On the validity of lipid dequenching assays for estimating virus fusion kinetics

Octadecylrhodamine (R18) has often been used to measure membrane fusion of enveloped viruses by fluorescence dequenching. In order to see whether non-specific R18 exchange between non-fused membranes occurs we have measured fusion of influenza virus with erythrocyte membranes by utilizing dequenching of the non-exchangeable lipid analogue N-(lissamine-rhodamine B-sulfonyl)diacylphosphatidylethanolamine (N-Rh-PE). Rather low concentration of N-Rh-PE (< 0.1 mol%) were required to assess fusion since self-quenching in the influenza virus membrane was more efficient in comparison to R18. For both markers we observed the same kinetics as well as the same extent of fluorescence dequenching upon triggering low pH-induced fusion. Non-specific marker transfer was not observed. Haemolysis was not affected by either type of fluorophore. Our results confirm that R18 is a valuable tool to investigate membrane fusion of enveloped viruses in a quantitative manner. Differences in the efficiency of self-quenching of both markers are discussed.