Specific Movement of Cell Membranes Fused with HVJ (Sendai Virus)

Abstract

The cell-fusion phenomenon with hemagglutinating virus of Japan (Sendai virus) (HVJ) (1-5) seems to offer a suitable experimental system for studies on the biological nature of animal-cell membranes (6). Ehrlich ascites tumor cells rapidly agglutinate on addition of HVJ at 00; this agglutination occurs with adsorption of HVJ virions onto the surface of the cells. When cell aggregates are incubated at 370, the viral envelopes react with the cell membranes, the membranes of adjacent cells in cell aggregates fuse together within a few minutes, and spherical fused cells appear within 30 min. The efficiency of cell fusion is a function of the concentration of HVJ added (7).In the course of these reactions, some abnormal conditions for the cells are produced. At an early stage, the cell-membrane structure becomes unstable due to reaction with the virus; decrease in the membrane potential and membrane resistance, with leak of ions through the cell membranes, can be detected electrophysiologically. The instability is reversed with progress of cell fusion (8). At about the same stage of the reaction, fusion of viral envelopes with cell membranes occurs (9); glycoproteins and lipids originating from the envelope are explanted into the cell membranes. Within 30 min of the cellfusion process, the original cell-surface area decreases, the degree of decrease depending on the degree of fusion of the cells; In the present paper, a specific movement of fused cell membranes is deduced from observations on the movement of viral antigens integrated into the membranes of fused cells. MATERIALS AND METHODSAs Ehrlich ascites tumor cells, cells passaged in the abdomen of ddO mice or cells of an established line passaged in vitro were used. Results with the two were similar in the present experiments. The cells were washed and suspended in 9 volumes of balanced salt solution (140 mM NaCl; 54 mM KCl; 0.34 mM Na2HPO4; 0.44 mM KH2PO4; buffered with 10 mM Tris * HCl at pH 7.6) containing 2 mM CaCI2. HVJ, Z strain, was passaged in embryonated eggs, purified by differential centrifugation (2), and then, suspended in balanced salt solution at a concentration of 2000 hemagglutinating units. The virus was inactivated by UV-irradiation before use. A mixture of equal volumes (0.5 ml each) of tumor cells and HVJ was incubated at 00 for 5 min and then the mixture was incubated in a water bath at 370 for 30 min with shaking.After completion of the cell-fusion reaction, cells were washed to remove free virus at 00, and then cultured in minimal essential medium supplemented by 10% calf serum at 37°.When inhibitors were used, they were dissolved in the medium. After culture, cells were washed again and exposed to antiserum against HVJ envelope developed in rabbit at 00 for 20 min. Then the cells were washed at 0°, and mixed with fluorescein isothiocyanate-labeled anti-rabbit IgG 7S antibody (Hyland Biochemicals). After incubation for 20 min at 00, the cells were washed and observed under a microscope with incident-light fluorescence (Leitz). In ...

Suppression of Enveloped Virus Production with a Substance from Silkworm Faeces.

“…In general, it has been considered that HVJ enters into the host cell with envelope fusion between viral envelope and the cell membrane. After envelope fusion, the cell surface is diffusely stained with fluorescence (16). Accordingly, the observations suggest that envelope fusion occurred at the beginning of incubation at 37°C.…”

Modification of HVJ Envelopes Integrated in Fused Cell Membrane Structure during the Course of Cell Fusion

“…In our system the cap formation is also observed with anti-HVJ envelope antibody at the same site as that without antibody. Cap formation of antigens of cell origin on the fused ETC could be observed at the same site when the fused cells are exposed to anti-ETC antibody then cultured (10). The biological meaning of these caps will be summarized in near future.…”

“…Cycloheximide at a concentration of 1 mg/ml showed little inhibitory effect on both the HA-disappearance and cap formation. We suggested the necessity of protein synthesis for the cell membrane movement on the basis of the cycloheximide effect in a previous paper (10). But this concentration used was extremely high when compared with the concentration used for inhibition of protein synthesis in cells.…”

Ligand-independent cap formation: redistribution of surface receptors on mouse lymphocytes and thymocytes in hypertonic medium.

“…Segregation of crosslinked receptors from other receptors is a characteristic of ligand-dependent cap formation. For instance, when surface immunoglobulins (Ig) on mouse lymphocytes are crosslinked with antibody directed against immunoglobulin (anti-Ig) and then capped, other surface molecules (e.g., alloantigens such as H-2 antigens) still remain distributed randomly on the cell surface (2).In contrast to the common cap formation described above, exceptional cases have been reported to be independent of ligands, including cap formation of viral antigens of tumor cells (3,4) and that observed on lymphocytes stimulated with lipopolysaccharide (5). The former, however, occurs only after prolonged incubation (up to 4 hr) in tissue culture medium (3) and the latter has been observed mainly on stimulated B-lymphocytes that were undergoing movement (5).…”