Bioscience Reports, Vol. 12, No. 3, 1992

Changes in Membrane Permeability During Semliki Forest Virus Induced Cell Fusion Markus Lanzrein, ~ Nicole K~isermann, ~ and Christoph Kempf ~'2 Received April 6, 1992

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH ( "-

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Fig. 5. Effectsof various anionic inhibitors on low pH induced 14C-AIBrelease from SFV-infected Aedes ceils. Infected cells in confluent monolayers at 16h.p.i. were loaded with 14C-AIB, washed and incubated in PBS pHT.4 (7.4) or PBS pH 5.6 containing 1 mM Aurintricarboxylic acid (ATA), 1 mM Glycyrrhycine (Glyc), 1 mM Trypanblue (TB), 1 mM Trypanred (TB), 1 mM Diisocyanato-stilben-2,2'disulfonic acid (DIDS) 200 ftg/ml Dextransulfate Mr 500 (Dx) or no compound (5.6). After 10 rain, the release was measured by removing aliquots of the incubation-medium and determining the radioactivity. Total values were obtained by lysing the cells in 0.1% Triton X-100. Mean values • standard deviation of triplicate values are shown.

SFV and Membrane Permeability

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(e.g. dextran sulphate) and anionic dyes against HIV might consist in specific binding to gp 120 which would result in inhibition of virus adsorption and subsequent fusion of the envelope with the target membrane (Baba et al. 1988; Schols et al., 1989; Schols et al., 1990; Pal et al., 1991). It was shown for fusion of various enveloped viruses with a target membrane that one of'the initial events is an often irreversible conformational change within the spike proteins. Therefore, it is tempting to speculate that an interaction between sulfonated compounds and viral envelope proteins might interfere with the required changes of the glycoproteins conformation and thus prevent membrane fusion. In respect to the data presented above this would mean that the observed low pH induced permeability changes might also be linked to a conformational change of viral spike proteins in the cell membrane. Therefore, SFV infected, AIB loaded cells were first exposed to pH 5.6 and then Trypan blue was added. As depicted in Fig. 6a subsequent addition of Trypan blue was unable to block the low pH induced permeability change. Conclusively, Trypan blue has to be present at the time of lowering the pH in order to perform its inhibitory effect. Thus, Trypan blue might inhibit fusion and the permeability change by preventing the low pH induced irreversible conformational change of SFV spike proteins. To test this hypothesis, the extracellular pH of SFV infected, AIB loaded cells was lowered in presence

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Time (rain) Fig. 6. Effect of Trypanblue on low pH induced ~4C-AlB-release from SFV-infected Aedes cells. Infected cells in confluent monolayers at 16 h.p.i. were loaded with laC-AIB, washed and release was measured under the following conditions: P B S p H 7 . 4 (control, ~ ) ; V - - V : PBSpH5.6, 1 mM Trypanblue was added after 1 rain (arrow 1); ~ - - ~ : P B S p H 5 . 6 containing 1 mM Trypanblue, after 5 min (arrow 2) cells were washed four times with PBS pH 7.4 and PBS pH 7.4 was then added, after 20 min (arrow 3) the pH was lowered to 5.6 again by exchanging the PBS buffer. Total values were obtained by lysing the cells in 0.1% Triton X-100. Mean values • standard deviation of triplicate values are shown.

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of Trypan blue. After 10 minutes the cells were extensively washed with PBS, pH 7.4, in order to remove the dye. Then, in a third step the ph was lowered to 5.6 again. If trypan blue inhibited the irreversible conformational change the cells should not exhibit an enhanced permeability after removal of the dye but the permeability change should be inducible by lowering the pH again. As shown in Fig. 6 AIB release is blocked at low pH in presence of Trypan blue and removing the dye concomitantly with increasing the pH to 7.4 yielded no significant enhancement of the permeability. However, reverting back the pH to 5.6 in absence of Trypan blue resulted in a dramatic efflux of AIB. Thus, these data support the above mentioned speculation. Summarizing all the results the following statements can be made: i) SFV induced cell-cell FFWI is a leaky process, ii) the observed permeability change is not a prerequisite for membrane fusion and iii) the permeability change is most probabily linked to the acid induced conformational change of viral envelope proteins.

DISCUSSION

Exposure of Semliki Forest virus infected cells to mildly acidic pH (5.6) results in polykaryon formation. As previously reported this fusion process correlated with dynamic changes of the plasma membranes properties (Kempf et al., 1988a). In this report we present data showing that conditions which provoke cell-cell fusion also lead to a dramatic increase in cell permeability towards low molecular weight molecules. The data presented strongly suggest that the permeability change does not represent a the formation of lesions resulting in a partial breakdown of the lipid bilayer but rather the opening of a proteinacious pore of limited size. The diameter of this putative pore can be estimated from its molecular weight cut-off point (Michel et al., 1988) which lies between 700 to 900 D. Thus the pore diameter would be roughly 1 to 1.5 nm. These results are very similar to the data described for Sendai virus induced permeability changes where a cut off limit of