Influenza virus enters host cells by fusion of viral and endosomal membranes mediated by the influenza hemagglutinin (HA). The pathway of HA-catalyzed fusion has been widely investigated in influenza virus membrane fusion with liposomes. In this chapter we describe methodology for studying the virus-liposome fusion system using a combination of fluorescence dequenching assays and cryo-electron tomography (cryo-ET). In particular, the fluorescence dequenching is used to monitor the efficiency of membrane fusion between whole influenza viruses labeled with a lipophilic dye (DiD) in the membrane and liposomes labeled with a water-soluble dye (sulforhodamine B). By simultaneously monitoring the two fluorescent signals, we can determine the relative time scales of liposomal content leakage or transfer vs. lipid merging. In addition, cryo-ET offers a means of imaging three-dimensional snapshots of different stages of virus-liposome fusion such as prefusion, fusion intermediates, and postfusion.
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