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As life extended into eukaryota, a great host of strategies emerged in the pursuit of cellular life. Some cells have been successful in solitude, some moved into cooperatives (i.e., multicellular organisms), but one additional strategy emerged. Throughout eukaryotes, many of the diverse multicellular cooperatives took life in partnership one step further. These cells came together and lost their singularity in the expanse of syncytial life. Recently in our search for this elusive “how”, we discovered the intriguing peculiarity of a nuclear, RNA‐binding protein living a second life as a fusion manager at the surface of developing osteoclasts, ushering them into syncytia 1. It is from here that we will develop several thoughts about the advantages of multinucleated cells and discuss how these fusing cells pass through several hallmarks of cell death. We will propose that cell fusion shares much with cell death because cell fusion is a death of sorts for the cells that undergo it – a death of the life that was and the beginning of new life in a community without borders. [ABSTRACT FROM AUTHOR]

Simple Summary: Uveal melanoma (UM) is a rare type of cancer of the eye that develops from a cell type called melanocytes, which are present in the uveal tract. These patients have a high risk of liver metastasis, usually many years after the initial diagnosis, which is life threatening. We discovered a population of cells in the primary tumor called dual-nature hybrid cells (DNCs) similar to previously discovered cells in the blood (i.e., circulating hybrid cells, CHCs) with shared characteristics of both cancer cells and white blood cells. In this paper, we determined the types of white blood cells capable of participating in the formation of a hybrid cell with a cancer cell, and we re-created these DNCs in a cell culture. The study of the cells of this tumor, particularly DNCs, is crucial to better understanding dissemination and uncovering a possible target for treatment of this systemic disease. Background: Metastasis, occurring years after primary diagnosis, represents a poor prognosis in uveal melanoma (UM)-affected individuals. The nature of cells involved in this process is under debate. Circulating hybrid cells that have combined tumor and immune cell features found in blood were predictive of metastasis and may correspond to dual-nature cells (DNC) in the primary tumor. Herein, we sought to determine the presence of DNCs in primary UM tumors, the cell types involved in their genesis, and their ability to be formed in vitro. Methods: UM lesions (n = 38) were immunolabeled with HMB45 in combination with immune-cell-specific antibodies. In parallel, we co-cultured UM cells and peripheral blood mononuclear cells (PBMCs) to analyze DNC formation. Results: HMB45+/CD45+ DNCs were present in 90% (26/29) of the tumors, HMB45+/CD8+ DNCs were present in 93% (26/28), and HMB45+/CD68+ DNCs were present in 71% (17/24). DNCs formed with CD8+ and CD68+ cells were positively correlated to the infiltration of their respective immune cells. Notably, UM cells were prone to hybridize with PBMCs in vitro. Conclusions: This phenotypical characterization of DNCs in UM demonstrates that CD8+ T-cells and macrophages are capable of DNC formation, and they are important for better understanding metastatic dissemination, thus paving the path towards novel therapeutic avenues. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection. [ABSTRACT FROM AUTHOR]

ABSTRACTSARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell–cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that “first-generation” Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while “second-generation” Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Oncolytic viruses have two anticancer functions: direct oncolysis and elicitation of antitumor immunity. We previously developed a novel fusogenic oncolytic vaccinia virus (FUVAC) from a non‐fusogenic vaccinia virus (VV) and, by remodeling the tumor immune microenvironment, we demonstrated that FUVAC induced stronger oncolysis and antitumor immune responses compared with non‐fusogenic VV. These functions depend strongly on cell–cell fusion induction. However, FUVAC tends to have decreased fusion activity in cells with low virus replication efficacy. Therefore, another combination strategy was required to increase cell–cell fusion in these cells. Histone deacetylase (HDAC) inhibitors suppress the host virus defense response and promote viral replication. Therefore, in this study, we selected an HDAC inhibitor, trichostatin A (TSA), as the combination agent for FUVAC to enhance its fusion‐based antitumor potential. TSA was added prior to FUVAC treatment of murine tumor B16‐F10 and CT26 cells. TSA increased the replication of both FUVAC and parental non‐fusogenic VV. Moreover, TSA enhanced cell–cell fusion and FUVAC cytotoxicity in these tumor cells in a dose‐dependent manner. Transcriptome analysis revealed that TSA‐treated tumors showed altered expression of cellular component‐related genes, which may affect fusion tolerance. In a bilateral tumor‐bearing mouse model, combination treatment of TSA and FUVAC significantly prolonged mouse survival compared with either treatment alone or in combination with non‐fusogenic VV. Our findings demonstrate that TSA is a potent enhancer of cell–cell fusion efficacy of FUVAC. [ABSTRACT FROM AUTHOR]

ZEB1 plays a pivotal role in epithelial-to-mesenchymal transition (EMT), (cancer) cell stemness and cancer therapy resistance. The M13HS tumor hybrids, which were derived from spontaneous fusion events between the M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg breast cancer cells, express ZEB1 and exhibit prospective cancer stem cell properties. To explore a possible correlation between the ZEB1 and stemness/ EMT-related properties in M13HS tumor hybrids, ZEB1 was knocked-out by CRISPR/Cas9. Colony formation, mammosphere formation, cell migration, invasion assays, flow cytometry and Western blot analyses were performed for the characterization of ZEB1 knock-out cells. The ZEB1 knock-out in M13HS tumor cells was not correlated with the down-regulation of the EMT-related markers N-CADHERIN (CDH2) and VIMENTIN and up-regulation of miR-200c-3p. Nonetheless, both the colony formation and mammosphere formation capacities of the M13HS ZEB1 knock-out cells were markedly reduced. Interestingly, the M13HS-2 ZEB1-KO cells harbored a markedly higher fraction of ALDH1-positive cells. The Transwell/ Boyden chamber migration assay data indicated a reduced migratory activity of the M13HS ZEB1-knock-out tumor hybrids, whereas in scratch/ wound-healing assays only the M13SH-8 ZEB1-knock-out cells possessed a reduced locomotory activity. Similarly, only the M13HS-8 ZEB1-knock-out tumor hybrids showed a reduced invasion capacity. Although the ZEB1 knock-out resulted in only moderate phenotypic changes, our data support the role of ZEB1 in EMT and stemness. [ABSTRACT FROM AUTHOR]

ABSTRACTThe rapid evolution of SARS-CoV-2 variants presents a constant challenge to the global vaccination effort. In this study, we conducted a comprehensive investigation into two newly emerged variants, BA.2.87.1 and JN.1, focusing on their neutralization resistance, infectivity, antigenicity, cell-cell fusion, and spike processing. Neutralizing antibody (nAb) titers were assessed in diverse cohorts, including individuals who received a bivalent mRNA vaccine booster, patients infected during the BA.2.86/JN.1-wave, and hamsters vaccinated with XBB.1.5-monovalent vaccine. We found that BA.2.87.1 shows much less nAb escape from WT-BA.4/5 bivalent mRNA vaccination and JN.1-wave breakthrough infection sera compared to JN.1 and XBB.1.5. Interestingly, BA.2.87.1 is more resistant to neutralization by XBB.1.5-monovalent-vaccinated hamster sera than BA.2.86/JN.1 and XBB.1.5, but efficiently neutralized by a class III monoclonal antibody S309, which largely fails to neutralize BA.2.86/JN.1. Importantly, BA.2.87.1 exhibits higher levels of infectivity, cell-cell fusion activity, and furin cleavage efficiency than BA.2.86/JN.1. Antigenically, we found that BA.2.87.1 is closer to the ancestral BA.2 compared to other recently emerged Omicron subvariants including BA.2.86/JN.1 and XBB.1.5. Altogether, these results highlight immune escape properties as well as biology of new variants and underscore the importance of continuous surveillance and informed decision-making in the development of effective vaccines.IMPORTANCEThis study investigates the recently emerged SARS-CoV-2 variants, BA.2.87.1 and JN.1, in comparison to earlier variants and the parental D614G. Varied infectivity and cell-cell fusion activity among these variants suggest potential disparities in their ability to infect target cells and possibly pathogenesis. BA.2.87.1 exhibits lower nAb escape from bivalent mRNA vaccinee and BA.2.86/JN.1-infected sera than JN.1 but is relatively resistance to XBB.1.5-vaccinated hamster sera, revealing distinct properties in immune reason and underscoring the significance of continuing surveillance of variants and reformulation of vaccines. Antigenic differences between BA.2.87.1 and other earlier variants yield critical information not only for antibody evasion but also for viral evolution. In conclusion, this study furnishes timely insights into the spike biology and immune escape of the emerging variants BA.2.87.1 and JN.1, thus guiding effective vaccine development and informing public health interventions.

Syncytium formation, i.e., cell-cell fusion resulting in the formation of multinucleated cells, is a hallmark of infection by paramyxoviruses and other pathogenic viruses. This natural mechanism has historically been a diagnostic marker for paramyxovirus infection in vivo and is now widely used for the study of virus-induced membrane fusion in vitro. However, the role of syncytium formation in within-host dissemination and pathogenicity of viruses remains poorly understood. The diversity of henipaviruses and their wide host range and tissue tropism make them particularly appropriate models with which to characterize the drivers of syncytium formation and the implications for virus fitness and pathogenicity. Based on the henipavirus literature, we summarized current knowledge on the mechanisms driving syncytium formation, mostly acquired from in vitro studies, and on the in vivo distribution of syncytia. While these data suggest that syncytium formation widely occurs across henipaviruses, hosts, and tissues, we identified important data gaps that undermined our understanding of the role of syncytium formation in virus pathogenesis. Based on these observations, we propose solutions of varying complexity to fill these data gaps, from better practices in data archiving and publication for in vivo studies, to experimental approaches in vitro.

ABSTRACTThe SARS-CoV-2 Omicron subvariants have dominated the pandemic due to their high transmissibility and immune evasion conferred by the spike mutations. The Omicron subvariants can spread by cell-free virus infection and cell–cell fusion, the latter of which is more effective but has not been extensively investigated. In this study, we developed a simple and high-throughput assay that provides a rapid readout to quantify cell–cell fusion mediated by the SARS-CoV-2 spike proteins without using live or pseudotyped virus. This assay can be used to identify variants of concern and to screen for prophylactic and therapeutic agents. We further evaluated a panel of monoclonal antibodies (mAbs) and vaccinee sera against D614G and Omicron subvariants, finding that cell–cell fusion is substantially more resistant to mAb and serum inhibition than cell-free virus infection. Such results have important implications for the development of vaccines and antiviral antibody drugs against cell–cell fusion induced by SARS-CoV-2 spikes.

ABSTRACTPorcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that has been reported to infect a variety of animals and even humans. Cell–cell fusion has been identified as an alternative pathway for the cell-to-cell transmission of certain viruses, but the ability of PDCoV to exploit this transmission model, and the relevant mechanisms, have not been fully elucidated. Herein, we provide evidence that cell-to-cell transmission is the main mechanism supporting PDCoV spread in cell culture and that this efficient spread model is mediated by spike glycoprotein-driven cell–cell fusion. We found that PDCoV efficiently spread to non-susceptible cells via cell-to-cell transmission, and demonstrated that functional receptor porcine aminopeptidase N and cathepsins in endosomes are involved in the cell-to-cell transmission of PDCoV. Most importantly, compared with non-cell-to-cell infection, the cell-to-cell transmission of PDCoV was resistant to neutralizing antibodies and immune sera that potently neutralized free viruses. Taken together, our study revealed key characteristics of the cell-to-cell transmission of PDCoV and provided new insights into the mechanism of PDCoV infection.

ABSTRACTContinuous emergence of the Omicron variant, along with its subvariants, has caused an increasing number of infections, reinfections, and vaccine-breakthrough infections, seriously threatening human health. Recently, several new Omicron subvariants, such as BA.5, BA.2.75, BA.4.6, and BF.7, bearing distinct mutation profiles in their spike (S) proteins, have significantly increased their capacity to evade vaccine-induced immunity and have shown enhanced infectivity and transmissibility, quickly becoming dominant sublineages. In this study, we found the S proteins of these Omicron subvariants to have 2- to 4-fold more efficient membrane fusion kinetics than that of the original Omicron variant (BA.1), indicating that these novel Omicron subvariants might possess increased pathogenicity. We also identified that peptide-based pan-CoV fusion inhibitors, EK1 and EK1C4, showed equal efficacy against membrane fusion mediated by S proteins of the noted Omicron subvariants and infection by their pseudoviruses. Additionally, either immune sera induced by wild-type (WT) SARS-CoV-2 RBD-based vaccine or BA.2 convalescent sera showed potent synergism with EK1 against both WT SARS-CoV-2 and various Omicron subvariants, further suggesting that EK1-based fusion inhibitors are promising candidates for development as clinical antiviral agents against the currently circulating Omicron subvariants.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Fusion-associated small transmembrane (FAST) proteins can promote cell fusion, alter membrane permeability and trigger apoptosis to promote virus proliferation in orthoreoviruses. However, it is unknown whether FAST proteins perform these functions in aquareoviruses (AqRVs). Non-structural protein 17 (NS17) carried by grass carp reovirus Honghu strain (GCRV-HH196) belongs to the FAST protein family, and we preliminarily explored its relevance to virus infection. NS17 has similar domains to FAST protein NS16 of GCRV-873, comprising a transmembrane domain, a polybasic cluster, a hydrophobic patch and a polyproline motif. It was observed in the cytoplasm and the cell membrane. Overexpression of NS17 enhanced the efficiency of cell-cell fusion induced by GCRV-HH196 and promoted virus replication. Overexpression of NS17 also led to DNA fragmentation and reactive oxygen species (ROS) accumulation, and it triggered apoptosis. The findings illuminate the functions of NS17 in GCRV infection, and provide a reference for the development of novel antiviral strategies.

Long COVID, also called post-acute sequelae of SARS-CoV-2, is characterized by a multitude of lingering symptoms, including impaired cognition, that can last for many months. This symptom, often called "brain fog", affects the life quality of numerous individuals, increasing medical complications as well as healthcare expenditures. The etiopathogenesis of SARS-CoV-2-induced cognitive deficit is unclear, but the most likely cause is chronic inflammation maintained by a viral remnant thriving in select body reservoirs. These viral sanctuaries are likely comprised of fused, senescent cells, including microglia and astrocytes, that the pathogen can convert into neurotoxic phenotypes. Moreover, as the enteric nervous system contains neurons and glia, the virus likely lingers in the gastrointestinal tract as well, accounting for the intestinal symptoms of long COVID. Fusogens are proteins that can overcome the repulsive forces between cell membranes, allowing the virus to coalesce with host cells and enter the cytoplasm. In the intracellular compartment, the pathogen hijacks the actin cytoskeleton, fusing host cells with each other and engendering pathological syncytia. Cell–cell fusion enables the virus to infect the healthy neighboring cells. We surmise that syncytia formation drives cognitive impairment by facilitating the "seeding" of hyperphosphorylated Tau, documented in COVID-19. In our previous work, we hypothesized that the SARS-CoV-2 virus induces premature endothelial senescence, increasing the permeability of the intestinal and blood–brain barrier. This enables the migration of gastrointestinal tract microbes and/or their components into the host circulation, eventually reaching the brain where they may induce cognitive dysfunction. For example, translocated lipopolysaccharides or microbial DNA can induce Tau hyperphosphorylation, likely accounting for memory problems. In this perspective article, we examine the pathogenetic mechanisms and potential biomarkers of long COVID, including microbial cell-free DNA, interleukin 22, and phosphorylated Tau, as well as the beneficial effect of transcutaneous vagal nerve stimulation. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell–cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike's fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike's fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Cell fusion takes place in all domains of life and contributes greatly to the formation of complex multicellular structures. In particular, many fungi, such as the filamentous Neurospora crassa, rely on conspecific somatic cell fusion to drive the unicellular-to-multicellular transition and formation of the interconnected mycelial syncytium. This can, however, lead to the transmission of infectious elements and deleterious genotypes that have a negative impact on the organismal fitness. Accumulating evidence obtained from natural populations suggests that N. crassa has evolved various self/non-self or allorecognition systems to avoid fusion between genetically non-identical spores or hyphae at all costs. Here we present an overview of the recent advances made in the field of fungal allorecognition, describe its genetic basis, and comment on its evolutionary meaning. These data pinpoint the multilayered complexity of the cooperative social behaviors undertaken by a model eukaryotic microbe.

The ubiquitously-expressed proteolytic enzyme furin is closely related to the pathogenesis of SARS-CoV-2 and therefore represents a key target for antiviral therapy. Based on bioinformatic analysis and pseudovirus tests, we discovered a second functional furin site located in the spike protein. Furin still increased the infectivity of mutated SARS-CoV-2 pseudovirus in 293T-ACE2 cells when the canonical polybasic cleavage site (682–686) was deleted. However, K814A mutation eliminated the enhancing effect of furin on virus infection. Furin inhibitor prevented infection by 682–686-deleted SARS-CoV-2 in 293T-ACE2-furin cells, but not the K814A mutant. K814A mutation did not affect the activity of TMPRSS2 and cathepsin L but did impact the cleavage of S2 into S2′ and cell–cell fusion. Additionally, we showed that this functional furin site exists in RaTG13 from bat and PCoV-GD/GX from pangolin. Therefore, we discovered a new functional furin site that is pivotal in promoting SARS-CoV-2 infection.

SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell–cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.

Viruses can induce the fusion of infected and neighboring cells, leading to the formation of syncytia. Cell–cell fusion is mediated by viral fusion proteins on the plasma membrane of infected cells that interact with cellular receptors on neighboring cells. Viruses use this mechanism to spread rapidly to adjacent cells or escape host immunity. For some viruses, syncytium formation is a hallmark of infection and a known pathogenicity factor. For others, the role of syncytium formation in viral dissemination and pathogenicity remains poorly understood. Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. Clinical HCMV isolates have broad cell tropism but differ in their ability to induce cell–cell fusions, and little is known about the molecular determinants. We developed a system to analyze HCMV glycoprotein B (gB) variants in a defined genetic background. HCMV strains TB40/E and TR were used as vectors to compare the fusogenicity of six gB variants from congenitally infected fetuses with those from three laboratory strains. Five of them conferred the ability to induce the fusion of MRC-5 human embryonic lung fibroblasts to one or both backbone strains, as determined by a split GFP–luciferase reporter system. The same gB variants were not sufficient to induce syncytia in infected ARPE-19 epithelial cells, suggesting that additional factors are involved. The system described here allows a systematic comparison of the fusogenicity of viral envelope glycoproteins and may help to clarify whether fusion-promoting variants are associated with increased pathogenicity. [ABSTRACT FROM AUTHOR]

Wild‐type herpes simplex virus (HSV) strains infrequently mediate cell–cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell–cell fusion induced by wild‐type HSV strains. The established system clarified that the HSV‐1 envelope glycoprotein B and its N‐glycosylation at asparagine at position 141 were required for efficient cell–cell fusion. This study provides a link between cell–cell fusion induced by wild‐type HSV‐1 and viral pathogenesis in vivo. [ABSTRACT FROM AUTHOR]

Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies.

Mammalian placenta formation requires continuous fusion of trophoblasts. Human endogenous retrovirus-derived proteins syncytin-1 and syncytin-2 mediate cell–cell fusion of placental cytotrophoblasts to form syncytiotrophoblasts in primates, which is required for normal placenta function and fetal development. Syncytins are post-translationally cleaved by the endoprotease furin into surface (SU) and transmembrane (TM) subunits for activation. Little is currently known about the molecular mechanisms of syncytin-mediated cell–cell fusion, and their functions have not been well studied in vitro. Here, we express tagged syncytin-2 in mammalian HEK293T cells and demonstrate that the tagging greatly influences the cleavage and fusogenic activity of syncytin-2. By detecting the N-terminal tagged SU, we find that it is released into the extracellular space during the fusion process. Furthermore, when N-linked glycosylation and disulfide bond formation are blocked, the cleavage and fusogenic activity of syncytin-2 are inhibited. Finally, we were able to purify functional syncytin-2 from HEK293T cells and incorporate it into proteoliposomes. These findings lay a solid foundation for interogating the molecular mechanisms of syncytin-2-mediated cell–cell fusion in vitro. [ABSTRACT FROM AUTHOR]

In this review, we consider the role of cell-cell fusion in cancer development and progression through an evolutionary lens. We begin by summarizing the origins of fusion proteins (fusogens), of which there are many distinct classes that have evolved through convergent evolution. We then use an evolutionary framework to highlight how the persistence of fusion over generations and across different organisms can be attributed to traits that increase fitness secondary to fusion; these traits map well to the expanded hallmarks of cancer. By studying the tumor microenvironment, we can begin to identify the key selective pressures that may favor higher rates of fusion compared to healthy tissues. The paper concludes by discussing the increasing number of research questions surrounding fusion, recommendations for how to answer them, and the need for a greater interest in exploring cell fusion and evolutionary principles in oncology moving forward. [Display omitted] • Cell-cell fusion is a known phenomenon that characterizes human physiology as well as the pathological hallmarks of cancer. • Many of the known biochemical pathways that up-regulate fusion (via fusogen production) have been previously associated with tumorigenesis. • Several of these triggers of cell-cell fusion are key components of the tumor microenvironment (TME). • There is growing evidence that cell-cell fusion enables cancer hallmarks, and could become a cancer hallmark in its own right. [ABSTRACT FROM AUTHOR]

In addition to CD4+ T lymphocytes, cells of the myeloid lineage such as macrophages, dendritic cells (DCs), and osteoclasts (OCs) are emerging as important target cells for HIV‐1, as they likely participate in all steps of pathogenesis, including sexual transmission and early virus dissemination in both lymphoid and nonlymphoid tissues where they can constitute persistent virus reservoirs. At least in vitro, these myeloid cells are poorly infected by cell‐free viral particles. In contrast, intercellular virus transmission through direct cell‐to‐cell contacts may be a predominant mode of virus propagation in vivo leading to productive infection of these myeloid target cells. HIV‐1 cell‐to‐cell transfer between CD4+ T cells mainly through the formation of the virologic synapse, or from infected macrophages or dendritic cells to CD4+ T cell targets, have been extensively described in vitro. Recent reports demonstrate that myeloid cells can be also productively infected through virus homotypic or heterotypic cell‐to‐cell transfer between macrophages or from virus‐donor‐infected CD4+ T cells, respectively. These modes of infection of myeloid target cells lead to very efficient spreading in these poorly susceptible cell types. Thus, the goal of this review is to give an overview of the different mechanisms reported in the literature for cell‐to‐cell transfer and spreading of HIV‐1 in myeloid cells. [ABSTRACT FROM AUTHOR]

The messenger RNA (mRNA) vaccines for COVID-19, Pfizer-BioNTech and Moderna, were authorized in the US on an emergency basis in December of 2020. The rapid distribution of these therapeutics around the country and the world led to millions of people being vaccinated in a short time span, an action that decreased hospitalization and death but also heightened the concerns about adverse effects and drug-vaccine interactions. The COVID-19 mRNA vaccines are of particular interest as they form the vanguard of a range of other mRNA therapeutics that are currently in the development pipeline, focusing both on infectious diseases as well as oncological applications. The Vaccine Adverse Event Reporting System (VAERS) has gained additional attention during the COVID-19 pandemic, specifically regarding the rollout of mRNA therapeutics. However, for VAERS, absence of a reporting platform for drugvaccine interactions left these events poorly defined. For example, chemotherapy, anticonvulsants, and antimalarials were documented to interfere with the mRNA vaccines, but much less is known about the other drugs that could interact with these therapeutics, causing adverse events or decreased efficacy. In addition, SARS-CoV-2 exploitation of host cytochrome P450 enzymes, reported in COVID-19 critical illness, highlights viral interference with drug metabolism. For example, patients with severe psychiatric illness (SPI) in treatment with clozapine often displayed elevated drug levels, emphasizing drug-vaccine interaction. [ABSTRACT FROM AUTHOR]

The messenger RNA (mRNA) vaccines for COVID-19, Pfizer-BioNTech and Moderna, were authorized in the US on an emergency basis in December of 2020. The rapid distribution of these therapeutics around the country and the world led to millions of people being vaccinated in a short time span, an action that decreased hospitalization and death but also heightened the concerns about adverse effects and drug-vaccine interactions. The COVID-19 mRNA vaccines are of particular interest as they form the vanguard of a range of other mRNA therapeutics that are currently in the development pipeline, focusing both on infectious diseases as well as oncological applications. The Vaccine Adverse Event Reporting System (VAERS) has gained additional attention during the COVID-19 pandemic, specifically regarding the rollout of mRNA therapeutics. However, for VAERS, absence of a reporting platform for drug-vaccine interactions left these events poorly defined. For example, chemotherapy, anticonvulsants, and antimalarials were documented to interfere with the mRNA vaccines, but much less is known about the other drugs that could interact with these therapeutics, causing adverse events or decreased efficacy. In addition, SARS-CoV-2 exploitation of host cytochrome P450 enzymes, reported in COVID-19 critical illness, highlights viral interference with drug metabolism. For example, patients with severe psychiatric illness (SPI) in treatment with clozapine often displayed elevated drug levels, emphasizing drug-vaccine interaction.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell–cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell–cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell–cell fusion in Vero cells. [ABSTRACT FROM AUTHOR]

Herein we analyze two special routes of the multinucleated cells' formation – the fusion of mononuclear cells and the formation of cell-in-cell structures – in the healthy tissues and in tumorigenesis. There are many theories of tumorigenesis based on the phenomenon of emergence of the hybrid cancer cells. We consider the phenomena, which are rarely mentioned in those theories: namely, cellularization of syncytium or coenocytes, and the reversible or irreversible somatogamy. The latter includes the short-term and the long-term vegetative (somatic) cells' fusions in the life cycles of unicellular organisms. The somatogamy and multinuclearity have repeatedly and independently emerged in various groups of unicellular eukaryotes. These phenomena are among dominant survival and biodiversity sustaining strategies in protists and we admit that they can likely play an analogous role in cancer cells. [ABSTRACT FROM AUTHOR]

Pregnancy complications occur frequently and are particularly prevalent during the first trimester. They are caused by a multitude of factors, including karyotypic, genetic or environmental conditions, congenital infections and inflammation. The molecular mechanisms leading to placental complications under inflammatory conditions remain unclear. In this review, we discuss how uncontrolled inflammation, triggered by viral infections or other diseases can lead to placental complications. We first highlight the importance of syncytins, ancestral retroviral genes co-opted by mammals including humans, millions of years ago for the process of placenta formation. We then focus on recent advances elucidating how interferon-induced transmembrane (IFITM) proteins, antiviral proteins rendering cells refractory to viral infections, interfere with placental development.

Abstract Background Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell–cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. Results We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell–cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. Conclusions We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

ABSTRACT Sexual reproduction facilitates infection by the production of both a lineage advantage and infectious sexual spores in the ubiquitous human fungal pathogen Cryptococcus deneoformans. However, the regulatory determinants specific for initiating mating remain poorly understood. Here, we identified a velvet family regulator, Cva1, that strongly promotes sexual reproduction in C. deneoformans. This regulation was determined to be specific, based on a comprehensive phenotypic analysis of cva1Δ under 26 distinct in vitro and in vivo growth conditions. We further revealed that Cva1 plays a critical role in the initiation of early mating events, including sexual cell-cell fusion, but is not important for the late sexual development stages or meiosis. Thus, Cva1 specifically contributes to mating activation. Importantly, a novel mating-responsive protein, Cfs1, serves as the key target of Cva1 during mating, since its absence nearly blocks cell-cell fusion in C. deneoformans and its sister species C. neoformans. Together, our findings provide insight into how C. deneoformans ensures the regulatory specificity of mating. IMPORTANCE The human fungal pathogen C. deneoformans is a model organism for studying fungal sexual reproduction, which is considered to be important to infection. However, the specific regulatory determinants for activation of sexual reproduction remain poorly understood. In this study, by combining transcriptomic and comprehensive phenotypic analysis, we identified a velvet family regulator Cva1 that specifically and critically elicits early mating events, including sexual cell-cell fusion. Significantly, Cva1 induces mating through the novel mating-responsive protein Cfs1, which is essential for cell-cell fusion in C. deneoformans and its sister species C. neoformans. Considering that Cva1 and Cfs1 are highly conserved in species belonging to Cryptococcaeceae, they may play conserved and specific roles in the initiation of sexual reproduction in this important fungal clade, which includes multiple human fungal pathogens.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR 690 /S motif at the S2′ cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR 690 /S motif at the S2′ cleavage site induced low-level cell–cell fusion, while H855Q substitution hindered cell–cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines. • S2 subunit mutations are associated with the strain HV80 Vero cell adaptation. • The RRRR 690 /S motif at the S2′ cleavage site triggers the infection. • The Q855H substitution enhances infection efficiency through cell-cell fusion. • The H855Q substitution also blocks the cleavage event with S20 product. [ABSTRACT FROM AUTHOR]

Viruses can induce the fusion of infected and neighboring cells, leading to the formation of syncytia. Cell–cell fusion is mediated by viral fusion proteins on the plasma membrane of infected cells that interact with cellular receptors on neighboring cells. Viruses use this mechanism to spread rapidly to adjacent cells or escape host immunity. For some viruses, syncytium formation is a hallmark of infection and a known pathogenicity factor. For others, the role of syncytium formation in viral dissemination and pathogenicity remains poorly understood. Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. Clinical HCMV isolates have broad cell tropism but differ in their ability to induce cell–cell fusions, and little is known about the molecular determinants. We developed a system to analyze HCMV glycoprotein B (gB) variants in a defined genetic background. HCMV strains TB40/E and TR were used as vectors to compare the fusogenicity of six gB variants from congenitally infected fetuses with those from three laboratory strains. Five of them conferred the ability to induce the fusion of MRC-5 human embryonic lung fibroblasts to one or both backbone strains, as determined by a split GFP–luciferase reporter system. The same gB variants were not sufficient to induce syncytia in infected ARPE-19 epithelial cells, suggesting that additional factors are involved. The system described here allows a systematic comparison of the fusogenicity of viral envelope glycoproteins and may help to clarify whether fusion-promoting variants are associated with increased pathogenicity.

CS/ICs have raised great expectations in cancer research and therapy, as eradication of this key cancer cell type is expected to lead to a complete cure. Unfortunately, the biology of CS/ICs is rather complex, since no common CS/IC marker has yet been identified. Certain surface markers or ALDH1 expression can be used for detection, but some studies indicated that cancer cells exhibit a certain plasticity, so CS/ICs can also arise from non-CS/ICs. Another problem is intratumoral heterogeneity, from which it can be inferred that different CS/IC subclones must be present in the tumor. Cell–cell fusion between cancer cells and normal cells, such as macrophages and stem cells, has been associated with the generation of tumor hybrids that can exhibit novel properties, such as an enhanced metastatic capacity and even CS/IC properties. Moreover, cell–cell fusion is a complex process in which parental chromosomes are mixed and randomly distributed among daughter cells, resulting in multiple, unique tumor hybrids. These, if they have CS/IC properties, may contribute to the heterogeneity of the CS/IC pool. In this review, we will discuss whether cell–cell fusion could also lead to the origin of different CS/ICs that may expand the overall CS/IC pool in a primary tumor. [ABSTRACT FROM AUTHOR]

Variants of SARS‐CoV‐2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high‐frequency naturally occurring amino acid changes in the receptor‐binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2‐overexpressing 293T cells of different species, LLC‐MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high‐frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high‐frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies. Highlights: Infectivity increased by adding three VOCs of V367F, S494P, or A520S.The infectivity of the three VOCs with multiple high‐frequency mutations decreased.Almost all of the possible variants of the three VOCs did not show severe antigenic drift. [ABSTRACT FROM AUTHOR]

ABSTRACT The 380-to-393-amino-acid glycoprotein I (gI) encoded by herpes simplex virus 1 (HSV-1) is a critical mediator for viral cell-to-cell spread and syncytium formation. Here we report a previously unrecognized aberrant form of gI in HSV-1-infected cells. Production of this molecule is independent of cell type and viral strains. It had an unexpected gel migration size of approximately 23 kDa, was packaged into viral particles, and could be coimmunoprecipitated by antibodies to both N and C termini of gI. Deep sequencing failed to detect alternative RNA splicing, and the in vitro transcribed full-length mRNA gave rise to the 23 kDa protein in transfected cells. Combined mass spectrometry and antibody probing analyses detected peptide information across different regions of gI, suggesting the possibility of a full-length gI but with abnormal migration behavior. In line with this notion, the HA insertion mutagenesis revealed a stable fold in the gI extracellular region aa.38-196 resistant to denaturing conditions, whereas small deletions within this region failed the antibodies to detect the fast, but not the slow-moving species of gI. It is also intriguing that the structure could be perturbed to some extent by a gBsyn mutation, leading to exposure or shielding of the gI epitopes. Thus, the HSV-1 gI apparently adopts a very stable fold in its natural form, rendering it an unusual biophysical property. Our findings provide novel insight into the biological properties of HSV gI and have important implications in understanding the viral spread and pathogenesis. IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but its behavior during infection has remained poorly defined. Along with the classic 66 kDa product, here we report a previously unrecognized, approximately 23 kDa form of gI. Biochemical and genetics analyses revealed that this molecule represents the full-length form of gI but adopts a stable fold in its extracellular domain that is resistant to denatured conditions, thus contributing to the aberrant migration rate. Our results revealed a novel property of HSV-1 gI and have important implications in understanding viral pathogenesis.

SARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell-cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1 614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that "first-generation" Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while "second-generation" Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell-cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cellto-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell-cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spikemediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis. [ABSTRACT FROM AUTHOR]

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. [ABSTRACT FROM AUTHOR]

Cell fusion takes place in all domains of life and contributes greatly to the formation of complex multicellular structures. In particular, many fungi, such as the filamentous Neurospora crassa, rely on conspecific somatic cell fusion to drive the unicellular-to-multicellular transition and formation of the interconnected mycelial syncytium. This can, however, lead to the transmission of infectious elements and deleterious genotypes that have a negative impact on the organismal fitness. Accumulating evidence obtained from natural populations suggests that N. crassa has evolved various self/non-self or allorecognition systems to avoid fusion between genetically non-identical spores or hyphae at all costs. Here we present an overview of the recent advances made in the field of fungal allorecognition, describe its genetic basis, and comment on its evolutionary meaning. These data pinpoint the multilayered complexity of the cooperative social behaviors undertaken by a model eukaryotic microbe.

Fertilization is a multistep process that culminates in the fusion of sperm and egg, thus marking the beginning of a new organism in sexually reproducing species. Despite its importance for reproduction, the molecular mechanisms that regulate this singular event, particularly sperm–egg fusion, have remained mysterious for many decades. Here, we summarize our current molecular understanding of sperm–egg interaction, focusing mainly on mammalian fertilization. Given the fundamental importance of sperm–egg fusion yet the lack of knowledge of this process in vertebrates, we discuss hallmarks and emerging themes of cell fusion by drawing from well-studied examples such as viral entry, placenta formation, and muscle development. We conclude by identifying open questions and exciting avenues for future studies in gamete fusion. [ABSTRACT FROM AUTHOR]

Background: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell–cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. Results: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell–cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. Conclusions: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein. [ABSTRACT FROM AUTHOR]

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell–cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell–cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell–cell fusion in Vero cells.