SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

Epithelial mechanics are maintained by inhibiting cell fusion with age in Drosophila.

A characteristic of normal aging and age-related diseases is the remodeling of the cellular organization of a tissue through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that abdominal epithelium of the adult fruit fly becomes polyploid with age through generation of multinucleated cells by cell fusion. Inhibition of fusion does not improve the lifespan of the fly, but does enhance its biomechanical fitness, a measure of the healthspan of the animal. Remarkably, Drosophila can maintain their epithelial tension and abdominal movements with age when cell fusion is inhibited. Epithelial cell fusion also appears to be dependent on a mechanical cue, as knockdown of Rho kinase, E-cadherin or α-catenin is sufficient to induce multinucleation in young animals. Interestingly, mutations in α-catenin in mice result in retina pigment epithelial multinucleation associated with macular disease. Therefore, we have discovered that polyploid cells arise by cell fusion and contribute to the decline in the biomechanical fitness of the animal with age.

Isolation of Primary Porcine Bronchial Epithelial Cells for Nipah Virus Infections.

The Malaysian strain of Nipah virus (NiV) first emerged in 1998/99 and caused a major disease outbreak in pigs and humans. While humans developed fatal encephalitis due to a prominent infection of brain microvessels, NiV-infected pigs mostly suffered from an acute respiratory disease and efficiently spread the infection via airway secretions. To elucidate the molecular basis of the highly productive NiV replication in porcine airways in vitro, physiologically relevant cell models that have maintained functional characteristics of airway epithelia in vivo are needed. Here, we describe in detail the method of isolating bronchial epithelial cells (PBEpC) from pig lungs that can be used for NiV infection studies. After the dissection of primary bronchia and removal of the mucus and protease digestion, bronchi segments are cut open and epithelial cells are scraped off and seeded on collagen-coated cell culture flasks. With this method, it is possible to isolate about 2 × 106 primary cells from the primary bronchi of one pig lung which can be cryopreserved or further subcultured. PBEpC form polarized monolayers on Transwell membrane inserts as controlled by immunostainings of epithelial marker proteins. NiV infection causes rapid formation of syncytia, allowing productive NiV infections in living PBEpC cultures to be monitored by phase-contrast microscopy.

Ultrastructural study confirms the formation of single and heterotypic syncytial cells in bronchoalveolar fluids of COVID-19 patients.

BACKGROUND: SARS-CoV-2 was reported to induce cell fusions to form multinuclear syncytia that might facilitate viral replication, dissemination, immune evasion, and inflammatory responses. In this study, we have reported the types of cells involved in syncytia formation at different stages of COVID-19 disease through electron microscopy. METHODS: Bronchoalveolar fluids from the mild (n = 8, SpO2 > 95%, no hypoxia, within 2-8 days of infection), moderate (n = 8, SpO2 90% to ≤ 93% on room air, respiratory rate ≥ 24/min, breathlessness, within 9-16 days of infection), and severe (n = 8, SpO2 < 90%, respiratory rate > 30/min, external oxygen support, after 17th days of infection) COVID-19 patients were examined by PAP (cell type identification), immunofluorescence (for the level of viral infection), scanning (SEM), and transmission (TEM) electron microscopy to identify the syncytia. RESULTS: Immunofluorescence studies (S protein-specific antibodies) from each syncytium indicate a very high infection level. We could not find any syncytial cells in mildly infected patients. However, identical (neutrophils or type 2 pneumocytes) and heterotypic (neutrophils-monocytes) plasma membrane initial fusion (indicating initiation of fusion) was observed under TEM in moderately infected patients. Fully matured large-size (20-100 µm) syncytial cells were found in severe acute respiratory distress syndrome (ARDS-like) patients of neutrophils, monocytes, and macrophage origin under SEM. CONCLUSIONS: This ultrastructural study on the syncytial cells from COVID-19 patients sheds light on the disease's stages and types of cells involved in the syncytia formations. Syncytia formation was first induced in type II pneumocytes by homotypic fusion and later with haematopoetic cells (monocyte and neutrophils) by heterotypic fusion in the moderate stage (9-16 days) of the disease. Matured syncytia were reported in the late phase of the disease and formed large giant cells of 20 to 100 µm.

Divergent regulation of auxin responsive genes in root-knot and cyst nematodes feeding sites formed in Arabidopsis.

Cysts (CNs) and root-knot nematodes (RKNs) induce specialized feeding cells, syncytia, and giant cells (GCs), respectively, within plant roots. The plant tissues around the GCs usually by respond forming a root swelling called a gall that contains the GCs. The ontogenesis of feeding cells is different. GC formation is a process of new organogenesis from vascular cells, which are still not well characterized, that differentiate into GCs. In contrast, syncytia formation involves the fusion of adjacent cells that have already differentiated. Nonetheless, both feeding sites show an auxin maximum pertinent to feeding site formation. However, data on the molecular divergences and similarities between the formation of both feeding sites regarding auxin-responsive genes are still scarce. We studied genes from the auxin transduction pathways that are crucial during gall and lateral root (LR) development in the CN interaction by using promoter-reporter (GUS/LUC)transgenic lines, as well as loss of function lines of Arabidopsis. The promoters pGATA23 and several deletions of pmiR390a were active in syncytia, as were in galls, but pAHP6 or putative up-stream regulators as ARF5/7/19 were not active in syncytia. Additionally, none of these genes seemed to play a key role during cyst nematode establishment in Arabidopsis, as the infection rates in loss of function lines did not show significant differences compared to control Col-0 plants. Furthermore, the presence of only canonical AuxRe elements in their proximal promoter regions is highly correlated with their activation in galls/GCs (AHP6, LBD16), but those promoters active in syncytia (miR390, GATA23) carry AuxRe overlapping core cis-elements for other transcription factor families (i.e., bHLH, bZIP). Strikingly, in silico transcriptomic analysis showed very few genes upregulated by auxins common to those induced in GCs and syncytia, despite the high number of upregulated IAA responsive genes in syncytia and galls. The complex regulation of auxin transduction pathways, where different members of the auxin response factor (ARF) family may interact with other factors, and the differences in auxin sensitivity, as indicated by the lower induction of the DR5 sensor in syncytia than galls, among other factors, may explain the divergent regulation of auxin responsive genes in the two types of nematode feeding sites.

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages.

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.

Spike-mediated viral membrane fusion is inhibited by a specific anti-IFITM2 monoclonal antibody.

The early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies.

Persistent SARS-CoV-2 infection in patients seemingly recovered from COVID-19.

SARS-CoV-2 infection is clinically heterogeneous, ranging from asymptomatic to deadly. A few patients with COVID-19 appear to recover from acute viral infection but nevertheless progress in their disease and eventually die, despite persistent negativity at molecular tests for SARS-CoV-2 RNA. Here, we performed post-mortem analyses in 27 consecutive patients who had apparently recovered from COVID-19 but had progressively worsened in their clinical conditions despite repeated viral negativity in nasopharyngeal swabs or bronchioalveolar lavage for 11-300 consecutive days (average: 105.5 days). Three of these patients remained PCR-negative for over 9 months. Post-mortem analysis revealed evidence of diffuse or focal interstitial pneumonia in 23/27 (81%) patients, accompanied by extensive fibrotic substitution in 13 cases (47%). Despite apparent virological remission, lung pathology was similar to that observed in acute COVID-19 individuals, including micro- and macro-vascular thrombosis (67% of cases), vasculitis (24%), squamous metaplasia of the respiratory epithelium (30%), frequent cytological abnormalities and syncytia (67%), and the presence of dysmorphic features in the bronchial cartilage (44%). Consistent with molecular test negativity, SARS-CoV-2 antigens were not detected in the respiratory epithelium. In contrast, antibodies against both spike and nucleocapsid revealed the frequent (70%) infection of bronchial cartilage chondrocytes and para-bronchial gland epithelial cells. In a few patients (19%), we also detected positivity in vascular pericytes and endothelial cells. Quantitative RT-PCR amplification in tissue lysates confirmed the presence of viral RNA. Together, these findings indicate that SARS-CoV-2 infection can persist significantly longer than suggested by standard PCR-negative tests, with specific infection of specific cell types in the lung. Whether these persistently infected cells also play a pathogenic role in long COVID remains to be addressed. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Multiple Sites on Glycoprotein H (gH) Functionally Interact with the gB Fusion Protein to Promote Fusion during Herpes Simplex Virus (HSV) Entry.

Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789Y in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs. IMPORTANCE Enveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known. This study examined the gH/gL-gB interaction using HSV-1 mutants that displayed reduced virus entry due to changes in gB. The mutant viruses were grown over time to select for additional mutations that could partially restore entry. Two mutations in gH (H789Y and S830N) were identified. The positions of the mutations in gH/gL may represent sites that contribute to gB activation during virus entry.

ADAM10 and ADAM17 promote SARS-CoV-2 cell entry and spike protein-mediated lung cell fusion.

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

Cell-Cell Fusion Mediated by Viruses and HERV-Derived Fusogens in Cancer Initiation and Progression.

Cell fusion is a well-known, but still scarcely understood biological phenomenon, which might play a role in cancer initiation, progression and formation of metastases. Although the merging of two (cancer) cells appears simple, the entire process is highly complex, energy-dependent and tightly regulated. Among cell fusion-inducing and -regulating factors, so-called fusogens have been identified as a specific type of proteins that are indispensable for overcoming fusion-associated energetic barriers and final merging of plasma membranes. About 8% of the human genome is of retroviral origin and some well-known fusogens, such as syncytin-1, are expressed by human (cancer) cells. Likewise, enveloped viruses can enable and facilitate cell fusion due to evolutionarily optimized fusogens, and are also capable to induce bi- and multinucleation underlining their fusion capacity. Moreover, multinucleated giant cancer cells have been found in tumors derived from oncogenic viruses. Accordingly, a potential correlation between viruses and fusogens of human endogenous retroviral origin in cancer cell fusion will be summarized in this review.

An easy method for developing fusion enabled SARS-CoV2 virus fusion mimic (SCFM), bypassing the need of Bio Safety Level (BSL) facility.

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.

COVID-19: the CaMKII-like system of S protein drives membrane fusion and induces syncytial multinucleated giant cells.

The SARS-CoV-2 S protein on the membrane of infected cells can promote receptor-dependent syncytia formation, relating to extensive tissue damage and lymphocyte elimination. In this case, it is challenging to obtain neutralizing antibodies and prevent them through antibodies effectively. Considering that, in the current study, structural domain search methods are adopted to analyze the SARS-CoV-2 S protein to find the fusion mechanism. The results show that after the EF-hand domain of S protein bound to calcium ions, S2 protein had CaMKII protein activities. Besides, the CaMKII_AD domain of S2 changed S2 conformation, facilitating the formation of HR1-HR2 six-helix bundles. Apart from that, the Ca2+-ATPase of S2 pumped calcium ions from the virus cytoplasm to help membrane fusion, while motor structures of S drove the CaATP_NAI and CaMKII_AD domains to extend to the outside and combined the viral membrane and the cell membrane, thus forming a calcium bridge. Furthermore, the phospholipid-flipping-ATPase released water, triggering lipid mixing and fusion and generating fusion pores. Then, motor structures promoted fusion pore extension, followed by the cytoplasmic contents of the virus being discharged into the cell cytoplasm. After that, the membrane of the virus slid onto the cell membrane along the flowing membrane on the gap of the three CaATP_NAI. At last, the HR1-HR2 hexamer would fall into the cytoplasm or stay on the cell membrane. Therefore, the CaMKII_like system of S protein facilitated membrane fusion for further inducing syncytial multinucleated giant cells.

Headless Henipaviral Receptor Binding Glycoproteins Reveal Fusion Modulation by the Head/Stalk Interface and Post-receptor Binding Contributions of the Head Domain.

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we demonstrate quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct C-terminal stalk lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3- to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

Communal living: the role of polyploidy and syncytia in tissue biology.

Multicellular organisms are composed of tissues with diverse cell sizes. Whether a tissue primarily consists of numerous, small cells as opposed to fewer, large cells can impact tissue development and function. The addition of nuclear genome copies within a common cytoplasm is a recurring strategy to manipulate cellular size within a tissue. Cells with more than two genomes can exist transiently, such as in developing germlines or embryos, or can be part of mature somatic tissues. Such nuclear collectives span multiple levels of organization, from mononuclear or binuclear polyploid cells to highly multinucleate structures known as syncytia. Here, we review the diversity of polyploid and syncytial tissues found throughout nature. We summarize current literature concerning tissue construction through syncytia and/or polyploidy and speculate why one or both strategies are advantageous.

Virus-Mediated Cell-Cell Fusion.

Cell-cell fusion between eukaryotic cells is a general process involved in many physiological and pathological conditions, including infections by bacteria, parasites, and viruses. As obligate intracellular pathogens, viruses use intracellular machineries and pathways for efficient replication in their host target cells. Interestingly, certain viruses, and, more especially, enveloped viruses belonging to different viral families and including human pathogens, can mediate cell-cell fusion between infected cells and neighboring non-infected cells. Depending of the cellular environment and tissue organization, this virus-mediated cell-cell fusion leads to the merge of membrane and cytoplasm contents and formation of multinucleated cells, also called syncytia, that can express high amount of viral antigens in tissues and organs of infected hosts. This ability of some viruses to trigger cell-cell fusion between infected cells as virus-donor cells and surrounding non-infected target cells is mainly related to virus-encoded fusion proteins, known as viral fusogens displaying high fusogenic properties, and expressed at the cell surface of the virus-donor cells. Virus-induced cell-cell fusion is then mediated by interactions of these viral fusion proteins with surface molecules or receptors involved in virus entry and expressed on neighboring non-infected cells. Thus, the goal of this review is to give an overview of the different animal virus families, with a more special focus on human pathogens, that can trigger cell-cell fusion.

Syncytia formation by SARS-CoV-2-infected cells.

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.

PRMT5 Is Required for Bovine Leukemia Virus Infection In Vivo and Regulates BLV Gene Expression, Syncytium Formation, and Glycosylation In Vitro.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle and is closely related to human T-cell leukemia viruses. We investigated the role of a new host protein, PRMT5, in BLV infection. We found that PRMT5 is overexpressed only in BLV-infected cattle with a high proviral load, but not in those with a low proviral load. Furthermore, this upregulation continued to the lymphoma stage. PRMT5 expression was upregulated in response to experimental BLV infection; moreover, PRMT5 upregulation began in an early stage of BLV infection rather than after a long period of proviral latency. Second, siRNA-mediated PRMT5 knockdown enhanced BLV gene expression at the transcript and protein levels. Additionally, a selective small-molecule inhibitor of PRMT5 (CMP5) enhanced BLV gene expression. Interestingly, CMP5 treatment, but not siRNA knockdown, altered the gp51 glycosylation pattern and increased the molecular weight of gp51, thereby decreasing BLV-induced syncytium formation. This was supported by the observation that CMP5 treatment enhanced the formation of the complex type of N-glycan more than the high mannose type. In conclusion, PRMT5 overexpression is related to the development of BLV infection with a high proviral load and lymphoma stage and PRMT5 inhibition enhances BLV gene expression. This is the first study to investigate the role of PRMT5 in BLV infection in vivo and in vitro and to reveal a novel function for a small-molecule compound in BLV-gp51 glycosylation processing.

Multiscale moving boundary modelling of cancer interactions with a fusogenic oncolytic virus: The impact of syncytia dynamics.

Oncolytic viral therapies is one of the new promising strategies against cancer, due to the ability of oncolytic viruses to specifically replicate inside cancer cells and kill them. There is increasing evidence that a sub-class of viruses that contain fusion proteins (triggering the formation of syncytia) can lead to better oncolytic results. Since the details of the tumour dynamics following syncytia formation are not fully understood, in this study we consider a modelling and computational approach to describe the effect of a fusogenic oncolytic virus on the multiscale dynamics of a spreading tumour. We show that for the baseline parameter values considered here, small syncytia diffusion coefficient leads to tumour reduction. Further tumour reduction can be obtained when we increase the probability of syncytia formation, in the context of different viral burst rates and death rates for individually-infected tumour cells and syncytia structures. Finally, we show that the type of syncytia diffusion coefficient (i.e., constant or density dependent) also impacts the outcome of the oncolytic viral therapy.

Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane.

Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia viruses, is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. The transmembrane subunit of the BLV envelope glycoprotein, gp30, contains three completely conserved YXXL sequences that fit an endocytic sorting motif. The two N-terminal YXXL sequences are reportedly critical for viral infection. However, their actual function in the viral life cycle remains undetermined. Here, we identified the novel roles of each YXXL sequence. Syncytia formation ability was upregulated by a single mutation of the tyrosine (Tyr) residue in any of the three YXXL sequences, indicating that each YXXL sequence is independently able to regulate the fusion event. The alteration resulted from significantly high expression of gp51 on the cell surface, thereby decreasing the amount of gp51 in early endosomes and further revealing that the three YXXL sequences are independently required for internalization of the envelope (Env) protein, following transport to the cell surface. Moreover, the 2nd and 3rd YXXL sequences contributed to Env protein incorporation into the virion by functionally distinct mechanisms. Our findings provide new insights regarding the three YXXL sequences toward the BLV viral life cycle and for developing new anti-BLV drugs.