Early events of Semliki Forest virus-induced cell-cell fusion.

Insect cells (Aedes albopictus) were infected with Semliki Forest Virus. Cell-cell fusion was then induced by lowering the extracellular pH. The underlying processes were examined by monitoring the intercellular current flow, Ij. Experimentally, this involved the use of cell pairs in conjunction with a dual voltage-clamp method. This approach allowed us to monitor the kinetics of fusion at high temporal resolution. The fusion process began shortly after acidification (delay: 3-138 sec). Initially, Ij increased in a stepwise manner, later on it developed more gradually. Fusion between two cells reached a steady state within 7-70 sec. The steps in Ij are attributable to the formation of cytoplasmic connections between the cells, presumably involving proteinaceous fusion pores. The mean amplitude of Ij steps corresponds to a conductance of 300 pS, consistent with a pore radius of 1 nm. Cytoplasmic connections developed rapidly, i.e., Ij steps occurred within less than 1 msec. The absence of Ij flickering implies that formation of cytoplasmic connections, and hence SFV induced cell-cell fusion, is irreversible.

Changes in membrane permeability during Semliki Forest virus induced cell fusion.

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (less than 6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influx of protons into the cells (Kempf et al. Biosci. Rep. 7, 761-769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (M(r) less than 1000) was observed when infected cells were exposed to a pH less than 6.2. The pH dependence of the permeability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.