Virus-induced Cell-cell Fusion Research Articles

Establishment of a system to quantify wild-type herpes simplex virus-induced cell-cell fusion reveals a role of N-glycosylation of HSV-1 envelope glycoprotein B in cell-cell fusion.

Wild-type herpes simplex virus (HSV) strains infrequently mediate cell-cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell-cell fusion induced by wild-type HSV strains. The established system clarified that the HSV-1 envelope glycoprotein B and its N-glycosylation at asparagine at position 141 were required for efficient cell-cell fusion. This study provides a link between cell-cell fusion induced by wild-type HSV-1 and viral pathogenesis in vivo.

Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein.

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins.

Virus-Mediated Cell-Cell Fusion.

Cell-cell fusion between eukaryotic cells is a general process involved in many physiological and pathological conditions, including infections by bacteria, parasites, and viruses. As obligate intracellular pathogens, viruses use intracellular machineries and pathways for efficient replication in their host target cells. Interestingly, certain viruses, and, more especially, enveloped viruses belonging to different viral families and including human pathogens, can mediate cell-cell fusion between infected cells and neighboring non-infected cells. Depending of the cellular environment and tissue organization, this virus-mediated cell-cell fusion leads to the merge of membrane and cytoplasm contents and formation of multinucleated cells, also called syncytia, that can express high amount of viral antigens in tissues and organs of infected hosts. This ability of some viruses to trigger cell-cell fusion between infected cells as virus-donor cells and surrounding non-infected target cells is mainly related to virus-encoded fusion proteins, known as viral fusogens displaying high fusogenic properties, and expressed at the cell surface of the virus-donor cells. Virus-induced cell-cell fusion is then mediated by interactions of these viral fusion proteins with surface molecules or receptors involved in virus entry and expressed on neighboring non-infected cells. Thus, the goal of this review is to give an overview of the different animal virus families, with a more special focus on human pathogens, that can trigger cell-cell fusion.

Efficient Reovirus- and Measles Virus-Mediated Pore Expansion during Syncytium Formation Is Dependent on Annexin A1 and Intracellular Calcium

Orthoreovirus fusion-associated small transmembrane (FAST) proteins are dedicated cell-cell fusogens responsible for multinucleated syncytium formation and are virulence determinants of the fusogenic reoviruses. While numerous studies on the FAST proteins and enveloped-virus fusogens have delineated steps involved in membrane fusion and pore formation, little is known about the mechanics of pore expansion needed for syncytiogenesis. We now report that RNA interference (RNAi) knockdown of annexin A1 (AX1) expression dramatically reduced both reptilian reovirus p14 and measles virus F and H protein-mediated pore expansion during syncytiogenesis but had no effect on pore formation. A similar effect was obtained by chelating intracellular calcium, which dramatically decreased syncytiogenesis in the absence of detectable effects on p14-induced pore formation. Coimmunoprecipitation revealed calcium-dependent interaction between AX1 and p14 or measles virus F and H proteins, and fluorescence resonance energy transfer (FRET) demonstrated calcium-dependent p14-AX1 interactions in cellulo. Furthermore, antibody inhibition of extracellular AX1 had no effect on p14-induced syncytium formation but did impair cell-cell fusion mediated by the endogenous muscle cell fusion machinery in C2C12 mouse myoblasts. AX1 can therefore exert diverse, fusogen-specific effects on cell-cell fusion, functioning as an extracellular mediator of differentiation-dependent membrane fusion or as an intracellular promoter of postfusion pore expansion and syncytium formation following virus-mediated cell-cell fusion. Numerous enveloped viruses and nonenveloped fusogenic orthoreoviruses encode membrane fusion proteins that induce syncytium formation, which has been linked to viral pathogenicity. Considerable insights into the mechanisms of membrane fusion have been obtained, but processes that drive postfusion expansion of fusion pores to generate syncytia are poorly understood. This study identifies intracellular calcium and annexin A1 (AX1) as key factors required for efficient pore expansion during syncytium formation mediated by the reptilian reovirus p14 and measles virus F and H fusion protein complexes. Involvement of intracellular AX1 in syncytiogenesis directly correlates with a requirement for intracellular calcium in p14-AX1 interactions and pore expansion but not membrane fusion and pore formation. This is the first demonstration that intracellular AX1 is involved in pore expansion, which suggests that the AX1 pathway may be a common host cell response needed to resolve virus-induced cell-cell fusion pores.

Critical role of exosomes in sperm-egg fusion and virus-induced cell-cell fusion.

In mammals, two integral membrane proteins, sperm IZUMO1 and egg CD9, regulate sperm-egg fusion, and their roles are critical, but yet unclear. Recent studies, however, indicate interesting connections between the sperm-egg fusion and virus-induced cell-cell fusion. First, CD9-containing exosome-like vesicles, which are released from wild-type eggs, can induce the fusion between sperm and CD9-deficient egg, even though CD9-deficient eggs are highly refractory to the fusion with sperm. This finding provides strong evidence for the involvement of CD9-containing, fusion-facilitating vesicles in the sperm-egg fusion. Secondly, there are similarities between the generation of retroviruses in the host cells and the formation of small cellular vesicles, termed exosomes, in mammalian cells. The exosomes are involved in intercellular communication through transfer of proteins and ribonucleic acids (RNAs) including mRNAs and microRNAs. These collective studies provide an insight into the molecular mechanism of membrane fusion events.

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.

Mechanisms of Varicella-Zoster Virus Neuropathogenesis in Human Dorsal Root Ganglia

Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects sensory ganglia and reactivates from latency to cause herpes zoster. VZV replication was examined in human dorsal root ganglion (DRG) xenografts in mice with severe combined immunodeficiency using multiscale correlative immunofluorescence and electron microscopy. These experiments showed the presence of VZV genomic DNA, viral proteins, and virion production in both neurons and satellite cells within DRG. Furthermore, the multiscale analysis of VZV-host cell interactions revealed virus-induced cell-cell fusion and polykaryon formation between neurons and satellite cells during VZV replication in DRG in vivo. Satellite cell infection and polykaryon formation in neuron-satellite cell complexes provide mechanisms to amplify VZV entry into neuronal cell bodies, which is necessary for VZV transfer to skin in the affected dermatome during herpes zoster. These mechanisms of VZV neuropathogenesis help to account for the often severe neurologic consequences of herpes zoster.

Antibodies against analogous heptad repeat peptide HR212 of Newcastle Disease Virus inhibit virus-cell membrane fusion

Abstract Membrane fusion is a key step in enveloped virus entry. Highly conserved heptad repeat regions (HR1 and HR2) of Newcastle disease virus (NDV) fusion protein (F) are critical functional domains for viral membrane fusion. They display different conformations in the membrane fusion states and are viewed as candidate targets for neutralizing antibody responses. We previously reported that an analog of heptad repeat peptides HR2-HR1-HR2(HR212) and HR2 could inhibit NDV induced cell-cell membrane fusion. Here we show that HR212 can induce the production of highly potent antibody in immunized rabbits which could recognize full length peptides of both HR1 and HR2 and inhibit NDV hemagglutination and NDV entry. These suggest that either HR212 or its antibody could be an inhibitor of virus-induced cell-cell membrane fusion. *Supported by National Program on Key Basic Research Project (Grant No.2005CB523001)

Cell-cell fusion induced by the avian reovirus membrane fusion protein is regulated by protein degradation.

The p10 fusion-associated small transmembrane protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the protein for degradation following p10 insertion into an early membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral membrane fusion protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell fusion.

EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.

A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types.

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.

Two neutralizing anti-V3 monoclonal antibodies act by affecting different functions of human immunodeficiency virus type 1.

Monoclonal antibody (MAb) ICR41.1i (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3 , and MAb F58 (mouse IgG1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immunofluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.li had no effect on HIV-1-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41.1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, postattachment neutralization (PAN) was investigated. F58 mediated PAN at 21 degrees C and 35 degrees C. However, ICR41.1i gave PAN at 21 degrees C but not at 35 degrees C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.

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.

Early Events of Semliki Forest Virus-Induced Cell-Cell Fusion

Insect cells( Aedes albopictus) were infected with Semliki Forest Virus. Cell-cell fusion was then induced by lowering the extracellular pH. The underlying processes were examined by monitoring the intercellular current flow, I j. Experimentally, this involved the use of cell pairs in conjunction with a dual voltage-clamp method. This approach allowed us to monitor the kinetics of fusion at high temporal resolution. The fusion process began shortly after acidification (delay: 3-138 sec). Initially, I j increased in a stepwise manner, later on it developed more gradually. Fusion between two cells reached a steady state within 7-70 sec. The steps in I j are attributable to the formation of cytoplasmic connections between the cells, presumably involving proteinaceous fusion pores. The mean amplitude of I j steps corresponds to a conductance of 300 pS, consistent with a pore radius of 1 nm. Cytoplasmic connections developed rapidly, i.e., I j steps occurred within less than 1 msec. The absence of I j flickering implies that formation of cytoplasmic connections, and hence SFV induced cell-cell fusion, is irreversible.

Syncytium-inducing mutations localize to two discrete regions within the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B.

Herpes simplex virus type 1 glycoprotein B (gB) is essential for virus entry, an event involving fusion of the virus envelope with the cell surface membrane, and virus-induced cell-cell fusion, resulting in polykaryocyte, or syncytium, formation. The experiments described in this report employed a random mutagenesis strategy to develop a more complete genetic map of mutations resulting in the syn mutant phenotype. The results indicate that syn mutations occur within two essential and highly conserved hydrophilic, alpha-helical regions of the gB cytoplasmic domain. Region I is immediately proximal to the transmembrane domain and includes residues R796 to E816/817. Region II is localized centrally in the cytoplasmic domain and includes residues A855 and R858. Positively charged residues were particularly affected in both regions, suggesting that charge interactions may be required to suppress the syn mutant phenotype. No syn mutations were identified within the transmembrane domain. A virus containing a rate of entry (roe) mutation at residue A851, either within or immediately proximal to syn region II, was isolated. Since roe mutations have also been discovered in the external domain of gB, it appears likely that the external and cytoplasmic domains cooperate in virus penetration. Moreover, the observation that both roe and syn mutations occur in the cytoplasmic domain further suggests that gB functions in an analogous manner in both membrane fusion events. It might be predicted from these observations that membrane fusion involves transduction of a fusion signal along the gB molecule through the transmembrane domain. Communication between the external and cytoplasmic domain may thus be required for gB-mediated membrane fusion events.

Cell-to-cell spread of HIV-1 occurs within minutes and may not involve the participation of virus particles

Although virus infections have been classically studied with “cell-free” virion preparations, many animal viruses are able to spread both in vitro and in vivo by inducing cell-cell fusion. An efficient system to monitor the cell-to-cell spread of HIV-1 has been developed employing chronically infected H9 donor cells. Under appropriate conditions of cocultivation with uninfected cells, the synthesis of unintegrated viral DNA, monitored by Southern blot hybridization, occurred between 2 and 4 hr following infection; viral proteins were detected 8 to 12 hr following cocultivation and progeny virions were released into the medium by 16 hr. The use of metabolic inhibitors specific envelope/receptor antibodies revealed that the cell-to-cell spread of HIV required: (1) gpl20-CD4 interaction and (2) reverse transcription. Light and electron microscopy, fluorescent dye redistribution, and soluble CD4 competition experiments all demonstrated that the HIV-induced cell-cell fusion began within 10 to 30 min of cocultivation. Surprisingly, the electron microscopic analyses also suggested that budding or mature virus particles did not participate in this process. Thus the virus-induced cell-cell fusion observed is very likely the result of gp120/gp41 proteins, on the surface of infected cells, interacting with CD4 molecules on uninfected cells. These findings are of immediate importance in understanding the mechanism(s) of HIV-1 transmission in vivo and for the design of effective vaccines and antiviral agents.

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 (less than 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.

Fusion resistance and decreased infectability as major host cell determinants of coronavirus persistence

Mouse hepatitis virus persists in cultures of a subline (designated LM-K) of mouse LM cells but produces a lytic infection in L-2 cells. Persistence in the LM-K cells was not accompanied by production of is mutants or of soluble anti-MHV factors. Infectious center assay demonstrated an approximately 500-fold lower level of infectibility by MHV of the LM-K cells as compared to L-2 cells. On an infected cell basis, production levels of infectious progeny and viral RNA were comparable between the two cell lines. The extent of virus-induced cell-cell fusion, however, was markedly reduced in the LM-K cells. Cell-mixing experiments showed that both infected L-2 and LM-K cells have the capacity of fusing with neighboring uninfected L-2 cells but not with uninfected LM-K cells. This suggests that the decreased level of fusion observed in the LM-K infection is due not to absence of viral fusion protein at the cell surface, but rather to an inherent resistance of the LM-K cell membrane to MHV-induced fusion. It is believed that such fusion resistance in LM-K cells moderates virus dissemination throughout the culture, thereby contributing to a state of virus persistence.