribution is randomized by lipid scramblases.
As discussed in the text, the primary trigger of coagulation induced by viral infections is the extrinsic tenase (top left), which is a complex of TF (Tissue Factor) and FVIIa assembled on externalized PS in the presence of calcium ions. This tenase produces FXa to activate enough thrombin for generate the components of the intrinsic tenase, which increases FXa production and thus thrombin to yield enough fibrin for making a thrombus, which is a meshwork of polymerized and cross-linked fibrin with entrapped blood cells, primarily platelets, that is large and stiff enough to obstruct a blood vessel.
Note that TF is encrypted, that is unable to activate FVIIa, until it is de-encrypted by externalized PS (reviewed in (Grover and Mackman, 2018)).
Diamond, 2016), the patch exposed by sloughing of a syncytium made of more than several cells might be large enough to produce a thrombus.
Cell fusion and vaccines. The hypothesis that cell fusion induced by spike can trigger blood coagulation might also explain the rare cases of thrombosis following the injection of vaccines that use viral vectors or mRNA delivery vesicles to express SARS-CoV-2 spike in human cells. A current explanation for this phenomenon (Kowarz et al., 2021) states that expressing spike using adenoviral vectors, such as used in some of the vaccines, produces a soluble fragment of the protein that induces thrombosis through signaling pathways mediated by ACE2, a receptor of spike.
I would like to suggest that since spike transduced into cultured cells by viral vectors fuses cells even in undetectable amounts (Theuerkauf et al., 2021), it is possible the amounts produced in cells by the vehicles used in the vaccines would be sufficient to fuse some cells in humans. The extent of this fu-sion would depend on the level of spike expression, which is determined by the gene delivery vehicle used, the cell type of the cells transduced in the body, and whether these cells release extracellular vesicles that can fuse cells at sites distant to the site of injection (Rosell et al., 2021). Whether the resulting syncytia, if they arise, would cause any problems would depend on the number of syncytia, their size, and their access to blood. Should the hypothesis linking cell fusion to thrombosis be proven correct, or out of an abundance of caution, a solution would be to use spike mutants that are deficient in cell fusion or (and) in inducing scramblases.
Cell fusion and SARS-CoV-2 strains. While spike can fuse viruses to cells and cells to each other, the underlying mechanisms of these two activities are not identical. For example, sera from convalescent COVID-19 patients neutralize fusion of the virus to cells but fail to prevent the fusion of cells (Theuerkauf et al., 2021). Likewise, modifying the spike of SARS-CoV, which causes severe Figure 2. Syncytia induced by SARS-CoV-2 spike as a platform for triggering blood coagulation cascade. SARS-CoV-2 is covered by an envelope, which is fused to the cell membrane by spike once this protein binds one of its receptors an is activated by a membrane protease (both are not shown for simplicity). The infected cell produces viral components, including spike. Now, spike can fuse the membrane of the host cell with the membrane of an adjacent cell if that cell also has a spike receptor. Braga and colleagues (Braga et al., 2021) found that spike-induced cell fusion is associated with activation of TMEM16F, a scramblases that externalizes PS. This commentary proposes that PS externalized by spike enables the formation of the extrinsic tenase (Fig. 1), the key trigger of blood coagulation cascade during viral infections. (Theuerkauf et al., 2021), like extracellular vesicles released by infected cells. This mechanism is known as fusion from without, as the viral particle or a vesicle provides a bridge between the membranes. Syncytia produced by this mechanism would have no trace of SARS-CoV-2.

SARS-CoV-2 spike can also fuse cells if the virus is not infectious, or even if spike is incorporated into membrane vesicles
Note that TF is encrypted, that is unable to activate FVIIa, until it is de-encrypted by externalized PS (reviewed in (Grover and Mackman, 2018)). acute respiratory syndrome, to enable maturation of this protein by furin, a protease that also processes SARS-CoV-2 spike, increased the ability of SARS-CoV to fuse cells manyfold with little effect on virus-cell fusion (Follis, York and Nunberg, 2006). Finally, a single mutation in a porcine coronavirus spike enables this protein to cause cell-cell fusion at barely detectably amounts without affecting the ability of the virus to infect (Wanitchang et al., 2019). These observations mean that some strains of SARS-CoV-2 can be equally infective but differ in how well they fuse cells and thus in the consequences of this fusion.

Inflammation and fibrosis.
Besides mediating blood clotting, the coagulation cascade is interwoven with signaling pathways regulating inflammation, fibrosis and some other conditions associated with COVID-19 (Sriram and Insel, 2021). Therefore, if syncytia produced by spike trigger blood coagulation cascade, this activity would contribute to COVID-19 beyond inducing thrombosis.
Overall, the outlined possibilities suggest that syncytia produced by spike might be another wrench thrown into the gears of the human body by SARS-CoV-2. Yet, as we are about to discuss, the potential ability of the syncytia to trigger the blood coagulation cascade might be only one of many abnormalities associated with these cells.

Syncytia and other products of cell fusion are heterogeneous abnormal cell types with emergent properties.
In the lungs of COVID-19 patients, SARS-CoV-2 infects, and thus can fuse, ciliated cells in the airway, alveolar type 2 pneumocytes, and epithelial progenitors among others (Buchrieser et al., 2020). What are the properties of, say, syncytia that united pneumocytes with an epithelial progenitor? What happens if a leukocyte or another cell happens to join in? Because spike receptors ACE2 (Zhou et al., 2020), CD147 Neuropilin-1 (Cantuti-Castelvetri et al., 2020) are present in a variety of cell types, the diversity of cells SARS-CoV-2 can produce might be substantial.
Moreover, SARS-CoV-2 may be not the only cause of syncytia in COVID-19. For example, HERV-W ENV, the fusogen of an endogenous retrovirus, was detected in the leukocytes of COVID-19 patients at concentrations that exceeded that in the cells from healthy donors by orders of magnitude (Balestrieri et al., 2021). HERV-W ENV is also known as syncytin-1 because it mediates the fusion of trophoblasts into syncytiotrophoblast and can fuse other cells in culture if expressed ectopically (Blond et al., 2000). Should expression of syncytin-1 in leukocytes enable them to fuse with each other and other cells, the assortment of cell types in COVID-19 patients would increase even further.
Syncytia made by exogenous viruses are abnormal by definition because cell fusion in the body is normally restricted to a handful of physiological processes, such as fertilization, myogenesis, and the formation of osteoclasts, the cell that remodel bones (Brukman et al., 2019;Petrany and Millay, 2019).
What is known about the mechanisms of physiological fusionwhich is much less that one would expect given its function in the bodygives the impression that these mergers are planned and rehearsed down to detail to ensure that only the right cells fuse in a right time and at a right place and that, with the exception of fertilization and stem cell fusion, the resulting syncytia do not attempt to proliferate.
These sophisticated mechanisms, however, are overridden by many infectious viruses, including SARS CoV-2, that fuse cells randomly as long as the cells carry a cognate receptor (Hernández and Podbilewicz, 2017;Brukman et al., 2019). This randomness means that cell fusion induced by infectious viruses is a violent event that forcefully unites two or more finely tuned and specialized systems that just happened to be next to each other but may be quite different in their functions, gene expression patterns, cell cycle stage, age, activation status, and other features. All these features have to be reconciled in the syncytia and hybrids if they are to stay alive (Lazebnik, 2014).

What are the properties of abnormal syncytia?
The properties and the fates of non-physiological syncytia in the body remain practically unknown. However, observations made in experimental systems and by studying physiological syncytia provide some clues. One of them is that syncytia can become abnormal not only by combining distinct features of parental cells that are not found together in normal cell types, but also by having emergent properties that appear as a result of rec-onciling two underlying gene expression patterns (Koulakov and Lazebnik, 2012;Lazebnik, 2014).
For example, fusion of human bronchial epithelial cells to human multipotent stromal cells resulted in cells that appeared epithelial but failed to function properly because the two ion channel required to maintain bronchial and alveolar fluid balance were impaired due to changes in gene expression. One lacked a subunit, the other was improperly expressed (Curril et al., 2010). Likewise, bone marrow derived cells fused to hepatocytes in a mouse model of chronic liver damage yielded cells that differed in their gene expression patterns from both parental types and, unexpectedly, expressed cytokines and genes involved in neurotransmission and in the TGF-β pathway (Quintana-Bustamante et al., 2012). In an extreme case illustrating an enigmatic phenomenon called extinction, fusion of hepatoma cells to fibroblasts silenced hundreds of genes specific to either parental cell type, thus producing dedifferentiated cells (Bulla et al., 2010). Even fusion of cells belonging to the same cell type can produce syncytia with new properties, as it happens with osteoclasts, which resorb bone better than their mononuclear precursors (Yagi et al., 2005).
Given the outlined examples, it is not unreasonable to envision that some syncytia created by SARS CoV-2, associated viral infections, or induced endogenous viruses can produce cytokines or other signaling factors capable of deregulating tissue homeostasis either locally or even systemically, as it happens in COVID-19 during cytokine storm (Sriram and Insel, 2021). These cells might also become sanctuaries for the virus as it has been described for HIV (Symeonides et al., 2015;Bracq et al., 2018), or by evading immune surveillance, perhaps by fusing to immune cells.
What could abnormal syncytia do if they proliferate? While the published reports on COVID-19 discuss large syncytia, as these cells are the most noticeable products of cell fusion due to their size and numerous nuclei (thus often called multinucleated giant cells), they are not the only outcome of cell fusion. Cell fusion can produce binuclear or trinuclear cells, which are often more abundant in experimental systems than large syncytia but could go unnoticed in human tissues or not attributed to cell fusion because distinguishing them reliably from binuclear cells produced by failed mitosis is difficult or impossible with available tools (Gast et al., 2018). A syncytium, especially if it has only two or three nuclei, can also enter mitosis and produce mononuclear daughter cells, an outcome that might contribute to the pool of dysmorphic cells in the lung, another hallmark of COVID-19 (Bussani et al., 2020). Multipolar mitoses that are common in these mitoses are prone to produce aneuploid cells with chromosomal aberrations, adding another abnormal feature to the offspring of cell fusion (Duelli ets al., 2007), a phenomenon that may be particular relevant to patients with neoplastic lesions.
Fusion of proliferating cells introduces additional abnormalities by combining cells that are at different phases of the cell cycle. Because nuclei united in one cell can progress through the cell cycle independently, one nucleus can force the others to enter mitosis prematurely, causing fragmentation of their chromatin in a phenomenon known as chromatin pulverization (reviewed in (Stevens et al., 2010)). The resulting fragments can incorporate into the chromatin of the hybrid (Pantelias et al., 2019). The common features of chromosomal aberrations in cell hybrids and cancer cells (Ogle et al., 2004;Delespaul et al., 2019;Lartigue et al., 2020) along with the shared clonal heterogeneity of cancers and neoplastic cell hybrids, which enables their evolution (Miroshnychenko et al., 2021) has provided support for a long standing model that cell fusion is involved in cancer development, progression, recurrence, dormancy, and acquired drug resistance (reviewed in: (Duelli and Lazebnik, 2003;Parris, 2013;Shabo et al., 2020)). This model has been supported by recent reports of cell hybrids in human cancers (Gast et al., 2018;Laberge et al., 2019) and by multiple observations in animal models (reviewed in (Duelli and Lazebnik, 2003;Pawelek and Chakraborty, 2008;Noubissi and Ogle, 2016)). Whether any neoplastic hybrids found in humans are made by viral fusogens, as it has been suggested (Duelli et al., 2005;Parris, 2005;, is yet to be learned. This incomplete list of options that cell fusion can uniquely use to produce diverse abnormal cells suggests that drugs that target this largely unexplored biological process, including those identified by Braga and colleagues (Braga et al., 2021), might also be useful in conditions other than COVID-19. If true, discovering otherwise neglected syncytia in COVID-19 patients may help to dissect and control cell fusion and its consequences in 7 health and disease. After all, we tend to see only what we expect to see.