scholarly journals HIV-Envelope–Dependent Cell-Cell Fusion: Quantitative Studies

2009 ◽  
Vol 9 ◽  
pp. 746-763 ◽  
Author(s):  
Leonor Huerta ◽  
Nayali López-Balderas ◽  
Evelyn Rivera-Toledo ◽  
Guadalupe Sandoval ◽  
Guillermo Gómez-Icazbalceta ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Fusion ◽  
Relative Size ◽  
Resonance Energy ◽  
Biological Properties ◽  
Target Cells ◽  
Multinucleated Cells ◽  
Hiv Envelope ◽  
Cell Cell

Interactionin vitrobetween cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006,J. Virol. Methods138, 17–23; López-Balderas et al., 2007,Virus Res.123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

scholarly journals Antigenic Properties of the Human Immunodeficiency Virus Transmembrane Glycoprotein during Cell-Cell Fusion

2002 ◽  
Vol 76 (23) ◽  
pp. 12123-12134 ◽  
Author(s):  
Catherine M. Finnegan ◽  
Werner Berg ◽  
George K. Lewis ◽  
Anthony L. DeVico
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Fusion ◽  
Target Cells ◽  
Dye Transfer ◽  
Temperature Dependent ◽  
Gp41 Ectodomain ◽  
Immunodeficiency Virus ◽  
Hiv Envelope ◽  
Soluble Cd4 ◽  
Cell Cell

ABSTRACT 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 HIVHXB2 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°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.

FRET in the Analysis of In Vitro Cell–Cell Fusion by Flow Cytometry

2015 ◽  
pp. 217-227 ◽  
Author(s):  
Guillermo Gómez-Icazbalceta ◽  
Mirna Berenice Ruiz-Rivera ◽  
Edmundo Lamoyi ◽  
Leonor Huerta
Keyword(s):  
Flow Cytometry ◽  
Cell Fusion ◽  
Cell Cell

scholarly journals Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

2016 ◽  
Vol 6 ◽  
Author(s):  
Mai Izumida ◽  
Haruka Kamiyama ◽  
Takashi Suematsu ◽  
Eri Honda ◽  
Yosuke Koizumi ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Envelope Protein ◽  
Target Cells ◽  
Cell Cell

scholarly journals Virus-Mediated Cell-Cell Fusion

2020 ◽  
Vol 21 (24) ◽  
pp. 9644
Author(s):  
Héloïse Leroy ◽  
Mingyu Han ◽  
Marie Woottum ◽  
Lucie Bracq ◽  
Jérôme Bouchet ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Fusion Proteins ◽  
Target Cells ◽  
Special Focus ◽  
Human Pathogens ◽  
General Process ◽  
Tissue Organization ◽  
Donor Cells ◽  
Infected Cells ◽  
Cell Cell

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.

scholarly journals Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Victor K. Outlaw ◽  
Francesca T. Bovier ◽  
Megan C. Mears ◽  
Maria N. Cajimat ◽  
Yun Zhu ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Ex Vivo ◽  
Supportive Therapy ◽  
Medical Intervention ◽  
Heptad Repeat ◽  
Cell Monolayers ◽  
Viral Spread ◽  
Human Airway ◽  
Cell Cell

ABSTRACT The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

scholarly journals The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

2003 ◽  
Vol 77 (5) ◽  
pp. 3058-3066 ◽  
Author(s):  
Laurie J. Earp ◽  
Sue E. Delos ◽  
Robert C. Netter ◽  
Paul Bates ◽  
Judith M. White
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Low Ph ◽  
Target Cells ◽  
Dependent Manner ◽  
Neutral Ph ◽  
Subtype A ◽  
Avian Sarcoma ◽  
Virus Subtype ◽  
Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

scholarly journals Interleukin 4 induces cultured monocytes/macrophages to form giant multinucleated cells.

1988 ◽  
Vol 167 (2) ◽  
pp. 598-611 ◽  
Author(s):  
A McInnes ◽  
D M Rennick
Keyword(s):  
Bone Marrow ◽  
Inflammatory Response ◽  
Cell Fusion ◽  
Cell Cultures ◽  
Soft Agar ◽  
Interleukin 4 ◽  
Multinucleated Cells ◽  
Granulomatous Lesions

Giant multinucleated cells (GMCs) are associated with granulomatous lesions that form in response to various infectious and noninfectious agents. The present study shows that mouse IL-4 induces the in vitro formation of GMCs by factor-dependent bone marrow and alveolar monocytes via cell fusion. GMCs appear 2 d after incubation of cell cultures with 20 U/ml or more of IL-4. Anti-IL-4 mAbs block the appearance of GMCs in these cultures, indicating that IL-4 acts directly on monocytes to promote fusion and does not secondarily induce the production of other soluble fusion factors. In soft agar cultures, IL-4 also causes the aggregation of macrophages and diminishes their migration. The role of IL-4 in a granulomatous inflammatory response is discussed.

scholarly journals Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5

2019 ◽  
Vol 218 (9) ◽  
pp. 3117-3133 ◽  
Author(s):  
Frank van Drogen ◽  
Ranjan Mishra ◽  
Fabian Rudolf ◽  
Michal J. Walczak ◽  
Sung Sik Lee ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Cell Fusion ◽  
Polarized Growth ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Pheromone Signaling ◽  
Stress Signals ◽  
Cell Cell

Cells continuously adapt cellular processes by integrating external and internal signals. In yeast, multiple stress signals regulate pheromone signaling to prevent mating under unfavorable conditions. However, the underlying crosstalk mechanisms remain poorly understood. Here, we show that mechanical stress activates Pkc1, which prevents lysis of pheromone-treated cells by inhibiting polarized growth. In vitro Pkc1 phosphorylates conserved residues within the RING-H2 domains of the scaffold proteins Far1 and Ste5, which are also phosphorylated in vivo. Interestingly, Pkc1 triggers dispersal of Ste5 from mating projections upon mechanically induced stress and during cell–cell fusion, leading to inhibition of the MAPK Fus3. Indeed, RING phosphorylation interferes with Ste5 membrane association by preventing binding to the receptor-linked Gβγ protein. Cells expressing nonphosphorylatable Ste5 undergo increased lysis upon mechanical stress and exhibit defects in cell–cell fusion during mating, which is exacerbated by simultaneous expression of nonphosphorylatable Far1. These results uncover a mechanical stress–triggered crosstalk mechanism modulating pheromone signaling, polarized growth, and cell–cell fusion during mating.

In Vitro Cytotoxicity Study of the Nano-Hydroxyapatite/Bacterial Cellulose Nanocomposites

2009 ◽  
Vol 610-613 ◽  
pp. 1011-1016 ◽  
Author(s):  
Yan Mei Chen ◽  
Ting Fei Xi ◽  
Yu Dong Zheng ◽  
Yi Zao Wan
Keyword(s):  
Tissue Engineering ◽  
Bone Tissue Engineering ◽  
Bacterial Cellulose ◽  
Bone Tissue ◽  
Biological Response ◽  
Biological Properties ◽  
In Vitro Cytotoxicity ◽  
National Standard ◽  
Target Cells

The nanocomposite of nano-hydroxyapatite/bacterial cellulose (nHA/BC) obtained by depositing in simulated body fluid (SBF), incorporating their excellent mechanical and biological properties, is expected to have potential applications in bone tissue engineering. However, the biological response evaluation of biomaterials is required to provide useful information to improve their design and application. In this article, the in vitro cytotoxicity of composites nHA/BC as well as its degradation residues was studied. Scanning electron microscopy (SEM) was used to observe the morphology of original materials and their degradation residues. The degree of degradation was evalued by measuring the concentration of reducing sugar (glucose) by ultraviolet spectrophotometer. Bone-forming osteoblasts (OB) and infinite culture cell line L929 fibroblasts were used to measure the cytotocixity of materials with MTT assay. Both kinds of cells in infusion proliferate greatly in a normal form and their relative growth rate (RGR) exceeds by 75%, which shows the cytotoxicity of materials is graded as 0~1, according to the national standard. Nevertheless, bone-forming OB cells, as a kind of target cells, are more susceptive on the cytotoxicity than infinite culture fibroblast cells L929. The results suggest the nanocomposite of nHA/BC without cytotoxicity is greatly promising as a kind of scaffold materials for bone tissue engineering and tissue functional cells are more suited to evaluate the cytotoxicity of biomedical materials.

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