Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell-cell fusion

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins–a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion–the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.

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Endosomal Proteolysis by Cathepsins Is Necessary for Murine Coronavirus Mouse Hepatitis Virus Type 2 Spike-Mediated Entry

Journal of Virology ◽

10.1128/jvi.00442-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5768-5776 ◽

Cited By ~ 99

Author(s):

Zhaozhu Qiu ◽

Susan T. Hingley ◽

Graham Simmons ◽

Christopher Yu ◽

Jayasri Das Sarma ◽

...

Keyword(s):

Cell Fusion ◽

Ammonium Chloride ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Low Ph ◽

Ph Values ◽

Neutral Ph ◽

Cell Cell ◽

Murine Coronavirus

ABSTRACT Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.

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CD46 on glial cells can function as a receptor for viral glycoprotein-mediated cell-cell fusion

Glia ◽

10.1002/glia.20219 ◽

2005 ◽

Vol 52 (3) ◽

pp. 252-258 ◽

Cited By ~ 15

Author(s):

Riccardo Cassiani-Ingoni ◽

Heather L. Greenstone ◽

Donatella Donati ◽

Anna Fogdell-Hahn ◽

Elena Martinelli ◽

...

Keyword(s):

Cell Fusion ◽

Glial Cells ◽

Viral Glycoprotein ◽

Cell Cell

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Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection

Journal of Virology ◽

10.1128/jvi.01613-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 4

Author(s):

Stefan L. Oliver ◽

Edward Yang ◽

Ann M. Arvin

Keyword(s):

Varicella Zoster Virus ◽

Cell Fusion ◽

Cellular Responses ◽

Melanoma Cells ◽

Live Cell ◽

Effective Vaccine ◽

Viral Glycoprotein ◽

Varicella Zoster ◽

Multinucleated Cells ◽

Cell Cell

ABSTRACT The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.

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Characterization of the cold-sensitive murine hepatitis virus mutants rescued from latently infected cells by cell fusion

Virology ◽

10.1016/0042-6822(79)90567-1 ◽

1979 ◽

Vol 98 (2) ◽

pp. 448-455 ◽

Cited By ~ 27

Author(s):

Stephen A. Stohlman ◽

Alan Y. Sakaguchi ◽

Leslie P. Weiner

Keyword(s):

Cell Fusion ◽

Hepatitis Virus ◽

Murine Hepatitis Virus ◽

Latently Infected ◽

Cold Sensitive ◽

Infected Cells

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CD9-dependent regulation of Canine distemper virus-induced cell–cell fusion segregates with the extracellular domain of the haemagglutinin

Journal of General Virology ◽

10.1099/vir.0.81629-0 ◽

2006 ◽

Vol 87 (6) ◽

pp. 1635-1642 ◽

Cited By ~ 13

Author(s):

K. Singethan ◽

E. Topfstedt ◽

S. Schubert ◽

W. P. Duprex ◽

B. K. Rima ◽

...

Keyword(s):

Cell Fusion ◽

Measles Virus ◽

Canine Distemper Virus ◽

Transmembrane Protein ◽

Extracellular Domain ◽

Canine Distemper ◽

Viral Glycoprotein ◽

F Protein ◽

H Protein ◽

Cell Cell

Antibodies to CD9, a member of the tetraspan transmembrane-protein family, selectively inhibit Canine distemper virus (CDV)-induced cell–cell fusion. Neither CDV-induced virus–cell fusion nor cell–cell fusion induced by the closely related morbillivirus Measles virus (MV) is affected by anti-CD9 antibodies. As CDV does not bind CD9, an unknown, indirect mechanism is responsible for the observed inhibition of cell–cell fusion. It was investigated whether this effect was restricted to only one viral glycoprotein, either the haemagglutinin (H) or the fusion (F) protein, which form a fusion complex on the surface of virions and infected cells, or whether it is dependent on both in transient co-transfection assays. The susceptibility to CD9 antibodies segregates with the H protein of CDV. By exchanging portions of the H proteins of CDV and MV, it was determined that the complete extracellular domain, including the predicted stem structure (stem 1, barrel strand 1 and stem 2) and globular head domain, of the CDV-H protein mediates the effect. This suggests that interaction of the CDV-H protein with an unknown cellular receptor(s) is regulated by CD9, rather than F protein-mediated membrane fusion.

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Using Split Luciferase Assay and anti-HSV Glycoprotein Monoclonal Antibodies to Predict a Functional Binding Site Between gD and gH/gL

Journal of Virology ◽

10.1128/jvi.00053-21 ◽

2021 ◽

Author(s):

Doina Atanasiu ◽

Wan Ting Saw ◽

Tina M Cairns ◽

Roselyn J Eisenberg ◽

Gary H Cohen

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

Cell Fusion ◽

Neutralizing Antibodies ◽

Rational Design ◽

Active Form ◽

The Other ◽

Luciferase Assay ◽

Functional Sites ◽

Cell Cell

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. HSV entry begins with gD binding its receptor (nectin-1), which then activates gH/gL to enable the conversion of pre-fusion gB to its active form to promote membrane fusion. Virus-neutralizing monoclonal antibodies (Mabs) interfere with one or more of these steps and localization of their epitopes identifies functional sites on each protein. Utilizing this approach, we have identified the gH/gL binding face on gD and the corresponding gD binding site on gH/gL. Here, we used combinations of these Mabs to define the orientation of gD and gH/gL relative to each other. We reasoned that if two Mabs, one directed at gD and the other at gH/gL, block fusion more effectively than when either were used alone (additive), then their epitopes would be spatially distanced and binding of one would not directly interfere with binding of the other during fusion. However, if the two Mabs blocked fusion with equal or lesser efficacy that when either were used alone (indifferent), we propose that their epitopes would be in close proximity in the complex. Using a live cell fusion assay, we found that some Mab pairings blocked the fusion with different mechanisms while other had a similar mechanisms of action. Grouping the different combinations of antibodies into indifferent and additive groups, we present a model for the orientation of gD vis-à-vis gH/gL in the complex. Importance: Virus entry and cell-cell fusion mediated by HSV require four essential glycoproteins, gD, gH/gL, gB and a gD receptor. Virus-neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with essential steps in the fusion pathway. Thus, the epitopes of these Mabs overlap and point to critical, functional sites on their target proteins. Here, we combined gD and gH/gL antibodies to determine whether they work in an additive or non-additive (indifferent) fashion to block specific events in glycoprotein-driven cell-cell fusion. Identifying combinations of antibodies that have additive effects will help in the rational design of an effective therapeutic “polyclonal antibody” to treat HSV disease. In addition, identification of the exact contact regions between gD and gH/gL can inform the design of small molecules that would interfere with the gD-gH/gL complex formation, thus preventing the virus from entering the host cell.

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