Studies of membrane fusion. VI. Mechanism of the membrane fusion and cell swelling stages of Sendai virus-mediated cell fusion

1980 ◽  
Vol 43 (1) ◽  
pp. 103-118
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Fusion Event ◽  
Virus Envelope ◽  
Membrane Rupture ◽  
Membrane Tubules

The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

Studies of membrane fusion. V. Fusion of erythrocytes with non-haemolytic Sendai virus

1979 ◽  
Vol 36 (1) ◽  
pp. 85-96
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Erythrocyte Membrane ◽  
Sendai Virus ◽  
Lateral Diffusion ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Viral Membrane ◽  
Cytoplasmic Bridges ◽  
Viral Envelopes

The fusion of human erythrocytes with non-haemolytic ‘1-day’ Sendai virus has been studied by electron microscopy. The mechanism of viral envelope-cell fusion is the same as that described previously for haemolytic ‘3-day’ Sendai virus except that fusion is frequently arrested at an initial stage when 2 segments of smooth linear viral membrane fuse and become incorporated into the erythrocyte membrane. After longer periods of incubation at 37 degrees C, in addition to many partly fused virus particles, long (up to 4 micrometer) lengths of smooth linear viral membrane are seen within the erythrocyte membrane which arise by linear aggregation of shorter (approximately 0.25 micrometer long) segments of smooth linear membrane derived from individual fused viral envelopes. Cell-Cell fusion, as a result of the fusion of a viral envelope with 2 adjacent erythrocytes also occurs but, in the absence of cell swelling, fusion is arrested at this stage with cells joined by one (or more) small cytoplasmic bridges. Typical fused cells are produced if such cells are swollen with hypotonic buffer. These observations provide further evidence that membrane fusion and cell swelling are distinct events in cell fusion and that cell swelling is the driving force both for completing the incorporation of the viral envelope into the cell membrane and for expanding cells connected by small cytoplasmic bridges to form spherical fused cells. Little lateral diffusion of viral envelope components occurs in the absence of cell swelling; in fact, some aggregation of components occurs. Comparison with previous studies using haemolytic ‘3-day’ Sendai virus suggests that virally induced cell swelling perturbs membrane structure so as to allow the rapid lateral diffusion of integrated viral envelope components.

scholarly journals Studies of membrane fusion. IV. Fusion of HeLa cells with Sendai virus

1979 ◽  
Vol 36 (1) ◽  
pp. 73-84
Author(s):  
S. Knutton
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Hela Cells ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Plasma Membrane ◽  
Virus Particles ◽  
Cell Plasma

The Sendai virus-induced fusion of HeLa cells has been studied by freeze-fracture electron microscopy. Freeze-fracture observations confirm previous scanning electron-microscope studies (1977) and show that at 4 degrees C virus particles bind to the cell surface and that cell agglutination results from the crosslinking by virus particles of microvilli on adjacent cells. Incubation at 37 degrees C initiates a change in viral envelope structure and fusion of ‘altered’ virus particles with the cell plasma membrane. Fusion of a virus particle with two crosslinked cells is probably the membrane fusion event which initiates cell-cell fusion; fusion is completed as a result of virally induced cell swelling. Lateral diffusion of viral envelope components following virus-cell fusion and, in some instances, an aggregation of plasma membrane intramembrane particles occurs in swollen cells. These observations show that the mechanisms of viral envelope-cell and probably cell-cell fusion are the same as have been reported for erythrocytes. Although endocytosis of intact virus particles does occur, the specialized cell-mediated mechanism for fusion of the viral envelope with the cell plasma membrane suggests that this, and not viropexis, is the mechanism of Sendai virus infection.

The role of cell swelling and haemolysis in Sendai virus-induced cell fusion and in the diffusion of incorporated viral antigens

1980 ◽  
Vol 42 (1) ◽  
pp. 153-167
Author(s):  
S. Knutton ◽  
T. Bachi
Keyword(s):  
Cell Fusion ◽  
Erythrocyte Membrane ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Haemolytic Activity ◽  
Erythrocyte Ghosts ◽  
Viral Antigens ◽  
Cell Cell

The role of the haemolytic activity of Sendai virus in cell-cell fusion has been examined in monolayers of human erythrocytes and erythrocyte ghosts fused with either haemolytic or non-haemolytic virus. Morphological observations indicate that cell swelling and haemolysis is a distinct event in cell-cell fusion irrespective of whether it is virally induced or, in the case of non-haemolytic virus, experimentally induced. Osmotic swelling appears to be the driving force by which cells which have established sites of membrane fusion expand such sites to form poly-erythrocytes. Immunofluorescent labelling of viral antigens incorporated into the erythrocyte membrane as a result of viral envelope-cell fusion indicates that diffusion of antigens in the plane of the membrane is restricted in intact erythrocytes and resealed erythrocyte ghosts but not in haemolysed erythrocytes or unsealed ghosts. A perturbation of the erythrocyte membrane resulting from osmotic lysis appears to form a prerequisite for the lateral diffusion of viral elements.

scholarly journals Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes

2017 ◽  
Vol 28 (26) ◽  
pp. 3801-3814 ◽  
Author(s):  
Sunandini Chandra ◽  
Raju Kalaivani ◽  
Manoj Kumar ◽  
Narayanaswamy Srinivasan ◽  
Debi P. Sarkar
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Bioactive Molecules ◽  
Host Cells ◽  
Actin Dynamics ◽  
Envelope Glycoproteins ◽  
Animal Cells ◽  
Viral Envelopes

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

scholarly journals HIV Cell Fusion Assay

2013 ◽  
Vol 19 (1) ◽  
pp. 108-118 ◽  
Author(s):  
Elizabeth B. Smith ◽  
Robert A. Ogert ◽  
David Pechter ◽  
Artjohn Villafania ◽  
Susan J. Abbondanzo ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Chemokine Receptor ◽  
Viral Entry ◽  
Envelope Glycoprotein ◽  
Laser Scanning ◽  
Viral Envelope ◽  
Fusion Event ◽  
Cxcr4 Signaling ◽  
Hiv Entry

The health and disease-related biology of the CXCR4 chemokine receptor presents the challenge of finding a small molecule that can bind CXCR4 and block T-cell tropic human immunodeficiency virus type 1 (HIV-1) cell entry, while preserving the ability of CXCR4 to respond to its native ligand, CXCL12. HIV entry into the host cell involves the interaction of the viral envelope glycoprotein gp120 binding to CD4, followed by a rearrangement in gp120, and subsequent interaction with the chemokine receptor CXCR4 or CCR5. These initial events can be re-created in a cell fusion assay that represents a surrogate system, mimicking the early stages of viral entry via these host cell receptors. In the current study, a T-tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from the T-tropic HIV NL4-3 and HeLa cells expressing CD4 and CXCR4. Detection of the cell fusion event was based on a Gal4/VP16-activated β-lactamase signal and was measured by automated microscopy or laser scanning plate cytometry. Changes in morphology associated with cell fusion were combined with β-lactamase activity to generate results with robust assay statistics in both 384-well and 1536-well plates. Compounds were subsequently characterized by CXCR4 signaling assays to eliminate functional antagonists and allow the identification of a function-sparing HIV entry inhibitor.

Studies of membrane fusion. III. Fusion of erythrocytes with polyethylene glycol

1979 ◽  
Vol 36 (1) ◽  
pp. 61-72
Author(s):  
S. Knutton
Keyword(s):  
Polyethylene Glycol ◽  
Cell Fusion ◽  
Close Contact ◽  
Freeze Fracture ◽  
Cell Swelling ◽  
Adjacent Cell ◽  
Cell Contact ◽  
Cell Agglutination ◽  
High Concentrations ◽  
Freeze Fracture Electron Microscopy

Freeze-fracture electron microscopy has been used to investigate the mechanism of polyethylene glycol-induced cell fusion. Interaction of cells with the high concentrations of polyethylene glycol required for cell fusion results in cell agglutination with large planar areas of very close contact between adjacent cell membranes. An aggregation of intramembrane particles into large patches at the sites of cell-cell contact accompanies cell agglutination. Fusion occurs following the removal of most of the PEG when cells only remain in close contact at small (approximately 0.1 micrometer diameter) plaques of smooth membrane resulting in cells connected by one (or more) small cytoplasmic connexions. Expansion to form spherical fused cells occurs by a process of cell swelling.

scholarly journals A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

2008 ◽  
Vol 83 (4) ◽  
pp. 1727-1741 ◽  
Author(s):  
Anuja Krishnan ◽  
Santosh K. Verma ◽  
Prashant Mani ◽  
Rahul Gupta ◽  
Suman Kundu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Sendai Virus ◽  
Biological Activities ◽  
Viral Envelope ◽  
Attachment Protein ◽  
Histidine Residues ◽  
Human Parainfluenza Virus ◽  
Mimicking Peptides

ABSTRACT Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.

scholarly journals Development of a Novel High-Throughput Surrogate Assay to Measure HIV Envelope/CCR5/CD4-Mediated Viral/Cell Fusion Using BacMam Baculovirus Technology

2003 ◽  
Vol 8 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Stephen Jenkinson ◽  
David C. Mc Coy ◽  
Sandy A. Kerner ◽  
Robert G. Ferris ◽  
Wendell K. Lawrence ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
High Throughput ◽  
Viral Envelope ◽  
Host Cells ◽  
Luciferase Reporter ◽  
Viral Fusion ◽  
Fusion Event ◽  
Surrogate Assay ◽  
Cell Cell

The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.

scholarly journals Membrane Fusion Tropism and Heterotypic Functional Activities of the Nipah Virus and Hendra Virus Envelope Glycoproteins

2002 ◽  
Vol 76 (22) ◽  
pp. 11186-11198 ◽  
Author(s):  
Katharine N. Bossart ◽  
Lin-Fa Wang ◽  
Michael N. Flora ◽  
Kaw Bing Chua ◽  
Sai Kit Lam ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Measles Virus ◽  
Nipah Virus ◽  
Recombinant Vaccinia Virus ◽  
Hendra Virus ◽  
Envelope Glycoproteins ◽  
Emerging Viruses ◽  
Fusion Event ◽  
Virus Envelope ◽  
Zoonotic Infections

ABSTRACT Nipah virus (NiV) and Hendra virus (HeV) are novel paramyxoviruses from pigs and horses, respectively, that are responsible for fatal zoonotic infections of humans. The unique genetic and biological characteristics of these emerging agents has led to their classification as the prototypic members of a new genus within the Paramyxovirinae subfamily called Henipavirus. These viruses are most closely related to members of the genus Morbillivirus and infect cells through a pH-independent membrane fusion event mediated by the actions of their attachment (G) and fusion (F) glycoproteins. Understanding their cell biological features and exploring the functional characteristics of the NiV and HeV glycoproteins will help define important properties of these emerging viruses and may provide new insights into paramyxovirus membrane fusion mechanisms. Using a recombinant vaccinia virus system and a quantitative assay for fusion, we demonstrate NiV glycoprotein function and the same pattern of cellular tropism recently reported for HeV-mediated fusion, suggesting that NiV likely uses the same cellular receptor for infection. Fusion specificity was verified by inhibition with a specific antiserum or peptides derived from the α-helical heptads of NiV or HeV F. Like that of HeV, NiV-mediated fusion also requires both F and G. Finally, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but here we show that the NiV and HeV glycoproteins are capable of highly efficient heterotypic functional activity with each other. However, no heterotypic activity was observed with envelope glycoproteins of the morbilliviruses Measles virus and Canine distemper virus.

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