Induction of cell-cell fusion from without by human herpesvirus 6B

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.     

Antigenic properties of the human immunodeficiency virus envelope during cell-cell fusion

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4( degrees )C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.     

The first family of cell-cell fusion

Our understanding of the developmentally critical process of cell-cell fusion has been greatly advanced by the identification of the first family of cell-cell fusion proteins. Together, the two founding members of the FF family execute the majority of cell-cell fusion events in C. elegans.     

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.     

基于包膜蛋白和Tat蛋白筛选HIV-1细胞融合抑制剂的高效方法

旨在建立一个细胞-细胞融合系统,高效筛选对HIV-1病毒细胞-细胞间传播有抑制作用的药物。构建了p EGFP-Tat质粒,将p EGFP-Tat质粒和HIV-1包膜质粒共转染HEK-293T细胞,成为表达Tat蛋白和包膜蛋白的效应细胞,然后与表达CD4及辅助受体和β-半乳糖苷酶、荧光素酶双报告基因的靶细胞TZM-bl融合,建立了细胞-细胞融合系统,并进行条件优化,确定了最佳的融合体系。用阳性融合抑制剂maraviroc以及没有融合抑制作用的AZT和raltegravir作用于该体系,证明该系统可以特异性有效筛选具有细胞融合抑制作用的药物。用该系统测试了8个样本,发现两种样品对融合有一定的抑制作用。该方法背景值低,特异性强,可用来高效筛选具有切断HIV-1病毒细胞-细胞间传播作用的抗病毒药物。     

Characterization of molecules mediating cell-cell communication in human cardiac valve interstitial cells

Cell-cell interactions and adhesion determine cellular architectural organization, proliferation, signaling, differentiation, and death. We have identified the molecular components of different cell-cell junctions in human valve interstitial cells (ICs) both in situ and in culture. ICs were isolated, cultured, and phenotyped for cell surface and cytoplasmic markers by flow cytometry and immunocytochemistry. Western blotting was used to identify and quantify the molecular components of these cell-cell junctions in human valve ICs and compared with expression in smooth muscle and fibroblast cell types. N-cadherin and desmoglein were weakly detected on a low percentage of ICs, and the other classical cadherins were not detected. α- and β-catenin, but not γ-catenin, were expressed at equivalent levels by all valve ICs. Valve ICs did not express connexin-32 and-40; however, connexin-26 and-43 were equally expressed by a low percentage of ICs, demonstrating cell surface and cytoplasmic expression, and connexin-45 was weakly expressed. The other cell types also expressed N-cadherin, α- and β-catenin, desmoglein and connexin-43. The expression of these junctional molecules was predominantly by valve ICs on the inflow side of the valves. Human valve ICs have the ability to communicate with other valve ICs and mediate cell-cell adhesion via N-cadherin, connexin-26 and-43, and desmoglein. The junctions between valve ICs could support an interconnecting and coordinated cellular unit capable of controlling the functionality of the valve.     

Effects of hemagglutinin-neuraminidase protein mutations on cell-cell fusion mediated by human parainfluenza type 2 virus

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.     

Envelope glycoproteins of hantavirus can mediate cell-cell fusion independently

Hantaviruses (HTVs) are enveloped viruses and can induce low PH-dependent cell fusion. In this report we molecularly cloned viral glycoproteins (GPs) cDNA and nucleocapsid (NP) cDNA of two strains of Hantaan virus and one strain of Seoul virus and expressed in Vero E6 cells under control of a CMV promoter. The examinations of viral gene expressions were carried out by IFA and immune-precipitation. After treatment with low PH (PH 5.8) medium the syncytium were observed in the cells transfected with the GPs clones while in the cells transfected with the NP clones we did not find this phenomenon. Furthermore cotransfection of the NP and GPs did not enhance fusion activity. Treatment with anti-GP monoclonal antibodies could inhibit fusion activity whereas the antibodies against NP could not. These results indicated that GPs can mediate cell-cell fusion independently.     

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.     

Fluorescence dequenching kinetics of single cell-cell fusion complexes.

In an earlier paper which models the cell-cell (or virus-cell) fusion complex as two partial spherical vesicles joined at a narrow neck (Rubin, R. J., and Yi-der Chen. 1990. Biophys. J. 58:1157-1167), the redistribution by diffusion of lipid-like molecules through the neck between the two fused cell surfaces was studied. In this paper, we extend the study to the calculation of the kinetics of fluorescence increase in a single fusion complex when the lipid-like molecules are fluorescent and self-quenching. The formalism developed in this paper is useful in deducing fusion activation mechanisms from cuvette fluorescence measurements in cell-cell fusion systems. Two different procedures are presented: 1) an exact one which is based on the exact local density functions obtained from diffusion equations in our earlier study; and 2) an approximate one which is based on treating the kinetics of transfer of probes between the two fused cells as a two-state chemical reaction. For typical cell-cell fusion complexes, the fluorescence dequencing curves calculated from the exact and approximate procedures are very similar. Due to its simplicity, the approximate method should be very useful in future applications. The formalism is applied to a typical cell-cell fusion complex to study the sensitivity of dequenching curves to changes in various fusion parameters, such as the radii of the cells, the radius of the pore at the fusion junction, and the number of probes initially loaded to the complex.     

Lateral mobility of reconstituted Sendai virus envelope glycoproteins on human erythrocytes: correlation with cell-cell fusion

We have recently employed fluorescence photobleaching recovery (FPR) to demonstrate that the envelope glycoproteins of Sendai virions become laterally mobile on the surface of human erythrocytes following fusion [Henis, Y. I., Gutman, O., & Loyter, A. (1985) Exp. Cell Res. 160, 514-526]. In order to investigate whether this lateral mobilization is involved in the mechanism of virally mediated cell-cell fusion, or is merely a result of viral envelope-cell fusion, we have now performed FPR studies on erythrocytes fused with reconstituted Sendai virus envelopes (RSVE). These RSVE, which were prepared by solubilization of Sendai virions with Triton X-100 followed by removal of the detergent through adsorption to SM-2 Bio-beads, fused with human erythrocytes as efficiently as native virions but induced cell-cell fusion to a much lower degree. The fraction of the viral envelope glycoproteins that became laterally mobile in the erythrocyte membrane following fusion was markedly lower in the case of RSVE than in the case of native virions. The lower cell-cell fusion activity of the RSVE does not appear to be due to inactivation of the viral fusion protein, since the envelope-cell fusion and hemolytic activities of the RSVE were similar to those of native virions. Moreover, fusion with RSVE or with native virions resulted in the incorporation of rather similar amounts of viral glycoproteins into the cell membrane. Since the reduced fraction of laterally mobile viral glycoproteins correlates with the lower cell-cell fusion activity of the RSVE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion

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.     

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.     

A virus-encoded cell-cell fusion machine dependent on surrogate adhesins

The reovirus fusion-associated small transmembrane (FAST) proteins function as virus-encoded cellular fusogens, mediating efficient cell-cell rather than virus-cell membrane fusion. With ectodomains of only approximately 20-40 residues, it is unclear how such diminutive viral fusion proteins mediate the initial stages (i.e. membrane contact and close membrane apposition) of the fusion reaction that precede actual membrane merger. We now show that the FAST proteins lack specific receptor-binding activity, and in their natural biological context of promoting cell-cell fusion, rely on cadherins to promote close membrane apposition. The FAST proteins, however, are not specifically reliant on cadherin engagement to mediate membrane apposition as indicated by their ability to efficiently utilize other adhesins in the fusion reaction. Results further indicate that surrogate adhesion proteins that bridge membranes as close as 13 nm apart enhance FAST protein-induced cell-cell fusion, but active actin remodelling is required for maximal fusion activity. The FAST proteins are the first example of membrane fusion proteins that have specifically evolved to function as opportunistic fusogens, designed to exploit and convert naturally occurring adhesion sites into fusion sites. The capacity of surrogate, non-cognate adhesins and active actin remodelling to enhance the cell-cell fusion activity of the FAST proteins are features perfectly suited to the structural and functional evolution of these fusogens as the minimal fusion component of a virus-encoded cellular fusion machine. These results also provide a basis for reconciling the rudimentary structure of the FAST proteins with their capacity to fuse cellular membranes.     

Characterization of the monomer-dimer equilibrium of human cytomegalovirus protease by kinetic methods

Herpesviruses encode a serine protease that is essential for the maturation of infectious virions. This protease has a unique polypeptide backbone fold and contains a novel Ser-His-His catalytic triad. It exists in a monomer-dimer equilibrium in solution, but only the dimer form of the enzyme is catalytically active. The stability of this dimer is affected by the presence of anti-chaotropic agents. Most of the reported Kd values for this dimer (between 0.6 and 6 microM) are inconsistent with the fact that the protease is routinely assayed at 20-50 nM concentrations, as only monomeric species would be expected with such Kd values. We have characterized the monomer-dimer equilibrium of HCMV protease using a new method, which observes the exchange between dimers of the wild-type enzyme and the active-site Ser132Ala mutant in a titration experiment. The Kd of the dimer was determined to be 8 microM and 31 nM in the absence or presence of anti-chaotropic agents (10% glycerol and 0.5 M Na2SO4), respectively. Detailed kinetic analysis also showed that, in addition to the 260-fold stabilization of the dimer, the anti-chaotropic agents produced a 7-fold enhancement in the catalytic activity of the dimer.     

Characterization of a panel of insertion mutants in human cytomegalovirus glycoprotein B

Glycoprotein B (gB; gpUL55) of human cytomegalovirus (HCMV) plays a critical role in virus entry and cell-to-cell spread of infection. To define the structure-function relationships in gB, a panel of linker-insertion mutations was generated throughout the coding region. This strategy yielded a panel of 22 mutants with four amino acid insertions and 3 large truncation mutants. Assessment of the mutant proteins' biosynthetic properties and folding patterns analyzed in context with predicted secondary features revealed novel insights into gB's structure and trafficking properties. All of the insertion mutants were able to assemble into oligomers, suggesting that oligomerization is tolerant of small insertions and/or that multiple regions of the protein may be involved. Computer algorithm predictions of gB's secondary structure indicate that the furin-recognized cleavage site falls within an exposed loop. This loop may be particularly sensitive to structural alterations, since insertions upstream and downstream of the cleavage site rendered the mutant proteins cleavage defective. In addition, a strong correlation existed between terminal folding and cleavage of gB. Interestingly, terminal folding was not correlated with delivery to the cell surface but may influence the rate of transport to the cell surface. Nine mutants, containing insertions in both the extracellular and intracellular portions of gB, retained wild-type structural properties. This panel of characterized gB mutants, the first of this type for an HCMV protein, will be a useful tool in dissecting the role of gB during HCMV infection.     

Specific inhibition of human cytomegalovirus glycoprotein B-mediated fusion by a novel thiourea small molecule

A novel small molecule inhibitor of human cytomegalovirus (HCMV) was identified as the result of screening a chemical library by using a whole-virus infected-cell assay. Synthetic chemistry efforts yielded the analog designated CFI02, a compound whose potency had been increased about 100-fold over an initial inhibitor. The inhibitory concentration of CFI02 in various assays is in the low nanomolar range. CFI02 is a selective and potent inhibitor of HCMV; it has no activity against other CMVs, alphaherpesviruses, or unrelated viruses. Mechanism-of-action studies indicate that CFI02 acts very early in the replication cycle, inhibiting virion envelope fusion with the cell plasma membrane. Mutants resistant to CFI02 have mutations in the abundant virion envelope glycoprotein B that are sufficient to confer resistance. Taken together, the data suggest that CFI02 inhibits glycoprotein B-mediated HCMV virion fusion. Furthermore, CFI02 inhibits the cell-cell spread of HCMV. This is the first study of a potent and selective small molecule inhibitor of CMV fusion and cell-cell spread.     

Semliki Forest virus induced cell-cell fusion at neutral extracellular pH

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.