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1
Leroy, Héloïse, Mingyu Han, Marie Woottum, Lucie Bracq, Jérôme Bouchet, Maorong Xie, and Serge Benichou. "Virus-Mediated Cell-Cell Fusion." International Journal of Molecular Sciences 21, no. 24 (December 17, 2020): 9644. http://dx.doi.org/10.3390/ijms21249644.
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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.
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Hernandez, L. D., L. R. Hoffman, T. G. Wolfsberg, and J. M. White. "VIRUS-CELL AND CELL-CELL FUSION." Annual Review of Cell and Developmental Biology 12, no. 1 (November 1996): 627–61. http://dx.doi.org/10.1146/annurev.cellbio.12.1.627.
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Zhang, Chuyuan, Xinjie Meng, and Hanjun Zhao. "Comparison of Cell Fusions Induced by Influenza Virus and SARS-CoV-2." International Journal of Molecular Sciences 23, no. 13 (July 1, 2022): 7365. http://dx.doi.org/10.3390/ijms23137365.
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Virus–cell fusion is the key step for viral infection in host cells. Studies on virus binding and fusion with host cells are important for understanding the virus–host interaction and viral pathogenesis for the discovery of antiviral drugs. In this review, we focus on the virus–cell fusions induced by the two major pandemic viruses, including the influenza virus and SARS-CoV-2. We further compare the cell fusions induced by the influenza virus and SARS-CoV-2, especially the pH-dependent fusion of the influenza virus and the fusion of SARS-CoV-2 in the type-II transmembrane serine protease 2 negative (TMPRSS2-) cells with syncytia formation. Finally, we present the development of drugs used against SARA-CoV-2 and the influenza virus through the discovery of anti-fusion drugs and the prevention of pandemic respiratory viruses.
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Podbilewicz, Benjamin. "Virus and Cell Fusion Mechanisms." Annual Review of Cell and Developmental Biology 30, no. 1 (October 11, 2014): 111–39. http://dx.doi.org/10.1146/annurev-cellbio-101512-122422.
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Muggeridge, Martin I. "Characterization of cell–cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells." Journal of General Virology 81, no. 8 (August 1, 2000): 2017–27. http://dx.doi.org/10.1099/0022-1317-81-8-2017.
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The mechanisms by which herpes simplex viruses (HSV) mediate fusion between their envelope and the plasma membrane during entry into cells, and between the plasma membranes of adjacent infected and uninfected cells to form multinucleated giant cells, are poorly understood. Four viral glycoproteins (gB, gD, gH and gL) are required for virus–cell fusion, whereas these plus several others are required for cell–cell fusion (syncytium formation). A better understanding would be aided by the availability of a model system, whereby fusion could be induced with a minimal set of proteins, in the absence of infection. A suitable system has now been developed for HSV-2, using transfected COS7, 293 or HEp-2 cells. Insofar as the minimal set of HSV-2 proteins required to cause cell–cell fusion in this system is gB, gD, gH and gL, it would appear to resemble virus–cell fusion rather than syncytium formation. However, the ability of a mutation in gB to enhance the fusion of both transfected cells and infected cells, while having no effect on virus–cell fusion, points to the opposite conclusion. The differential effects of a panel of anti-HSV antibodies, and of the fusion-inhibitor cyclosporin A, confirm that the fusion of transfected cells shares some properties with virus–cell fusion and others with syncytium formation. It may therefore prove useful for determining how these processes differ, and for testing the hypothesis that some viral proteins prevent membrane fusion until the appropriate point in the virus life-cycle, with other proteins then overcoming this block.
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Markosyan, Ruben M., Shan Lu Liu, and Fredric S. Cohen. "Cell-Cell Fusion Mediated by the Fusion Protein of Ebola Virus." Biophysical Journal 106, no. 2 (January 2014): 707a. http://dx.doi.org/10.1016/j.bpj.2013.11.3921.
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Connolly, Sarah A., and Robert A. Lamb. "Paramyxovirus fusion: Real-time measurement of parainfluenza virus 5 virus–cell fusion." Virology 355, no. 2 (November 2006): 203–12. http://dx.doi.org/10.1016/j.virol.2006.07.021.
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Schmid, Erik, Andreas Zurbriggen, Uta Gassen, Bert Rima, Volker ter Meulen, and Jürgen Schneider-Schaulies. "Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion." Journal of Virology 74, no. 16 (August 15, 2000): 7554–61. http://dx.doi.org/10.1128/jvi.74.16.7554-7561.2000.
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ABSTRACT Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.
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Zhou, Momei, Vivek Kamarshi, Ann M. Arvin, and Stefan L. Oliver. "Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion." PLOS Pathogens 16, no. 11 (November 20, 2020): e1009022. http://dx.doi.org/10.1371/journal.ppat.1009022.
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Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.
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Tsurudome, Masato, Machiko Nishio, Morihiro Ito, Shunsuke Tanahashi, Mitsuo Kawano, Hiroshi Komada, and Yasuhiko Ito. "Effects of Hemagglutinin-Neuraminidase Protein Mutations on Cell-Cell Fusion Mediated by Human Parainfluenza Type 2 Virus." Journal of Virology 82, no. 17 (June 18, 2008): 8283–95. http://dx.doi.org/10.1128/jvi.00460-08.
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ABSTRACT The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.
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Marsh, M., and R. Bron. "SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface." Journal of Cell Science 110, no. 1 (January 1, 1997): 95–103. http://dx.doi.org/10.1242/jcs.110.1.95.
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Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha- and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.
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Melancon, Jeffrey M., Timothy P. Foster, and Konstantin G. Kousoulas. "Genetic Analysis of the Herpes Simplex Virus Type 1 UL20 Protein Domains Involved in Cytoplasmic Virion Envelopment and Virus-Induced Cell Fusion." Journal of Virology 78, no. 14 (July 15, 2004): 7329–43. http://dx.doi.org/10.1128/jvi.78.14.7329-7343.2004.
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ABSTRACT The herpes simplex virus type 1 UL20 protein (UL20p) is an important determinant for cytoplasmic virion morphogenesis and virus-induced cell fusion. To delineate the functional domains of the UL20 protein, we generated a panel of single and multiple (cluster) alanine substitutions as well as UL20p carboxyl-terminal truncations. The UL20 mutant genes could be broadly categorized into four main groups: Group I UL20 mutant genes complemented for both virus production and virus-induced cell fusion; Group II UL20 mutant genes did not complement for either virus-induced cell fusion or infectious virus production; Group III UL20 mutant genes complemented for virus-induced cell fusion to variable extents but exhibited substantially decreased ability to complement UL20-null infectious virus production; Group IV mutant genes complemented for infectious virus production but had variable effects on virus-induced cell fusion; this group included two mutants that efficiently complemented for gBsyn3, but not for gKsyn1, virus-induced cell fusion. In addition, certain recombinant viruses with mutations in either the amino or carboxyl termini of UL20p produced partially syncytial plaques on Vero cells in the absence of any other virally encoded syncytial mutations. These studies indicated that the amino and carboxyl termini of UL20p contained domains that functioned both in infectious virus production and virus-induced cell fusion. Moreover, the data suggested that the UL20p's role in virus-induced cell fusion can be functionally separated from its role in cytoplasmic virion morphogenesis and that certain UL20p domains that function in gB-syn3 virus-induced cell fusion are distinct from those functioning in gKsyn1 virus-induced cell fusion.
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Kempf, Christoph, Marcel R. Michel, Adames Omar, Pia Jentsch, and Andreas Morell. "Semliki Forest virus induced cell-cell fusion at neutral extracellular pH." Bioscience Reports 10, no. 4 (August 1, 1990): 363–74. http://dx.doi.org/10.1007/bf01117236.
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Semliki Forest virus-induced cell-cell fusion from within was considered to exclusively occur at mildly acidic pH (<6.2). Data of this study show that such cell fusion can also be triggered by transient acidification of the cytoplasm of infected cells at an extracellular, neutral pH. Results were obtained by utilizing NH4Cl pulses combined with covalent modification of cell surface proteins. The observation implies a revision of the current consensus regarding the mechanism of Semliki Forest virus induced cell-cell fusion. We propose a model in which at least two peptide segments of the viral spike protein E1 may be involved in triggering the fusion event.
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Huerta, Leonor, Nayali López-Balderas, Evelyn Rivera-Toledo, Guadalupe Sandoval, Guillermo Gómez-Icazbalceta, Carlos Villarreal, Edmundo Lamoyi, and Carlos Larralde. "HIV-Envelope–Dependent Cell-Cell Fusion: Quantitative Studies." Scientific World JOURNAL 9 (2009): 746–63. http://dx.doi.org/10.1100/tsw.2009.90.
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Interactionin vitrobetween cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006,J. Virol. Methods138, 17–23; López-Balderas et al., 2007,Virus Res.123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.
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Hoornweg, Tabitha E., Mareike K. S. van Duijl-Richter, Nilda V. Ayala Nuñez, Irina C. Albulescu, Martijn J. van Hemert, and Jolanda M. Smit. "Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells." Journal of Virology 90, no. 9 (February 24, 2016): 4745–56. http://dx.doi.org/10.1128/jvi.03184-15.
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ABSTRACTChikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 min after addition to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The average time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was observed predominantly from within Rab5-positive endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes.IMPORTANCESince its reemergence in 2004, chikungunya virus (CHIKV) has spread rapidly around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, no vaccine or specific antiviral treatment against CHIKV is available. A potential antiviral strategy is to interfere with the cell entry process of the virus. However, conflicting results with regard to the cell entry pathway used by CHIKV have been published. Here we applied a novel technology to visualize the entry behavior of single CHIKV particles in living cells. Our results show that CHIKV cell entry is extremely rapid and occurs via clathrin-mediated endocytosis. Membrane fusion from within acidic early endosomes is observed. Furthermore, the membrane fusion capacity of CHIKV is strongly promoted by cholesterol in the target membrane. Taking these findings together, this study provides detailed insight into the cell entry process of CHIKV.
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Weissenhorn, Winfried, Andreas Hinz, and Yves Gaudin. "Virus membrane fusion." FEBS Letters 581, no. 11 (February 16, 2007): 2150–55. http://dx.doi.org/10.1016/j.febslet.2007.01.093.
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Jackson, Julia O., and Richard Longnecker. "Reevaluating Herpes Simplex Virus Hemifusion." Journal of Virology 84, no. 22 (September 15, 2010): 11814–21. http://dx.doi.org/10.1128/jvi.01615-10.
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ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.
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Gaudin, Yves. "Rabies Virus-Induced Membrane Fusion Pathway." Journal of Cell Biology 150, no. 3 (August 7, 2000): 601–12. http://dx.doi.org/10.1083/jcb.150.3.601.
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Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fusion at lower membrane concentrations and this inhibition was compensated by the presence of oleic acid. However, under suboptimal fusion conditions, short chain LPCs, which were translocated in the inner leaflet of the membranes, considerably reduced the lag time preceding membrane merging, resulting in faster kinetics of fusion. This indicated that the rate limiting step for fusion is the formation of a fusion pore in a diaphragm of restricted hemifusion. The previously described cold-stabilized prefusion complex was also characterized. This intermediate is at a well-advanced stage of the fusion process when the hemifusion diaphragm is destabilized, but lipid mixing is still restricted, probably by a ring-like complex of glycoproteins. I provide evidence that this state has a dynamic character and that its lipid organization can reverse back to two lipid bilayers.
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Shmulevitz, Maya, Jennifer Corcoran, Jayme Salsman, and Roy Duncan. "Cell-Cell Fusion Induced by the Avian Reovirus Membrane Fusion Protein Is Regulated by Protein Degradation." Journal of Virology 78, no. 11 (June 1, 2004): 5996–6004. http://dx.doi.org/10.1128/jvi.78.11.5996-6004.2004.
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ABSTRACT The p10 fusion-associated small transmembrane protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the protein for degradation following p10 insertion into an early membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral membrane fusion protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell fusion.
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Kobayashi, Mariko, Michael C. Bennett, Theodore Bercot, and Ila R. Singh. "Functional Analysis of Hepatitis C Virus Envelope Proteins, Using a Cell-Cell Fusion Assay." Journal of Virology 80, no. 4 (February 15, 2006): 1817–25. http://dx.doi.org/10.1128/jvi.80.4.1817-1825.2006.
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ABSTRACT Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.
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Kielian, M., M. R. Klimjack, S. Ghosh, and W. A. Duffus. "Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus." Journal of Cell Biology 134, no. 4 (August 15, 1996): 863–72. http://dx.doi.org/10.1083/jcb.134.4.863.
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Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell-cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.
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Earnest, James T., Michael P. Hantak, Jung-Eun Park, and Tom Gallagher. "Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains." Journal of Virology 89, no. 11 (April 1, 2015): 6093–104. http://dx.doi.org/10.1128/jvi.00543-15.
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ABSTRACTCoronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases.IMPORTANCEEnveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus priming with greater precision. Implications of these findings extend to the use of virus entry antagonists, such as protease inhibitors, which might be most effective when localized to these microdomains.
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RUDY. "Virus-cell fusion targeted for drug development." Chemical & Engineering News 74, no. 20 (May 13, 1996): 45. http://dx.doi.org/10.1021/cen-v074n020.p045.
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Bär, Séverine, Ayato Takada, Yoshihiro Kawaoka, and Marc Alizon. "Detection of Cell-Cell Fusion Mediated by Ebola Virus Glycoproteins." Journal of Virology 80, no. 6 (March 15, 2006): 2815–22. http://dx.doi.org/10.1128/jvi.80.6.2815-2822.2006.
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ABSTRACT Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a β-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.
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Valansi, Clari, David Moi, Evgenia Leikina, Elena Matveev, Martín Graña, Leonid V. Chernomordik, Héctor Romero, Pablo S. Aguilar, and Benjamin Podbilewicz. "Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens." Journal of Cell Biology 216, no. 3 (January 30, 2017): 571–81. http://dx.doi.org/10.1083/jcb.201610093.
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Cell–cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell–cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus–cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and exoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion.
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Greengard, Olga, Natalia Poltoratskaia, Evgenia Leikina, Joshua Zimmerberg, and Anne Moscona. "The Anti-Influenza Virus Agent 4-GU-DANA (Zanamivir) Inhibits Cell Fusion Mediated by Human Parainfluenza Virus and Influenza Virus HA." Journal of Virology 74, no. 23 (December 1, 2000): 11108–14. http://dx.doi.org/10.1128/jvi.74.23.11108-11114.2000.
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ABSTRACT 4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.
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Cosset, François-Loic, Philippe Marianneau, Geraldine Verney, Fabrice Gallais, Noel Tordo, Eve-Isabelle Pécheur, Jan ter Meulen, Vincent Deubel, and Birke Bartosch. "Characterization of Lassa Virus Cell Entry and Neutralization with Lassa Virus Pseudoparticles." Journal of Virology 83, no. 7 (January 19, 2009): 3228–37. http://dx.doi.org/10.1128/jvi.01711-08.
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ABSTRACT The cell entry and humoral immune response of the human pathogen Lassa virus (LV), a biosafety level 4 (BSL4) Old World arenavirus, are not well characterized. LV pseudoparticles (LVpp) are a surrogate model system that has been used to decipher factors and routes involved in LV cell entry under BSL2 conditions. Here, we describe LVpp, which are highly infectious, with titers approaching those obtained with pseudoparticles displaying G protein of vesicular stomatitis virus and their the use for the characterization of LV cell entry and neutralization. Upon cell attachment, LVpp utilize endocytic vesicles for cell entry as described for many pH-dependent viruses. However, the fusion of the LV glycoproteins is activated at unusually low pH values, with optimal fusion occurring between pH 4.5 and 3, a pH range at which fusion characteristics of viral glycoproteins have so far remained largely unexplored. Consistent with a shifted pH optimum for fusion activation, we found wild-type LV and LVpp to display a remarkable resistance to exposure to low pH. Finally, LVpp allow the fast and quantifiable detection of neutralizing antibodies in human and animal sera and will thus facilitate the study of the humoral immune response in LV infections.
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Sharma, Nishi R., Prashant Mani, Neha Nandwani, Rajakishore Mishra, Ajay Rana, and Debi P. Sarkar. "Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion." Journal of Virology 84, no. 9 (February 17, 2010): 4366–82. http://dx.doi.org/10.1128/jvi.01940-09.
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ABSTRACT Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.
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Zavorotinskaya, Tatiana, Zhaohui Qian, John Franks, and Lorraine M. Albritton. "A Point Mutation in the Binding Subunit of a Retroviral Envelope Protein Arrests Virus Entry at Hemifusion." Journal of Virology 78, no. 1 (January 1, 2004): 473–81. http://dx.doi.org/10.1128/jvi.78.1.473-481.2004.
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ABSTRACT The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.
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Jeetendra, E., Kakoli Ghosh, Derek Odell, Jin Li, Hara P. Ghosh, and Michael A. Whitt. "The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity." Journal of Virology 77, no. 23 (December 1, 2003): 12807–18. http://dx.doi.org/10.1128/jvi.77.23.12807-12818.2003.
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ABSTRACT The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.
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Kelly, James T., Stacey Human, Joseph Alderman, Fatoumatta Jobe, Leanne Logan, Thomas Rix, Daniel Gonçalves-Carneiro, et al. "BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein Production to Inhibit Cell–Cell Fusion." Viruses 11, no. 8 (July 30, 2019): 692. http://dx.doi.org/10.3390/v11080692.
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The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype.
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Ogino, Michiko, Kumiko Yoshimatsu, Hideki Ebihara, Koichi Araki, Byoung-Hee Lee, Megumi Okumura, and Jiro Arikawa. "Cell Fusion Activities of Hantaan Virus Envelope Glycoproteins." Journal of Virology 78, no. 19 (October 1, 2004): 10776–82. http://dx.doi.org/10.1128/jvi.78.19.10776-10782.2004.
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ABSTRACT Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.
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Atanasiu, Doina, Wan Ting Saw, Roselyn J. Eisenberg, and Gary H. Cohen. "Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion." Journal of Virology 90, no. 23 (September 14, 2016): 10535–44. http://dx.doi.org/10.1128/jvi.01501-16.
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ABSTRACTReceptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion.IMPORTANCECell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when using deregulated forms of gD1and gH2/gL2, the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion.
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Pedersen, Simon Metz, Bodil Øster, Bettina Bundgaard, and Per Höllsberg. "Induction of Cell-Cell Fusion from Without by Human Herpesvirus 6B." Journal of Virology 80, no. 19 (October 1, 2006): 9916–20. http://dx.doi.org/10.1128/jvi.02693-05.
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ABSTRACT Human herpesvirus (HHV) 6A induce fusion from without (FFWO), whereas HHV-6B is believed to be ineffective in this process. Here, we demonstrate that HHV-6B induces rapid fusion in both epithelial cells and lymphocytes. The fusion was identified 1 h postinfection, could be inhibited by antibodies to HHV-6B gH and to the cellular receptor CD46, and was dependent on virus titer but independent of de novo protein synthesis and UV inactivation of the virus. Comparisons indicate that HHV-6A is only 10-fold more effective in inducing FFWO than HHV-6B. These data demonstrate that HHV-6B can induce FFWO in epithelial cells and lymphocytes.
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Harada, Yuichirou, Keiichi Yoshida, Natsuko Kawano, and Kenji Miyado. "Critical role of exosomes in sperm-egg fusion and virus-induced cell-cell fusion." Reproductive Medicine and Biology 12, no. 4 (May 24, 2013): 117–26. http://dx.doi.org/10.1007/s12522-013-0152-2.
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Gaudin,, Y., Christine Tuffereau,, Peter Durrer,, Josef Brunner,, Anne Flamand,, and Rob Ruigrok. "Rabies virus-induced membrane fusion." Molecular Membrane Biology 16, no. 1 (January 1999): 21–31. http://dx.doi.org/10.1080/096876899294724.
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Chouljenko, Vladimir N., Arun V. Iyer, Sona Chowdhury, Dmitry V. Chouljenko, and Konstantin G. Kousoulas. "The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress." Journal of Virology 83, no. 23 (September 30, 2009): 12301–13. http://dx.doi.org/10.1128/jvi.01329-09.
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ABSTRACT Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.
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Ren, Guijie, Yunlong Zhuang, Keli Tian, Huiyu Li, Xueqin Diao, Miaomiao Wang, Nan Zhou, and Zhiyu Wang. "Acidic amino acids increase fusion activity in the specific fusion domain of Newcastle disease virus fusion protein." Canadian Journal of Microbiology 59, no. 9 (September 2013): 641–44. http://dx.doi.org/10.1139/cjm-2013-0133.
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To explore the effects of amino acids Gln and Asn within the specific fusion domain of fusion (F) protein on the specific membrane fusion in Newcastle disease virus (NDV), the mutants Q204E–Q205E and N245D were constructed in the specific fusion domain of F protein. The mutant genes were co-expressed with homologous or heterologous hemagglutinin–neuraminidase (HN) in BHK21 cells. Cell fusion functions of mutants were analyzed with Giemsa staining and reporter gene methods. Cell surface expression efficiency was analyzed with immunofluorescence assay and fluorescence-activated cell sorter analysis. Co-immunoprecipitation was performed to analyze the interaction of mutant F proteins with the homotypic HN protein. Both Q204E–Q205E and N245D mutations caused increased cell–cell fusion activity when they were co-expressed with homotypic HN protein. The mutant F proteins had slight changes in cell surface expression compared with that of wild-type F protein. The interactions of Q204E–Q205E or N245D with their homotypic HN increased significantly (P < 0.01) compared with the wild-type F protein. Neither Q204–Q205E nor N245D caused cell fusion in the presence of heterologous HN protein. Our data suggested that the residues Q204, Q205, and N245 play a critical role in the regulation of cell fusion. They may decrease the interaction of wild-type NDV F and NDV HN to suppress the fusion activity for survival of the infected host, which may enable a persistent virus infection and long-term virus reproduction and spread.
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Herschke, F., S. Plumet, T. Duhen, O. Azocar, J. Druelle, D. Laine, T. F. Wild, C. Rabourdin-Combe, D. Gerlier, and H. Valentin. "Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response." Journal of Virology 81, no. 23 (September 26, 2007): 12859–71. http://dx.doi.org/10.1128/jvi.00078-07.
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ABSTRACT Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-β) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-β response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-α/β production, an amplified IFN-β response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-β gene deficiencies were trans complemented to induce IFN-β production. Production of IFN-β and IFN-α was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-α/β. The amplification of IFN-β production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-α/β production in infected cells, and this indicates that MGC contribute to the antiviral immune response.
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Takikawa, Shingo, Koji Ishii, Hideki Aizaki, Tetsuro Suzuki, Hitoshi Asakura, Yoshiharu Matsuura, and Tatsuo Miyamura. "Cell Fusion Activity of Hepatitis C Virus Envelope Proteins." Journal of Virology 74, no. 11 (June 1, 2000): 5066–74. http://dx.doi.org/10.1128/jvi.74.11.5066-5074.2000.
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ABSTRACT To examine the cell fusion activity of hepatitis C virus (HCV) envelope proteins (E1 and E2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68:5411–5422, 1994). The chimeric HCV E1 and E2 proteins, each consisting of the ectodomain of the E1 and E2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G glycoprotein, were expressed on the cell surface. Cells expressing the chimeric envelope proteins and T7 RNA polymerase were cocultured with the various target cell lines transfected with a reporter plasmid encoding the luciferase gene under the control of the T7 promoter. After cocultivation, the cell fusion activity was determined by the expression of luciferase in the cocultured cells. The induction of cell fusion requires both the chimeric E1 and E2 proteins and occurs in a low-pH-dependent manner. Although it has been shown that HCV E2 protein binds human CD81 (P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Science 282:938–941, 1998), the expression of human CD81 alone is not sufficient to confer susceptibility to cell fusion in the mouse cell line. Treatment of the target cells with pronase, heparinase, or heparitinase reduced the cell fusion activity induced by the chimeric envelope proteins. These results suggest (i) that both HCV E1 and E2 proteins are responsible for fusion with the endosomal membrane after endocytosis and (ii) that certain protein molecules other than human CD81 and some glycosaminoglycans on the cell surface are also involved in the cell fusion induced by HCV.
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Singethan, K., E. Topfstedt, S. Schubert, W. P. Duprex, B. K. Rima, and Jürgen Schneider-Schaulies. "CD9-dependent regulation of Canine distemper virus-induced cell–cell fusion segregates with the extracellular domain of the haemagglutinin." Journal of General Virology 87, no. 6 (June 1, 2006): 1635–42. http://dx.doi.org/10.1099/vir.0.81629-0.
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Antibodies to CD9, a member of the tetraspan transmembrane-protein family, selectively inhibit Canine distemper virus (CDV)-induced cell–cell fusion. Neither CDV-induced virus–cell fusion nor cell–cell fusion induced by the closely related morbillivirus Measles virus (MV) is affected by anti-CD9 antibodies. As CDV does not bind CD9, an unknown, indirect mechanism is responsible for the observed inhibition of cell–cell fusion. It was investigated whether this effect was restricted to only one viral glycoprotein, either the haemagglutinin (H) or the fusion (F) protein, which form a fusion complex on the surface of virions and infected cells, or whether it is dependent on both in transient co-transfection assays. The susceptibility to CD9 antibodies segregates with the H protein of CDV. By exchanging portions of the H proteins of CDV and MV, it was determined that the complete extracellular domain, including the predicted stem structure (stem 1, barrel strand 1 and stem 2) and globular head domain, of the CDV-H protein mediates the effect. This suggests that interaction of the CDV-H protein with an unknown cellular receptor(s) is regulated by CD9, rather than F protein-mediated membrane fusion.
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Lanzrein, M., N. Käsermann, R. Weingart, and C. Kempf. "Early Events of Semliki Forest Virus-Induced Cell-Cell Fusion." Virology 196, no. 2 (October 1993): 541–47. http://dx.doi.org/10.1006/viro.1993.1509.
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Okazaki, Katsunori. "Proteolytic cleavage of glycoprotein B is dispensable for in vitro replication, but required for syncytium formation of pseudorabies virus." Journal of General Virology 88, no. 7 (July 1, 2007): 1859–65. http://dx.doi.org/10.1099/vir.0.82610-0.
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Glycoprotein B (gB) is the most conserved glycoprotein among herpesviruses and it plays important roles in virus infectivity. In most herpesviruses, including pseudorabies virus (PRV), gB is cleaved by a cellular protease into two disulfide-linked subunits. In the present study, I found that the PRV gB generated in human colon carcinoma LoVo cells, which lack the ubiquitous protease furin, remained in the uncleaved form and the virus replicated in these cells without cell fusion. The uncleaved gB was converted into its subunits after furin digestion. The virus also replicated in Madin–Darby bovine kidney cells without cell fusion in the presence of a furin inhibitor, whereas distinct syncytia were formed in the absence of the inhibitor. LoVo cells constitutively expressing furin showed cell fusion when they were infected with the virus. Penetration kinetics assays revealed that the virus carrying uncleaved gB penetrated the cells at the same rate as the virus carrying cleaved gB. These results indicate that PRV gB is cleaved by furin and that the cleavage is dispensable for virus replication in vitro. Furthermore, gB cleavage is involved in syncytium formation but not in penetration kinetics, suggesting that different mechanisms operate between cell–cell fusion and virus–cell fusion by PRV.
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Marquardt, M. T., T. Phalen, and M. Kielian. "Cholesterol is required in the exit pathway of Semliki Forest virus." Journal of Cell Biology 123, no. 1 (October 1, 1993): 57–65. http://dx.doi.org/10.1083/jcb.123.1.57.
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The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.
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Bradel-Tretheway, Birgit G., Qian Liu, Jacquelyn A. Stone, Samantha McInally, and Hector C. Aguilar. "Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus." Journal of Virology 89, no. 14 (May 6, 2015): 7235–47. http://dx.doi.org/10.1128/jvi.00773-15.
Full textAbstract:
ABSTRACTHendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within theParamyxoviridaefamily. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses.IMPORTANCEViral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses.
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Lifson, J., S. Coutré, E. Huang, and E. Engleman. "Role of envelope glycoprotein carbohydrate in human immunodeficiency virus (HIV) infectivity and virus-induced cell fusion." Journal of Experimental Medicine 164, no. 6 (December 1, 1986): 2101–6. http://dx.doi.org/10.1084/jem.164.6.2101.
Full textAbstract:
Human immunodeficiency virus (HIV) envelope glycoprotein interactions with cell surface CD4 are involved in both virion infectivity and virally mediated cell fusion. D-mannose-specific lectins such as Con A specifically blocked virion infectivity and cell fusion. Studies with a recombinant vaccinia virus containing the HIV envelope gene demonstrated that Con A-mediated inhibition of HIV-induced fusion involved lectin binding to the viral envelope glycoprotein. These results indicate the importance of envelope glycosylation in the pathobiology of HIV infection, and suggest potential mechanisms for interfering with HIV infectivity and cytopathology.
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Lineberger, Janet E., Renee Danzeisen, Daria J. Hazuda, Adam J. Simon, and Michael D. Miller. "Altering Expression Levels of Human Immunodeficiency Virus Type 1 gp120-gp41 Affects Efficiency but Not Kinetics of Cell-Cell Fusion." Journal of Virology 76, no. 7 (April 1, 2002): 3522–33. http://dx.doi.org/10.1128/jvi.76.7.3522-3533.2002.
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ABSTRACT Human immunodeficiency virus (HIV) entry into a host cell requires the fusion of virus and cellular membranes that is driven by interaction of the viral envelope glycoproteins gp120 and gp41 (gp120/gp41) with CD4 and a coreceptor, typically either CXCR4 or CCR5. The stoichiometry of gp120/gp41:CD4:CCR5 necessary to initiate membrane fusion is not known. To allow an examination of early events in gp120/gp41-driven membrane fusion, we developed a novel real-time cell-cell fusion assay. Using this assay to study fusion kinetics, we found that altering the cell surface density of gp120/gp41 affected the maximal extent of fusion without dramatically altering fusion kinetics. Collectively, these observations are consistent with the view that gp120/gp41-driven membrane fusion requires the formation of a threshold number of fusion-active intercellular gp120/gp41:CD4:CCR5 complexes. Furthermore, the probability of reaching this threshold is governed, in part, by the surface density of gp120/gp41.
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Cross, Karen J., Laura M. Burleigh, and David A. Steinhauer. "Mechanisms of cell entry by influenza virus." Expert Reviews in Molecular Medicine 3, no. 21 (August 6, 2001): 1–18. http://dx.doi.org/10.1017/s1462399401003453.
Full textAbstract:
A wide range of viruses, including many human and animal pathogens representing various taxonomic groups, contain genomes that are enclosed in lipid envelopes. These envelopes are generally acquired in the final stages of assembly, as viruses bud from regions of the membrane of the infected cell at which virally encoded membrane proteins have accumulated. The viruses procure their membranes during this process and mature particles ‘pinch off’ from the cellular membranes. Under most circumstances, initiation of another round of infection is dependent on two critical functions supplied by the envelope proteins. The virus must bind to cell-surface receptors of a new host cell, and fusion of the viral and cellular membranes must occur to transfer the viral genome into the cell. Enveloped viruses have evolved a variety of mechanisms to execute these two basic functions. Owing to their relative simplicity, studies of binding and fusion using enveloped viruses and their components have contributed significantly to the overall understanding of receptor–ligand interactions and membrane fusion processes – fundamental activities involved in a plethora of biological functions.
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Oliver, Stefan L., Momei Zhou, and Ann M. Arvin. "Varicella-zoster virus: molecular controls of cell fusion-dependent pathogenesis." Biochemical Society Transactions 48, no. 6 (December 1, 2020): 2415–35. http://dx.doi.org/10.1042/bst20190511.
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Varicella–zoster virus (VZV) is the causative agent of chicken pox (varicella) and shingles (zoster). Although considered benign diseases, both varicella and zoster can cause complications. Zoster is painful and can lead to post herpetic neuralgia. VZV has also been linked to stroke, related to giant cell arteritis in some cases. Vaccines are available but the attenuated vaccine is not recommended in immunocompromised individuals and the efficacy of the glycoprotein E (gE) based subunit vaccine has not been evaluated for the prevention of varicella. A hallmark of VZV pathology is the formation of multinucleated cells termed polykaryocytes in skin lesions. This cell–cell fusion (abbreviated as cell fusion) is mediated by the VZV glycoproteins gB, gH and gL, which constitute the fusion complex of VZV, also needed for virion entry. Expression of gB, gH and gL during VZV infection and trafficking to the cell surface enables cell fusion. Recent evidence supports the concept that cellular processes are required for regulating cell fusion induced by gB/gH–gL. Mutations within the carboxyl domains of either gB or gH have profound effects on fusion regulation and dramatically restrict the ability of VZV to replicate in human skin. This loss of regulation modifies the transcriptome of VZV infected cells. Furthermore, cellular proteins have significant effects on the regulation of gB/gH–gL-mediated cell fusion and the replication of VZV, exemplified by the cellular phosphatase, calcineurin. This review provides the current state-of-the-art knowledge about the molecular controls of cell fusion-dependent pathogenesis caused by VZV.
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Pritzen, Cornelia, and Andreas Herrmann. "Are osmotic forces involved in influenza virus-cell fusion?" Bioscience Reports 8, no. 1 (February 1, 1988): 55–64. http://dx.doi.org/10.1007/bf01128972.
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The kinetics of the fusion process of unsealed and resealed erthyrocyte ghosts with influenza virus (A/PR8/34, A/Chile 1/83), were measured under hypotonic, isotonic and hypertonic conditions using a recently developed fluorescence assay (Hoekstra et al. (1984) Biochemistry23:5675–5681]. No correlation between the external osmotic pressure and kinetics and extent of fusion was observed. Influenza viruses fuse as effectively with unsealed ghosts as with resealed ghosts. It is concluded that osmotic forces as well as osmotic swelling of cells are not necessary for virus-cell membrane fusion.