Cholesterol Alters Physical Properties of the Target Membrane to Facilitate Influenza Membrane Fusion at the Single-Particle Level

Abstract

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including: initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. Because the same viral protein, hemagglutinin, is responsible for IAV binding and fusing to the host membrane, it has been difficult to investigate the role of cholesterol in the host lipid membrane on viral fusion without convolving the two processes. Here, we use synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles without binding hemagglutinin. To avoid potential issues with adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles, and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, while the rate of lipid mixing is independent of cholesterol composition. We also link the effect of cholesterol on IAV fusion to physical properties of the membrane.