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At least 127 records · Page 7Linked to original sources
PubMed1993

Characterization of avian reovirus-induced cell fusion: the role of viral structural proteins.

Cell fusion induced by avian reovirus was analyzed using virus strain FC and Vero cells. One-step growth curves showed that cell fusion was directly associated with viral replication. Cell fusion occurred most efficiently at basic pH (8.0-8.5) and fusion from without could not be demonstrated. Actinomycin D, at low concentrations, increased cell fusion, and cycloheximide prevented cell fusion, indicating that viral protein(s) were responsible for the induction of cell fusion. Immunofluorescence tests indicated that viral proteins were present on the infected cell surface. Radioimmuno-precipitation identified structural proteins mu 2C and sigma 2 as predominant viral protein species present on the infected cell surface. Cell fusion was inhibited by virus-specific antisera, suggesting that mu 2C and/or sigma 2 present on the infected cell surface were involved in the induction of cell fusion. Trypsin and chymotrypsin treatment of purified viruses cleaved both mu 2C and sigma 2 proteins, but generated different cleavage products with each protein. The addition of trypsin to the culture media following infection increased cell fusion, whereas chymotrypsin treatment decreased cell fusion. The opposite effects of trypsin and chymotrypsin on the cell fusion, together with the different specificities of these two proteases in cleavage of mu 2C and sigma 2 proteins, further suggest that the cell surface-associated mu 2C and/or sigma 2 are involved in the syncytium formation.

Alkalies↗
PubMed1992

Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion.

The role of residues in the conserved hydrophobic N-terminal fusion peptide of the paramyxovirus fusion (F) protein in causing cell-cell fusion was examined. Mutations were introduced into the cDNA encoding the simian virus 5 (SV5) F protein, the altered F proteins were expressed by using an eukaryotic vector, and their ability to mediate syncytium formation was determined. The mutant F proteins contained both single- and multiple-amino-acid substitutions, and they exhibited a variety of intracellular transport properties and fusion phenotypes. The data indicate that many substitutions in the conserved amino acids of the simian virus 5 F fusion peptide can be tolerated without loss of biological activity. Mutant F proteins which were not transported to the cell surface did not cause cell-cell fusion, but all of the mutants which were transported to the cell surface were fusion competent, exhibiting fusion properties similar to or better than those of the wild-type F protein. Mutant F proteins containing glycine-to-alanine substitutions had altered intracellular transport characteristics, yet they exhibited a great increase in fusion activity. The potential structural implications of this substitution and the possible importance of these glycine residues in maintaining appropriate levels of fusion activity are discussed.

Amino Acid Sequence↗
PubMed2006

Nuclear reprogramming by cell fusion.

The use of cell fusion to study exchange of information at the molecular level between the nucleus and the cytoplasm of cells during regulation of gene expression was pioneered by Harris and Ringertz more than three decades ago. The ability to make heterokaryons with cells from different species or genetic strains is especially useful because genetic differences in gene products allow the origin of trans-acting regulatory factors to be determined. Heterokaryons between adult nucleated erythroid cells of one species and embryonic/larval nucleated erythroid cells of another species, for example, show cross-induction between the two types of nuclei, resulting in reprogramming of the adult nucleus to embryonic/larval globin gene expression and/or reprogramming of the embryonic/larval cell nucleus to adult globin expression. These experiments provided definitive evidence that developmental program switching is mediated by trans-acting factors. Other possible uses of this cell fusion protocol in stem cell biology and transplantation of genetically engineered cells for tissue regeneration are briefly discussed.

Animals↗
PubMed1980

Cell fusion activity of bovine leukaemia virus.

Bovine leukaemia virus (BLV) induces two types of cell fusion. Cells inoculated at high multiplicity show cell fusion within 2 h. This type of fusion can be induced by non-infectious virus and does not require protein synthesis for induction. Low m.o.i. also induces cell fusion but this depends on production of infectious virus.

Animals↗
PubMed1994

Polymer-induced membrane fusion: potential mechanism and relation to cell fusion events.

Poly(ethylene glycol) (PEG) is used widely to mediate cell-cell fusion in the production of somatic cell hybrids and in the fusion injection of macromolecules into cultured cells from erythrocytes or liposomes. However, little is known about the mechanisms by which PEG induces fusion of cell membranes, making its use much more an art than a science. This article considers possible molecular events involved in biomembrane fusion and summarizes what we have learned about these in recent years from studies of fusion of well-defined model membranes. In addition, it recounts observations made over the past several years about the process of PEG-mediated fusion of model membranes. These observations have defined the process to an extent sufficient to allow us to propose a model for the molecular events involved in the process. It is suggested that dehydration leads to asymmetry in the lipid packing pressure in the two leaflets of the membrane bilayer leading to formation of a single bilayer septum at a point of close apposition of two membranes. The single bilayer septum then decays during formation of the initial fusion pore. Agents that enhance or alleviate the dehydration-induced asymmetric packing stress will favor or inhibit fusion. Although the proposed picture is consistent with much accumulated data, it is not yet proven; experiments must now be devised to test its details. Finally, the proposed model is discussed in terms of potential implications for the mechanisms available to a cell in controlling more complex in vivo cell fusion processes such as endocytosis, exocytosis, protein sorting/transport, and viral budding/infection.

Animals↗
PubMed2005

DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells.

Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Osteoclasts form large multinuclear giant cells by fusion of mononuclear osteoclasts. How cell fusion is mediated, however, is unclear. We identify the dendritic cell-specific transmembrane protein (DC-STAMP), a putative seven-transmembrane protein, by a DNA subtraction screen between multinuclear osteoclasts and mononuclear macrophages. DC-STAMP is highly expressed in osteoclasts but not in macrophages. DC-STAMP-deficient mice were generated, and osteoclast cell fusion was completely abrogated in homozygotes despite normal expression of osteoclast markers and cytoskeletal structure. As osteoclast multinucleation was restored by retroviral introduction of DC-STAMP, loss of cell fusion was directly attributable to a lack of DC-STAMP. Defects in osteoclast multinucleation reduce bone-resorbing activity, leading to osteopetrosis. Similar to osteoclasts, foreign body giant cell formation by macrophage cell fusion was also completely abrogated in DC-STAMP-deficient mice. We have thus identified an essential regulator of osteoclast and macrophage cell fusion, DC-STAMP, and an essential role of osteoclast multinucleation in bone homeostasis.

Animals↗
PubMed2005

Human immunodeficiency virus 1 (HIV-1) envelope-dependent cell-cell fusion modulation by HIV-positive sera is related to disease progression.

Fusion of CD4+ cells by HIV-1 envelope proteins (Env) is a mechanism of virus spread and cell damage. Production of antibodies able to influence cell-cell fusion in vivo may affect the course of the infection. The effect of sera from 49 HIV-1-positive patients was tested on an in vitro fusion assay using Env-expressing and normal Jurkat T cells labelled with DiI and DiO dyes, and flow cytometry for quantification of cell-cell fusion. Sera varied in their activity on fusion: 69.4 % inhibited, 24.5 % had no effect and 6.1 % enhanced cell fusion. Fusion activity correlated positively with the CD4+ T-cell count and inversely with the viral load. Removal of IgG or IgM from sera reduced or eliminated inhibition and enhancing activities, respectively. Antibodies with inhibitory activity predominate in early and intermediate stages of infection, whereas loss of inhibition or enhancement of fusion correlates with progression to AIDS.

Cell Fusion↗
PubMed2002

Comparison of microinjection (piezo-electric) and cell fusion for nuclear transfer success with different cell types in cattle.

Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.

Animals↗
PubMed2004

Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating.

Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion. The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood. We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen. The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region. The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion. A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions. In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p. The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling. Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion.

Fungal Proteins↗
PubMed1998

Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis.

Cell fusions produce multinucleate syncytia that are crucial to the structure of essential tissues in many organisms [1-5]. In humans the entire musculature, much of the placenta, and key cells in bones and blood are derived from cell fusion. Yet the developmental fusion of cell membranes has never been directly observed and is poorly understood. Similarity between viral fusion proteins and recently discovered cellular proteins implies that both cell-cell and virus-cell fusion may occur by a similar mechanism [6-8]. Paradoxically, however, fusion of enveloped viruses with cells involves an opening originating as a single pore [9-11], whereas electron microscopy studies of cell-cell fusion describe simultaneous breakdown of large areas of membrane [12, 13]. Here, we have shown that developmental cell fusion is indeed consistent with initiation by a virus-like, pore-forming mechanism. We examined live cell fusions in the epithelia of Caenorhabditis elegans embryos by a new method that integrates multiphoton, confocal, and electron microscopy. The fusion aperture always originated at a single point restricted to the apical adherens junction and widened slowly as a radial wavefront. The fusing membranes dispersed by vesiculation, rather than simple unfolding of the conjoined double bilayer. Thus, in these cells fusion appears to require two specialized sequential processes: formation of a unique primary pore and expansion of the opening by radial internalization of the interacting cell membranes.

Animals↗
PubMedDate not supplied

Induction in chimpanzees of antibodies inhibiting receptor-mediated cell fusion by HIV glycoprotein.

Cell fusion of HTLV-IIIB-infected EBV-transformed B cells and CD4+ T cells was inhibited by sera from eight of nine HIV infected chimpanzees. Syncytia formation was reduced by sixty percent relative to control after only 5 minutes of preincubation of the HIV infected cells with immune primate serum, indicating that these antibodies have high affinity for HIV protein on the surface of infected cells. Serum dilutions that blocked formation of syncytia irreversibly within 24 hrs also blocked expression of HIV antigens by the target CD4+ cells. Three of four animals inoculated with the LAV or HTLV-IIIB strain of HIV developed antibodies inhibiting CD4-dependent cell fusion by HIV glycoprotein (CFI-antibodies) 2-3 months after inoculation coincident with development of HIV specific IgG antibodies. Similar early CFI-antibody responses occurred in two second passage chimpanzees. In contrast, a chimpanzee infected with a third passage of LAV had a delayed CFI-antibody response, indicating that variants of HIV with divergent CFI epitopes did eventually emerge. Delayed development of CFI-antibodies (6-11 months after inoculation) relative to HIV specific IgG ELISA antibody was also seen in a chimpanzee on primary passage and a chimpanzee on second passage of HTLV-IIIB. No CFI-antibodies were detected in a chimpanzee following inoculation with human brain tissue, while antibodies to other structural proteins were recognized by immunoblotting. These results indicate that changes in CFI epitopes occur under immune pressure and that the appearance of CFI-antibodies depends on the time after infection and on the degree to which CFI epitopes of the inoculum strain diverge from those of the test strain.

Animals↗
PubMed2004

Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast.

To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2Delta. Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2Delta cell fusion defect. BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2Delta and rvs161Delta. Lrg1p contains a Rho-GAP homologous region. Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2Delta. Furthermore, lrg1Delta caused a more severe mating defect in combination with other cell fusion mutations. Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection. Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of beta(1-3)-glucan synthase in vitro. beta(1-3)-glucan deposition was increased in lrg1Delta strains and mislocalized to the tip of the mating projection in fus2Delta strains. High-copy LRG1 suppressed the mislocalization of beta(1-3) glucan in fus2Delta strains. We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Polyethylene glycol-mediated cell fusion.

Polyethylene glycol (PEG)-mediated cell fusion is a simple and efficient technique used widely for the production of somatic cell hybrids and for nuclear transfer in mammalian cloning. We describe a basic protocol of PEG-mediated cell fusion for the production of somatic cell hybrids. Fusion can be performed between adherent and suspension cells or between adherent cells or suspension cells. Either whole cells or microcells can be used as donors to fuse with recipient cells. Microcell fusion is particularly useful in transfer of a single or a limited number of chromosomes between various types of cells. Using this method, we have successfully introduced mammalian minichromosomes into a variety of vertebrate cells. The technique described here can be adapted for uses in other cell fusion involved research. This protocol, in principle, provides guidelines for further development of PEG mediated cell fusion technology.

Animals↗
PubMed1997

An anti-fusion regulatory protein-1 monoclonal antibody suppresses human parainfluenza virus type 2-induced cell fusion.

Fusion regulatory protein-1 (FRP-1) regulates virus-mediated cell fusion and induces poly-karyocyte formation of monocytes without any fusogen. We have recently reported that FRP-1 and the 4F2/CD98 heavy chain are identical molecules. Cell fusion in Newcastle disease virus (NDV)-infected HeLa cells was enhanced when cells were incubated with anti-FRP-1 MAb. Anti-FRP-1 MAbs also induced human immunodeficiency virus gp160-mediated cell fusion. However, HBJ127, an anti-FRP-1/4F2/CD98 MAb that enhanced cell fusion in NDV-infected cells, delayed human parainfluenza virus type 2 (HPIV-2)-induced cell fusion in HeLa cells, although these viruses belong to the same genus Rubulavirus. No anti-FRP-1 MAbs enhanced cell fusion in HPIV-2-infected HeLa cells. Anti-FRP-1 MAbs including HBJ127 showed no effect on virus growth and expression levels of virus-specific poly-peptides in HPIV-2-infected HeLa cells, indicating that the delay in cell fusion by an anti-FRP-1 MAb is not due to suppression of virus replication. When HeLa cells were transfected with an expression vector harbouring HPIV-2 HN and F genes, cell fusion was also suppressed by HBJI27, but the effect was weak in comparison with virus-infected cells. These data indicate anti-FRP-1 antibodies not only induce/enhance, but also inhibit/delay virus-induced cell fusion and therefore FRP-1 molecules are multifunctional.

Animals↗
PubMed2000

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA.

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.

Antiviral Agents↗
PubMed1986

Fluorescence photobleaching recovery as a method to quantitate viral envelope-cell fusion: application to study fusion of Sendai virus envelopes with cells.

A method to quantitate viral envelope-cell fusion at the single-cell level is presented. The method is based on the incorporation of nonquenching concentrations of a fluorescent lipid probe into the viral envelope; fluorescence photobleaching recovery (FPR) is then applied to measure the lateral mobilization of the probe in the cell membrane following fusion. In adsorbed (unfused) viral envelopes, the probe is constricted to the envelope and is laterally immobile on the micrometer scale of FPR. After fusion, the envelope lipids intermix with the plasma membrane, the probe becomes laterally mobile, and the fraction of fused viral envelopes can be extracted from the fraction of mobile probe molecules. The method has several advantages: (i) It clearly distinguishes fused from internalized envelopes, as probes in the latter are immobile in FPR studies; (ii) focusing the laser beam on specific regions of the cell enables region-specific measurements of the fusion level; (iii) one cell is measured at a time, enabling studies on the distribution of the fusion level within the cell population. The new method was employed to study fusion of reconstituted Sendai virus envelopes (RSVE) containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine with several cell types. Experiments with human erythrocytes demonstrated that the lateral mobilization measured is due to fusion and not the result of exchange processes. The extent of RSVE-erythrocyte fusion determined by FPR was similar to that measured by two other independent methods (fluorescence dequenching and removal of adsorbed RSVE by dithiothreitol).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1972

Cell fusion by canine distemper virus-infected cells.

AV3 cells (continuous human amnion) infected with the Onderstepoort strain of canine distemper virus produced cell fusion within 2 to 5 hr when added to AV3 cell monolayers. An apparent requirement for intact, infected cells was demonstrated by showing that (i) frozen-and-thawed infected cells failed to induce fusion, (ii) infected cells frozen in the presence of glycerol retained their ability to induce fusion, (iii) infected cells subjected to swelling in hypotonic buffer and homogenization lost their ability to fuse cells, and (iv) semipurified and concentrated virus preparations with infectivity titers as high as 10(7.5) mean tissue culture doses per ml failed to induce fusion within 5 hr. Preparations of intact, infected cells had a mean log(10) ratio of infectivity to fusion activity of 3.6. Treatment with beta-propiolactone rendered the active preparations free from detectable infectivity while they retained their ability to cause cell fusion. Cycloheximide did not block the formation of syncytia in assay cells. This type of cell fusion was neutralized by canine distemper virus immune antisera, and measles virus immune sera showed a slight degree of cross-neutralization. Other cell lines, HEp-2, MA 139 (embryonic ferret lung), MA 104 (embryonic rhesus monkey kidney), and Vero (African green monkey kidney) were also susceptible.

Amnion↗