HVJ (Sendai virus)-induced envelope fusion and cell fusion are blocked by monoclonal anti-HN protein antibody that does not inhibit hemagglutination activity of HVJ.
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Biochemical studies on cell fusion. I. Lipid composition of fusion-resistant cells.
A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.
Animals↗
[Electrostimulated cell fusion in cell engineering].
A survey of studies on reconstructions of animal and plant cells which apply a new physical method--electrostimulated fusion, is presented. Effects of different factors of the medium on the efficiency of electrofusion is discussed. A detailed account is given of the authors' studies on zygotes reconstruction by combined methods of microsurgery and electrostimulated cell fusion. Advantages of the latter as compared to the widely distributed methods of fusion by polyethylenglycol and Sendai virus are considered. This physical method can play an important role in the progress of cellular engineering.
Animals↗
Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion.
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.
Animals↗
Assessment of fusion cells from patient-derived ovarian carcinoma cells and dendritic cells as a vaccine for clinical use.
PURPOSE: To evaluate a protocol that allowed the successful generation of DC and OVCA cells, fusion of these two cell types and assessment of stimulatory ability of the fusion cells for clinical use. PATIENTS AND METHODS: Ovarian cancer (OVCA) cells and dendritic cells (DC) were isolated or generated from 22 patients with OVCA and subsequently fused with PEG. The stimulatory ability of fusion cells including T cell proliferation and induction of cytotocic T lymphocytes (CTL) was assessed. In addition, the impact of radiation, freezing and thawing of the fusion cells was evaluated. RESULTS: OVCA cells derived from 22 patients were successfully fused with autologous DC. The created heterokaryons expressed tumor-associated antigens, such as MUC1 and CA-125, and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, CD4 and CD8 T cells derived from patients with ovarian cancer were stimulated by fusion cells and produced IFN-gamma as demonstrated with intracellular staining. Significantly, T cells primed by fusion cells produced MHC class I-dependent lysis of autologous ovarian tumor cells. One cycle of fusion-cell stimulation can maintain the CTL activity up to 25 days. CONCLUSIONS: The fusion of human OVCA cells and DC created immunogenic cells capable of stimulating CD4 and CD8 T cells. The effects of the processes required for preparing a vaccine for clinical use, including freezing and thawing and irradiation, do not interfere with the immunogenic properties of the fusion cells.
CD4-Positive T-Lymphocytes↗
Divalent cations, phospholipid asymmetry and osmotic swelling in electrically-induced lysis, cell fusion and giant cell formation with human erythrocytes.
We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength. Divalent cations (1.1 mM) protected the cells against haemolysis, in the order Mn2+ > Ca2+ > Ba2+ > Mg2+ >> Zn2+, whereas about 99% of the cells lysed immediately on breakdown in the presence of Na+ or K+ (2.1 mM), or Al3+ (0.95 mM). The lengths of pearl chains of fused erythrocytes formed was similarly greatest with Mn2+ and decreased progressively with Ba2+, Zn2+, Ca2+ and Mg2+. No cell fusion occurred with Na+, K+, or Al3+. It is suggested that interactions with phosphatidylserine, which is exposed at the cell surface by electrical breakdown, may enable Mn2+, Ba2+ and Ca2+ ions to inhibit cell lysis (via membrane resealing) and facilitate cell fusion. Following electrically-induced cell fusion, erythrocytes round-up into giant cells. It has previously been proposed that Ca2+ ions accelerate the rounding-up process. However, data are presented which show that, as with erythrocytes treated with Sendai virus, the formation of rounded, giant cells following cell fusion depends on the osmotic swelling properties of permeabilised erythrocytes. Osmotic swelling may also have induced any hemi-fused cells present to fuse completely. Zn2+ ions anomalously enabled erythrocytes to round-up very rapidly into giant cells following electrical breakdown. This phenomenon may result from an interaction of Zn2+ ions with cysteine groups in membrane proteins, which decreases the immediate loss of ions that occurs when erythrocytes are subjected to electrical breakdown in low-ionic-strength media.
Cadmium↗
Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion.
Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.
Animals↗
Recombinant wild-type and edmonston strain measles viruses bearing heterologous H proteins: role of H protein in cell fusion and host cell specificity.
Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but not IC-B H protein, was expressed. To analyze the role of H protein in the context of viral infection, a recombinant IC-B virus bearing Ed H protein (IC/Ed-H) and a recombinant Ed virus bearing IC-B H protein (Ed/IC-H) were generated from cloned cDNAs. IC/Ed-H replicated efficiently in Vero cells and induced small syncytia in Vero cells, indicating that Ed H protein conferred replication ability in Vero cells on IC/Ed-H. On the other hand, Ed/IC-H also replicated well in Vero cells and induced small syncytia, although parental Ed induced large syncytia in Vero cells. These results indicated that an MV protein(s) other than H protein was likely involved in determining cell fusion and host cell specificity of MV in the case of our recombinants. SLAM (CDw150), a recently identified cellular receptor for wild-type MV, was not expressed in Vero cells, and a monoclonal antibody against CD46, a cellular receptor for Ed, did not block replication or syncytium formation of Ed/IC-H in Vero cells. It is therefore suggested that Ed/IC-H entered Vero cells through another cellular receptor.
Animals↗
An alpha-helical domain within the carboxyl terminus of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is associated with cell fusion and resistance to heparin inhibition of cell fusion.
Previous studies from our laboratory indicated that a 28-amino-acid carboxyl-terminal truncation of gB caused extensive virus-induced cell fusion (Baghian et al., 1993, J Virol 67, 2396-2401). We tested the ability of additional truncations and mutations within gB to cause cell fusion in the recently established virus-free cell fusion assay (Turner et al., 1998, J. Virol. 72, 873-875). Deletion of the carboxyl-terminal 28 amino acids of gB (gBDelta28), which removed part of the predicted alpha-helical structure H17b, caused extensive cell fusion. A gB truncation specified by gBDelta36, which removed the entire H17b domain, caused as much cell fusion as the gBDelta28 truncation. Similarly, gB(A874P) containing a substitution of an Ala with Pro within H17b caused cell fusion. Heparin, a gB-specific inhibitor of virus-induced cell fusion, inhibited both wild-type gB and gB(syn3)-mediated cell fusion. In contrast, fusion of cells transfected with gB(Delta28), gB(Delta36), or gB(A874P) was resistant to heparin inhibition of cell fusion. We concluded the following: (1) The predicted alpha-helical structure of H17b within the carboxyl terminus of gB is involved in both virus-induced and virus-free cell fusion. (2) Heparin is a specific inhibitor of gB-mediated fusion in both systems. (3) Resistance to heparin inhibition of gB-mediated cell fusion is associated with the predicted alpha-helical structure H17b within the carboxyl terminus of gB.
Amino Acid Sequence↗
The kinetics of in vivo priming of CD4 and CD8 T cells by dendritic/tumor fusion cells in MUC1-transgenic mice.
Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro. However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4+ and CD8+ T cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c. into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation of T cells and induced MUC1-specific CD8+ CTL. Moreover, CD4+ T cells activated by FC/MUC1 were multifunctional effectors that produced IL-2, IFN-gamma, IL-4, and IL-10. These findings indicate that both CD4+ and CD8+ T cells can be primed in vivo by FC/MUC1 immunization.
Adoptive Transfer↗
Distinct morphological phenotypes of cell fusion mutants.
Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Delta fus2Delta, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus- zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.
COP9 Signalosome Complex↗
Fluorescence photobleaching recovery to probe virus-cell fusion and virus-mediated cell fusion.
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Animals↗
Fus2 localizes near the site of cell fusion and is required for both cell fusion and nuclear alignment during zygote formation.
Zygote formation occurs through tightly coordinated cell and nuclear fusion events. Genetic evidence suggests that the FUS2 gene product promotes cell fusion during zygote formation in Saccharomyces cerevisiae, functioning with the Fus1 plasma membrane protein at or before cell wall and plasma membrane fusion. Here we report the sequence of the FUS2 gene, localization of Fus2 protein, and show that fus1 and fus2 mutants have distinct defects in cell fusion. FUS2 encodes a unique open reading frame of 617 residues that only is expressed in haploid cells in response to mating pheromone. Consistent with a role in cell fusion, Fus2 protein localizes with discrete structures that could be of cytoskeletal or vesicular origin that accumulate at the tip of pheromone-induced shmoos and at the junction of paired cells in zygotes. Fus2 is predicted to be a coiled-coil protein and fractionates with a 100,000 g pellet, suggesting that it is associated with cytoskeleton, membranes, or other macromolecular structures. Fus2 may interact with structures involved in the alignment of the nuclei during cell fusion, because fus2 mutants have strong defects in karyogamy and fail to orient microtubules between parental nuclei in zygotes. In contrast, fus1 mutants show no karyogamy defects. These, and other results suggest that Fus2 defines a novel cell fusion function and subcellular structure that is also required for the alignment of parental nuclei before nuclear fusion.
Amino Acid Sequence↗
Characterization of cell fusion in XC cells induced by Suncus murinus mammary tumor virus.
Syncytium formation in a rat tumor cell line (XC) induced by Suncus murinus mammary tumor virus (Sm-MTV) was studied. Multinucleate giant cells containing 20-30 nuclei were formed in a monolayer of XC cells by cocultivation with X-ray-irradiated Sm-MTV producing cells (Sm-MT-1). By fluorescent antibody staining. Sm-MTV antigens were demonstrated in the cytoplasm of syncytia, and budding particles and intracytoplasmic A particles were found in syncytial giant cells by electron microscopy. Cell-free supernatant of Sm-MT-1 was capable of inducing syncytia at a much lower incidence than cocultivation of Sm-MT-1 cells. Syncytium formation was completely inhibited when anti-Sm-MTV bovine serum was added to the coculture medium. Pretreatment of XC cells with actinomycin-D caused a partial reduction of Sm-MTV-induced cell fusion, but syncytium formation did occur at a reduced rate even when cellular RNA synthesis was completely inhibited. Dexamethasone increased virus production in Sm-MT-1 cells, resulting in the enhancement of Sm-MTV mediated syncytium formation. Sm-MTV was found to have a unique characteristic of cell fusion activity on XC cells with striking enhancement by dexamethasone.
Animals↗
Evidence for a multistep mechanism for cell-cell fusion by herpes simplex virus with mutations in the syn 3 locus using heparin derivatives during fusion from within.
Addition of heparin-Na+ as well as related substances of high and intermediate MW (Arteparon and polyanion SP54) 3 h after infection inhibit fusion from within (FFWI) induced by HSV strains with mutations in the syn 3 locus only. The concentration of heparin-Na+ required to inhibit FFWI is 10-fold higher (1 mg/ml) than that needed to inhibit adsorption. Instead of fusion, cell rounding is observed. The effect is readily reversible. A low MW heparin disaccharide is ineffective. Neomycin, at a concentration of 8 mM, inhibits FFWI induced by all HSV-1 but not HSV-2 strains, whereas adsorption is inhibited at 3 mM. We conclude from our observations that cell-cell fusion (FFWI) induced by syn 3 locus mutants of HSV-1 depends on a multistep mechanism. One may be constituted by pre-existing cell-cell connections or microfusions leading to cell rounding, whereas another may be active using newly appearing cell bridges during FFWI; also the three-dimensional structure of the cell membrane may be of importance. Moreover, the molecular mechanisms of FFWI induced by mutations in the syn 3 locus compared to the other 5 syn loci should be different.
Animals↗
Control of virus-induced cell fusion by host cell lipid composition.
Virus-induced cell fusion has been examined in a series of stable cell lines which were originally selected for resistance to the fusogenic effects of polyethylene glycol (PEG). For a wide variety of viruses, including murine hepatitis virus (a coronavirus), vesicular stomatitis virus (a rhabdovirus), and two paramyxoviruses (Sendai virus and SV5), susceptibility to virus-induced fusion was found to be inversely correlated with susceptibility to PEG-induced fusion. This phenomenon was observed both for cell fusion occurring in the course of viral infection and for fusion induced "from without" by the addition of high titers of noninfectious or inactivated virus. The fusion-altered cell lines (fusible by virus but not by PEG) are characterized by their unusual lipid composition, including marked elevation of saturated fatty acids and the presence of an unusual ether-linked neutral lipid. To test the association between lipid composition and fusion, acyl chain saturation was manipulated by supplementing the culture medium with exogenous fatty acids. In such experiments, it was possible to control the responses of these cells to both viral and chemical fusogens. Increasing the cellular content of saturated fatty acyl chains increased the susceptibility of cells to viral fusion and decreased susceptibility to PEG-induced fusion, whereas lowering fatty acid saturation had the opposite effect. Thus, parallel cultures of cells can be either driven toward the PEG-fusible/virus-fusion-resistant phenotype of the parental cells or rendered susceptible to viral fusion but resistant to PEG-induced fusion, solely by the alteration of cellular lipids. The ability of cellular lipid composition to regulate virus-induced membrane fusion suggests a possible role for lipids in viral infection and pathogenesis.
Animals↗
Effect of water soluble polymer, polyethyleneglycol, and glass-forming compounds on cell fusion.
The cell fusion of Molt T-cells was investigated in the presence of water soluble polymer, polyethyleneglycol (PEG) and glass-forming monomers. In cell fusion with PEG only, inactivation of the cell occurred within 2 min. However, in the presence of PEG and water soluble polymer, most of the fused and unfused cells lived even after fusion times of more than 10 min. It was observed that water soluble polymer prevented the inactivation of cells during fusion as a protectant. As the result, ratio of fused cells increased in the presence of water soluble polymers. Some glass-forming monomers used as new fusogens such as M-23G (n = 23) and M-50G (n = 50) monomers having long oxyethylene chains in the methoxypolyethyleneglycol methacrylate, CH3O-(-CH2CH2-O)n-CO-C(CH3) = CH2 as well as PEG #2000 indicated large promoting and protecting effects on cell fusion.
Cell Fusion↗
Nanog promotes transfer of pluripotency after cell fusion.
Through cell fusion, embryonic stem (ES) cells can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ES cells. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain undifferentiated ES cell propagation. Here we report that in fusions between ES cells and neural stem (NS) cells, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ES cell characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ES cells; however, fewer colonies are obtained than from ES x NS cell fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NS x ES cell fusions elevated Nanog enables primary hybrids to develop into ES cell colonies with identical frequency to homotypic ES x ES fusion products. This means that in hybrids, increased Nanog is sufficient for the NS cell epigenome to be reset completely to a state of pluripotency. We conclude that Nanog can orchestrate ES cell machinery to instate pluripotency with an efficiency of up to 100% depending on the differentiation status of the somatic cell.
Animals↗