Cholesterol and Influenza Viral Fusion Mechanisms: Using Sterol Analogues to Probe Mechanism

Abstract

Fusion between the viral envelope and target cell membrane is a crucial step for infection of influenza and other enveloped viruses. The influenza envelope protein hemagglutinin provides a necessary driving force for fusion. However, both viral and target membrane composition can also have a significant impact on infectivity. Here, we examine the effect of sterol composition on viral membrane fusion kinetics. We have previously shown that changes to cholesterol concentration in either synthetic target liposomes or viral membranes can alter both hemi-fusion and fusion kinetics. Increasing cholesterol in target liposomes caused a monotonic increase in fusion rate, while depleting viral cholesterol from the starting cholesterol:phospholipid ratio of 1:1 had a more complex effect. To further characterize what chemical properties of cholesterol are responsible for the observed changes and begin to dissect the responsible mechanism, we examined the effect of other related sterols. We tested seven cholesterol analogues, measuring fusion kinetics when each sterol was used in place of cholesterol in target liposomes or when cholesterol was extracted from the viral membrane and replaced by each sterol tested. As measured by fluorescence dequenching kinetics, fusion between X-31 influenza virus and target vesicles containing 20% sterol did not significantly change hemi-fusion or fusion rates among the sterols tested. Most sterols tested in the viral envelope had a minimal effect on fusion kinetics compared with cholesterol. However, replacing viral envelope cholesterol with cholesteryl sulfate significantly slowed both hemi-fusion and fusion. We are currently working to identify the chemical basis for this effect.