Multiphasic effects of cholesterol on influenza fusion kinetics reflect multiple mechanistic roles

The envelope lipid composition of influenza virus differs from that of the cellular plasma membrane from which it buds. Viruses also appear to fuse preferentially to specific membrane compartments, suggesting that the lipid environment may influence permissiveness for fusion. Here, we investigated the influence of the membrane environment on fusion, focusing on cholesterol composition. Strikingly, manipulating cholesterol levels in the viral membrane had different effects on fusion kinetics compared with analogous changes to the target membrane. Increasing cholesterol content in target vesicles increased lipid- and contents-mixing rates. Moderate cholesterol depletion from the viral membrane sped fusion rates, whereas severe depletion slowed the process. The pleiotropic effects of cholesterol include alterations in both membrane-bending moduli and lateral organization. Because influenza virions have demonstrated cholesterol-dependent lateral organization, to separate these effects, we deliberately selected a target vesicle composition that does not support lateral heterogeneity. We therefore postulate that the monotonic response of fusion kinetics to target membrane cholesterol reflects bending and curvature effects, whereas the multiphasic response to viral cholesterol levels reflects the combined effects of lateral organization and material properties.