scholarly journals Extensive Syncytium Formation Mediated by the Reovirus FAST Proteins Triggers Apoptosis-Induced Membrane Instability

2005 ◽  
Vol 79 (13) ◽  
pp. 8090-8100 ◽  
Author(s):  
Jayme Salsman ◽  
Deniz Top ◽  
Julie Boutilier ◽  
Roy Duncan
Keyword(s):  
Cell Fusion ◽  
Fusion Reaction ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Syncytium Formation ◽  
Replication Cycle ◽  
Progeny Virus ◽  
Effector Caspase ◽  
Fast Proteins ◽  
Cell Cell

ABSTRACT The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by nonenveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively nonleaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ectodomain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic nonenveloped viruses, with nonleaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.

scholarly journals Cell-Cell Fusion Induced by the Avian Reovirus Membrane Fusion Protein Is Regulated by Protein Degradation

2004 ◽  
Vol 78 (11) ◽  
pp. 5996-6004 ◽  
Author(s):  
Maya Shmulevitz ◽  
Jennifer Corcoran ◽  
Jayme Salsman ◽  
Roy Duncan
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Protein ◽  
Syncytium Formation ◽  
Replication Cycle ◽  
Stable Form ◽  
Avian Reovirus ◽  
Membrane Fusion Protein ◽  
Cell Cell

ABSTRACT 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.

scholarly journals Cell–cell fusion induced by reovirus FAST proteins enhances replication and pathogenicity of non-enveloped dsRNA viruses

2019 ◽  
Vol 15 (4) ◽  
pp. e1007675 ◽  
Author(s):  
Yuta Kanai ◽  
Takahiro Kawagishi ◽  
Yusuke Sakai ◽  
Ryotaro Nouda ◽  
Masayuki Shimojima ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Fast Proteins ◽  
Dsrna Viruses ◽  
Cell Cell

scholarly journals Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

2020 ◽  
Vol 118 (1) ◽  
pp. e2007526118
Author(s):  
Ka Man Carmen Chan ◽  
Ashley L. Arthur ◽  
Johannes Morstein ◽  
Meiyan Jin ◽  
Abrar Bhat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Cell Fusion ◽  
Conformational Changes ◽  
Binding Motif ◽  
Mechanical Pressure ◽  
Actin Nucleation ◽  
Viral Spread ◽  
Fast Proteins ◽  
Cell Cell

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP–mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

scholarly journals Entry of Vaccinia Virus and Cell-Cell Fusion Require a Highly Conserved Cysteine-Rich Membrane Protein Encoded by the A16L Gene

2006 ◽  
Vol 80 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Suany Ojeda ◽  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Syncytium Formation ◽  
Redox System ◽  
Cysteine Residues ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Cell Cell

ABSTRACT The vaccinia virus A16L open reading frame encodes a 378-amino-acid protein with a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that is conserved in all sequenced members of the poxvirus family. The A16 protein was expressed late in infection and incorporated into intracellular virus particles with the N-terminal segment of the protein exposed on the surface. The cysteine residues were disulfide bonded via the poxvirus cytoplasmic redox system. Unsuccessful attempts to isolate a mutant virus with the A16L gene deleted suggested that the protein is essential for replication. To study the role of the A16 protein, we made a recombinant vaccinia virus that has the Escherichia coli lac operator system regulating transcription of the A16L gene. In the absence of inducer, A16 synthesis was repressed and plaque size and virus yield were greatly reduced. Nevertheless, virus morphogenesis occurred and normal-looking intracellular and extracellular virus particles formed. Purified virions made in the presence and absence of inducer were indistinguishable, though the latter had 60- to 100-fold-lower specific infectivity. A16-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, A16-deficient virions were unable to induce low-pH-triggered syncytium formation. The phenotype of the inducible A16L mutant was similar to those of mutants in which synthesis of the A21, A28, H2, or L5 membrane protein was repressed, indicating that at least five conserved viral proteins are required for entry of poxviruses into cells as well as for cell-cell fusion.

scholarly journals Rational Site-Directed Mutations of the LLP-1 and LLP-2 Lentivirus Lytic Peptide Domains in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41 Indicate Common Functions in Cell-Cell Fusion but Distinct Roles in Virion Envelope Incorporation

2003 ◽  
Vol 77 (6) ◽  
pp. 3634-3646 ◽  
Author(s):  
Vandana Kalia ◽  
Surojit Sarkar ◽  
Phalguni Gupta ◽  
Ronald C. Montelaro
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Syncytium Formation ◽  
Immunodeficiency Virus ◽  
Arginine Residues ◽  
Hiv 1 ◽  
Cell Cell

ABSTRACT Two highly conserved cationic amphipathic α-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses . Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.

scholarly journals Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus.

1996 ◽  
Vol 134 (4) ◽  
pp. 863-872 ◽  
Author(s):  
M Kielian ◽  
M R Klimjack ◽  
S Ghosh ◽  
W A Duffus
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Infectious Clone ◽  
Low Ph ◽  
Spike Protein ◽  
Initial Reaction ◽  
Target Membrane ◽  
Cell Cell

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell-cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.

scholarly journals The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Edward Yang ◽  
Ann M. Arvin ◽  
Stefan L. Oliver
Keyword(s):  
Varicella Zoster Virus ◽  
Cell Fusion ◽  
Postherpetic Neuralgia ◽  
Positive Charge ◽  
Syncytium Formation ◽  
Wild Type ◽  
Painful Condition ◽  
Varicella Zoster ◽  
Multinuclear Cells ◽  
Cell Cell

ABSTRACT The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (−1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a −0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt. IMPORTANCE Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed, as the current vaccine is not recommended for immunocompromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue, which has been proposed to be a factor contributing to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.

scholarly journals Timing of Galectin-1 Exposure Differentially Modulates Nipah Virus Entry and Syncytium Formation in Endothelial Cells

2014 ◽  
Vol 89 (5) ◽  
pp. 2520-2529 ◽  
Author(s):  
Omai B. Garner ◽  
Tatyana Yun ◽  
Olivier Pernet ◽  
Hector C. Aguilar ◽  
Arnold Park ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Virus Infection ◽  
Host Cell ◽  
Cell Fusion ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Progeny Virus ◽  
Human Endothelial Cells ◽  
Opposing Effects

ABSTRACTNipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome.IMPORTANCENipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by “bridging” the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell.

scholarly journals A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 229 ◽  
Author(s):  
María García-Murria ◽  
Neus Expósito-Domínguez ◽  
Gerard Duart ◽  
Ismael Mingarro ◽  
Luis Martinez-Gil
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
High Throughput Screening ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Viral Proteins ◽  
Viral Fusion ◽  
Multinucleated Cells ◽  
Virus Entry Inhibitors ◽  
Cell Cell

Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so-called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicellular Complementation (BiMuC), provides an adjustable platform to qualitatively and quantitatively investigate the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.

scholarly journals Overexpression of Thiol/Disulfide Isomerases Enhances Membrane Fusion Directed by the Newcastle Disease Virus Fusion Protein

2008 ◽  
Vol 82 (24) ◽  
pp. 12039-12048 ◽  
Author(s):  
Surbhi Jain ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Membrane Fusion ◽  
Disease Virus ◽  
Cell Fusion ◽  
Newcastle Disease ◽  
Syncytium Formation ◽  
F Protein ◽  
Disulfide Exchange ◽  
Disulfide Isomerases ◽  
Cell Cell

ABSTRACT Newcastle disease virus (NDV) fusion (F) protein directs membrane fusion, which is required for virus entry and cell-cell fusion. We have previously shown that free thiols are present in cell surface-expressed NDV F protein and that blocking the production of free thiols by thiol-disulfide exchange inhibitors inhibited the membrane fusion mediated by F protein (J Virol. 81:2328-2339, 2007). Extending these observations, we evaluated the role of the overexpression of two disulfide bond isomerases, protein disulfide isomerase (PDI) and ERdj5, in cell-cell fusion mediated by NDV glycoproteins. The overexpression of these isomerases resulted in significantly increased membrane fusion, as measured by syncytium formation and content mixing. The overexpression of these isomerases enhanced the production of free thiols in F protein when expressed without hemagglutination-neuraminidase (HN) protein but decreased free thiols in F protein expressed with HN protein. By evaluating the binding of conformation-sensitive antibodies, we found that the overexpression of these isomerases favored a postfusion conformation of surface-expressed F protein in the presence of HN protein. These results suggest that isomerases belonging to the PDI family catalyze the production of free thiols in F protein, and free thiols in F protein facilitate membrane fusion mediated by F protein.

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