Detection of cell-cell fusion mediated by Ebola virus glycoproteins

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 beta-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.     

Association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces.

We previously observed that cell fusion caused by human parainfluenza virus type 2 or type 3 requires the expression of both the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the same virus type, indicating that a type-specific interaction between F and HN is needed for the induction of cell fusion. In the present study we have further investigated the fusion properties of F and HN proteins of parainfluenza virus type 1 (PI1), type 2 (PI2), and type 3 (PI3), Sendai virus (SN), and simian virus 5 (SV5) by expression of their glycoprotein genes in HeLa T4 cells using the vaccinia virus-T7 transient expression system. Consistent with previous results, cell fusion was observed in cells transfected with homotypic F/HN proteins; with one exception, coexpression of any combination of F and HN proteins from different viruses did not result in cell fusion. The only exception was found with the closely related PI1 HN and SN HN glycoproteins, either of which could interact with SN F to induce cell fusion upon coexpression as previously reported. By specific labeling and coprecipitation of proteins expressed on the cell surface, we observed that anti-PI2 HN antiserum coprecipitated PI2 F when the homotypic PI2 F and PI2 HN were coexpressed, but not the F proteins of other paramyxoviruses when heterotypic F genes were coexpressed with PI2 HN, suggesting that the homotypic F and HN proteins are physically associated with each other on cell surfaces. Furthermore, we observed that PI3 F was found to cocap with PI3 HN but not with PI2 HN, also indicating a specific association between the homotypic proteins. These results indicate that the homotypic F and HN glycoproteins are physically associated with each other on the cell surface and suggest that such association is crucial to cell fusion induced by paramyxoviruses.     

Electron microscopic study of measles virus infection: cell fusion and hemadsorption.

Virus-induced cell fusion has been studied after infection of Vero cells with measles virus. Scanning and transmission electron microscopy were combined with immunoperoxidase labeling of measles antigens to correlate viral production and distribution of virus-induced erythrocyte binding sites with progress of fusion. Release of infectious virus started before syncytia were detected and decreased while the number and size of syncytia were increasing. Most virions were seen budding from mononucleated cells or from the periphery of syncytia where cells were being recruited. Moving inward, the surfaces of syncytia where cells were being recruited. Moving inward, the surfaces of syncytia were covered with numerous ridges containing viral antigen, but few viral buds were seen, suggesting that syncytia might be sites of defective viral formation. Hemadsorption occurred predominantly within the confines of syncytia. Erythrocytes were scattered sparsely over immature syncytia but were densely packed in the center of mature syncytia. Active binding sites for erythrocytes were located on cell villi and ridges covered with measles antigens. Hemadsorption was completely inhibited in measles virus-infected cultures pretreated with virus-specific immunoglobulin G for 1 h at 4 degrees C. However, when these cultures were shifted to 37 degrees C, hemadsorbing sites were recovered at the periphery of enlarging syncytia. Virus-induced sites for erythrocyte adsorption were found to move centripetally on syncytium membranes as fusion progressed.     

Cell fusion activities of Hantaan virus envelope glycoproteins

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.     

Glycoprotein gI of pseudorabies virus promotes cell fusion and virus spread via direct cell-to-cell transmission.

Mutants of pseudorabies virus defective in either glycoprotein gI or gIII are only slightly less virulent for mice and chickens than is wild-type virus, while mutants defective in both gI and gIII are avirulent. To clarify the reason for the lack of virulence of the gI- gIII- mutants, we have analyzed in some detail the interactions of these mutants with their hosts. The results obtained showed that the gI glycoprotein is an accessory protein that promotes cell fusion. This conclusion is based on the findings that in some cell types, syncytium formation is significantly reduced in mutants deficient in gI. Furthermore, despite efficient replication, gI- mutants form significantly smaller plaques on some cell types. Finally, while wild-type and gI- virus are neutralized similarly by antisera, the size of the plaques formed by gI- mutants, but not by wild-type virus, is reduced by the presence of neutralizing antibodies in the overlay. Passive immunization of mice with neutralizing antipseudorabies virus sera is also considerably more effective in protecting them against challenge with gI- mutants than in protecting them against challenge with wild-type virus. These results show that gI- mutants are deficient in their ability to form syncytia and to spread directly by cell-to-cell transmission and that these mutants spread mainly by adsorption of released virus to uninfected cells. Wild-type virus and gIII- mutants, however, spread mainly via direct cell-to-cell transmission both in vivo and in vitro. We postulate that the lack of virulence of the gIII- gI- virus is attributable to its inability to spread by either mode, the defect in gIII affecting virus spread by adsorption of released virus and the defect in gI affecting cell-to-cell spread. Although a gI- gIII- mutant replicates as well as a gIII- mutant, it will be amplified much less well. Our results with in vitro systems show that this is indeed the case.     

Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.     

Purified glycoproteins of influenza virus stimulate cell-mediated cytotoxicity in vivo

Previously, we reported that purified surface influenza viral glycoproteins can induce cell-mediated cytotoxicity (CMC) in vitro. Both neuraminidase (NA) and hemagglutinin (HA) were equally good stimulators, on an equimolar basis. In order to broaden the scope of these observations, we examined whether these glycoproteins stimulate natural killer (NK) activity in vivo. Biologically active preparations of glycoproteins NA and HA were purified from virus A/USSR/90/77 (H1N1) and recombinant virus A/USSR/92/77 (H1) x A/Prague/1/56 (N7), respectively. The studies were carried out using the optimal doses of NA and HA. In a 4-hour NK assay, using NK-sensitive YAC-1 cells as targets, both viral glycoproteins stimulated the NK activity of splenocytes of BALB/c and C3H mice. This stimulation was independent of the route of administration (intravenous or intraperitoneal) of the antigen. The observed NK activity was viral antigen-specific and could be modulated to levels comparable to those observed with the standard stimulator, polyinosinic acid-polycytidylic acid, by the use of an appropriate synthetic adjuvant, stearyl tyrosinate. Direct and indirect evidences suggest that the enhanced CMC is due to NK cells. These observations imply that enhancement of NK activity is the intrinsic property of influenza NA and HA.     

Cell fusion by the envelope glycoproteins of persistent measles viruses which caused lethal human brain disease.

Measles virus (MV) rarely induces lethal diseases of the human central nervous system characterized by reduced expression of the viral envelope proteins and by lack of viral budding. The MV envelope contains two integral membrane proteins, termed fusion (F) protein and hemagglutinin (H) protein, and a membrane-associated matrix (M) protein. Previously, analysis of MV genes from autopsy material indicated that the M protein and the F protein intracellular domain are often drastically altered by mutations. Here, we present evidence that truncation of the F protein intracellular domain does not impair fusion function, and we suggest that this alteration interferes with viral budding. Unexpectedly, certain combinations of functional F and H proteins were unable to induce syncytium formation, an observation suggesting that specific F-H protein interactions are required for cell fusion. We also found that three of four H proteins of persistent MVs are defective in intracellular transport, oligosaccharide modification, dimerization, and fusion helper function. Thus, MVs replicating in the brain at the terminal stage of infection are typically defective in M protein and in the two integral membrane proteins. Whereas the M protein appears dispensable altogether, partial preservation of F-protein function and H-protein function seems to be required, presumably to allow local cell fusion. Certain subtle alterations of the F and H proteins may be instrumental for disease development.     

Cell-cell fusion induced by measles virus amplifies the type I interferon response

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.     

In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins.     

Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV-1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pH-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the glycoproteins. Infection of cells expressing gB or gD glycoproteins with HSV-1 inhibited the low pH-induced cell fusion. The results suggest that although the glycoproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.     

Interplay between lipids and viral glycoproteins during hemolysis and fusion by influenza virus

Since mixtures of lipids alone are known to elicit membrane fusion without participation of fusion proteins, the role of viral lipids in the so-called virus-induced hemolysis and cell fusion has been investigated, using as a model the fowl plague virus (influenza A/FPV/Rostock/H7N1). The experiments were planned in a way that allowed quantitative modification of viral lipids without changing envelope glycoproteins. Under the conditions employed, cholesterol oxidase of Nocardia erythropolis and phospholipase C of Bacillus cereus were shown to completely modify their substrates in the virus without altering virus-associated hemagglutinating and neuraminidase activities. It was found with such enzyme treatment that virus-induced hemolysis and cell fusion are greatly influenced by cholesterol and phospholipids of the envelope. It became clear, that hemolysis and fusion are differently dependent on the nature of lipid components even though mediated by the same viral glycoproteins.     

The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins

Presumptive myoblasts from explants of chick embryo pectoral muscle proliferate, differentiate, and fuse to form multinucleate myotubes. One event critical to multinucleate cell formation is the specific adhesion of myoblasts before union of their membranes. In the studies reported here five known inhibitors of myotube formation--trifluoperazine, sodium butyrate, chloroquine, 1,10 phenanthroline, and tunicamycin--were tested for their effect on the Ca++-dependent myoblast adhesion step. The first four inhibitors of myotube formation do not perturb myoblast adhesion but rather block fusion of aggregated cells, which suggests that these agents perturb molecular events required for the union of the lipid bilayers. By contrast, tunicamycin exerts its effect by inhibiting the myoblast adhesion step, thereby blocking myotube formation. The effect of tunicamycin can be blocked by a protease inhibitor, however, which implies that the carbohydrate residues protect the glycoproteins from proteolytic degradation rather than participate directly in cell-cell adhesion. Whereas trypsin treatment of myoblasts in the absence of Ca++ destroys the cells' ability to exhibit Ca++-dependent adhesion, the presence of Ca++ during trypsin treatment inhibits the enzyme's effect, which suggests that myoblast adhesion is mediated by a glycoprotein(s) that has a conformation affected by Ca++. Finally, myoblast adhesion is inhibited by an antiserum raised against fusion-competent myoblasts. The effect of the antiserum is blocked by a fraction from the detergent extract of pectoral muscle that binds to immobilized wheat germ agglutinin, which again suggests that glycoproteins mediate Ca++-dependent myoblast adhesion.     

Accumulation of Sendai virus glycoproteins in cell-cell contact regions and its role in cell fusion.

Lateral motion of the viral envelope proteins in the target cell membrane was shown recently to be essential for cell fusion by Sendai virus (Henis, Y. I., Herman-Barhom, Y., Aroeti, B., and Gutman, O. (1989) J. Biol. Chem. 264, 17119-17125). To explore the mechanism that gives rise to this requirement, we have now investigated the distribution of Sendai virus envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) on human erythrocytes in the course of fusion, using fluorescence microscopy and image analysis. In these studies, both the F and the HN proteins were found to accumulate in cell-cell contact regions, on the time scale of the fusion process. We propose that migration of the viral glycoproteins to cell contact regions and accumulation at the contact sites are essential parts of the fusion mechanism and form the basis to the requirement for their lateral motion in the fusion event.