Morphological changes in Ehrlich ascites tumor cells during the cell fusion reaction with HVJ (Sendai virus): II. Cluster formation of intramembrane particles in the early stage of cell fusion

The morphological events in the cell membrane of Ehrlich ascites tumor (EAT) cells associated with cell fusion caused by HVJ were investigated with freeze-fracture technique. When cell fusion was carried out at 37 °C, the EATC fusion was too rapid to allow identification of the sequential steps of membrane fusion and no deleterious changes in the plasma membrane could be detected. However, on lowering the incubation temperature from 37 to 28 °C, the process of cell fusion was slower and there was a distinct alteration in the plasma membrane. On incubation of cell aggregates with HVJ at 28 °C, the fusion reaction proceeded very slowly. On incubation for 10 min, fusion was initiated in a few cells, but most of the cells remained agglutinated with their cell membranes close to those of neighboring cells and often in direct contact in small localized regions. When cells in this stage were chilled and fixed at 4 °C, large clusters of intramembrane particles (IMPs) were seen all over the P face. On further incubation of the cells at 37 °C, cell fusion proceeded rapidly and the IMPs became randomly redistributed, indicating that clustering is a reversible phenomenon occurring in the early stage of cell fusion. This clustering was temperature-dependent. It was seen in cell fixations at 4 °C, but not at 28 °C without chilling, and it was prevented by inhibitors of cell fusion, such as cytochalasin D (CD) or glucose at high concentration. These findings suggest that certain structural changes in the plasma membrane that may induce thermotropic aggregation of IMP are required to initiate cell fusion.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus).

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : I. Interaction between cell membranes and virus in the early stage

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.