Fluorescent lipid probes in the study of viral membrane fusion

Abstract

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.