scholarly journals The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1974 ◽  
Vol 140 (3) ◽  
pp. 405-411 ◽  
Author(s):  
Charles A. Pasternak ◽  
Kingsley J. Micklem
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Sendai Virus ◽  
Membrane Integrity ◽  
Intracellular Metabolites ◽  
Nitrophenyl Phosphate ◽  
Biochemical Studies ◽  
Membrane Components ◽  
Interesting System

1. Phospholipids prelabelled with [14C]acetate, [32P]phosphate, [3H]- or [14C]-choline or [3H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [3H]fucose, [14C]galactose or [3H]glucosamine. Less than 1nmol of lysophosphatidylcholine/107 cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K+ leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca2+ into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G1 and S periods of the cell cycle. 5. Leakage is inhibited by Ca2+, but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.

scholarly journals Covalent attachment of enzyme as a membrane-label for viable eucaryotic cells.

1979 ◽  
Vol 83 (2) ◽  
pp. 511-515 ◽  
Author(s):  
N M Hogg ◽  
B Rotman
Keyword(s):  
Cell Surface ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Covalent Attachment ◽  
Histological Evidence ◽  
Coupling Procedure ◽  
Specific Affinity ◽  
Enzyme Label ◽  
Membrane Components

An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D-galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.

Movement of Ca(2+)-ATPase molecules within the sarcoplasmic/endoplasmic reticulum in skeletal muscle

1996 ◽  
Vol 109 (10) ◽  
pp. 2529-2537
Author(s):  
Z. Kaprielian ◽  
S.W. Robinson ◽  
D.M. Fambrough ◽  
P.D. Kessler
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Mouse Cells ◽  
Membrane Bound ◽  
Species Specific ◽  
Membrane Components ◽  
P Type

The endoplasmic reticulum undergoes rapid, microscopic changes in its structure, including extension and anastomosis of tubular elements. Such dynamism is expected to manifest itself also as rapid intermixing of membrane components, at least within subdomains of the endoplasmic reticulum. Here we present evidence of a similar dynamism in the sarcoplasmic reticulum of developing skeletal muscle. The sarcoplasmic reticulum is sometimes considered a specialized type of endoplasmic reticulum, but it appears to be a rather static set of membrane-bound elements, repetitively arranged to enwrap each sarcomere of each myofibril. Both endoplasmic reticulum and sarcoplasmic reticulum contain P-type Ca(2+)-ATPases that transport calcium from the cytosol into their lumen. In the experiments reported here, chicken and mouse cells were fused by polyethylene glycol, natural myogenic cell fusion, or Sendai virus. The redistribution of Ca(2+)-ATPase molecules between chick and mouse endoplasmic reticulum/sarcoplasmic reticulum was followed by immunofluorescence microscopy in which species-specific monoclonal antibodies to chick and mouse Ca(2+)-ATPases were used. Redistribution was time- and temperature-dependent but independent of protein synthesis as well as the method of cell fusion. Intermixing occurred on a time scale of tens of minutes at 37 degrees C. These results verify the dynamic nature of the sarcoplasmic reticulum and illustrate an aspect of the special relationship between endoplasmic reticulum and sarcoplasmic reticulum.

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.

Quantitative cell fusion: the fusion sensitivity (FS) potential

1981 ◽  
Vol 49 (1) ◽  
pp. 87-97
Author(s):  
D. Rohme
Keyword(s):  
Cell Line ◽  
Cell Fusion ◽  
Cell Lines ◽  
Dose Response ◽  
Sendai Virus ◽  
Biological Significance ◽  
Diploid Cell ◽  
Mammalian Cell Lines ◽  
Virus Dose ◽  
A Cell

The dose response of Sendai virus-induced cell fusion was studied in 10 mammalian cell lines, comprising 5 continuous and 5 diploid cell lines originating from 5 species. The extent of fusion was calculated using a parameter directly proportional to the number of fusion events (t-parameter). At lower levels of fusion the dose response was found to be based on the same simple kinetic rules in all cell lines and was defined by the formula: t = FS. FAU/(I + FS. FAU), where FS (fusion sensitivity) is a cell-specific constant of the fusion rate and FAU (fusion activity units) is the virus dose. The FS potential of a cell line was determined as the linear regression coefficient of the fusion index (t/(I - t)) on the virus dose. At higher levels of fusion, when the fusion extent reached cell-line-specific maximal levels, the dose response was not as uniform. In general, and particularly in the cases of the diploid cell lines, these maximal levels were directly proportional to the FS potentials. Thus, it was concluded that the FS potential is the basic quantitative feature, which expresses the cellular fusion efficiency. The fact that FS varied extensively between cell lines, but at the same time apparently followed certain patterns (being higher in continuous compared to diploid cell lines and being related to the species of origin of the cells), emphasizes it biological significance as well as its possible usefulness in studies of the efficiency of various molecular interactions in the cell membrane/cytoskeleton system.

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.

Phosphatase systems in Fasciolopsis buski Lankester, 1857 and Gastrodiscus aegyptiacus Cobbold, 1876

Parasitology ◽  
1973 ◽  
Vol 67 (2) ◽  
pp. 197-204 ◽  
Author(s):  
Madan M. Goil
Keyword(s):  
Enzyme Activity ◽  
Tartaric Acid ◽  
Maximum Activity ◽  
Nitrophenyl Phosphate ◽  
Biochemical Studies ◽  
Phosphate Hydrolysis ◽  
Fasciolopsis Buski ◽  
Enzyme I ◽  
Inhibited Enzyme ◽  
Acid Phosphomonoesterase

Biochemical studies on the non-specific phosphomonoesterases have demonstrated the presence of acid phosphomonoesterase with maximum activity at pH 4·0 in Gastrodiscus aegyptiacus (enzyme I) and at pH 4·5 in the case of Fasdolopsis buski (enzyme II). The Km for ρ-nitrophenyl phosphate hydrolysis was 0·66 mM for enzyme I and 1·1 mM for enzyme II. Different concentrations of fluoride, arsenate, tartrate, tartaric acid, cysteine and copper brought about inhibition of both enzymes and magnesium, iodoaeetate, iodoacetamide and EDTA had no influence on either enzyme activity. Cobalt activated both enzymes while zinc inhibited enzyme I and strongly stimulated enzyme II.

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.

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