Control of Membrane Fusion Mechanism by Lipid Composition: Predictions from Ensemble Molecular Dynamics

Membrane fusion is critical to biological processes such as viral infection, endocrine hormone secretion, and neurotransmission, yet the precise mechanistic details of the fusion process remain unknown. Current experimental and computational model systems approximate the complex physiological membrane environment for fusion using one or a few protein and lipid species. Here, we report results of a computational model system for fusion in which the ratio of lipid components was systematically varied, using thousands of simulations of up to a microsecond in length to predict the effects of lipid composition on both fusion kinetics and mechanism. In our simulations, increased phosphatidylcholine content in vesicles causes increased activation energies for formation of the initial stalk-like intermediate for fusion and of hemifusion intermediates, in accordance with previous continuum-mechanics theoretical treatments. We also use our large simulation dataset to quantitatively compare the mechanism by which vesicles fuse at different lipid compositions, showing a significant difference in fusion kinetics and mechanism at different compositions simulated. As physiological membranes have different compositions in the inner and outer leaflets, we examine the effect of such asymmetry, as well as the effect of membrane curvature on fusion. These predicted effects of lipid composition on fusion mechanism both underscore the way in which experimental model system construction may affect the observed mechanism of fusion and illustrate a potential mechanism for cellular regulation of the fusion process by altering membrane composition

Hemagglutinin Spatial Distribution Shifts in Response to Cholesterol in the Influenza Viral Envelope

AbstractInfluenza virus delivers its genome to the host cytoplasm via a process of membrane fusion mediated by the viral hemagglutinin protein. Optimal fusion likely requires multiple hemagglutinin trimers, so the spatial distribution of hemagglutinin on the viral envelope may influence fusion mechanism. We have previously shown that moderate depletion of cholesterol from the influenza viral envelope accelerates fusion kinetics even though it decreases fusion efficiency, both in a reversible manner. Here, we use electron cryo-microscopy to measure how the hemagglutinin lateral density in the viral envelope changes with cholesterol extraction. We extract this information by measuring the radial distribution function of electron density in >4000 viral images per sample, assigning hemagglutinin density by comparing images with and without anti-HA Fab bound. On average, hemagglutinin trimers move closer together: we estimate that the typical trimer-trimer spacing reduces from 94 to 84 Å when ∼90% of cholesterol is removed from the viral membrane. Upon restoration of viral envelope cholesterol, this spacing once again expands. This finding can qualitatively explain the observed changes to fusion kinetics: contemporary models from single-virus microscopy are that fusion requires the engagement of several hemagglutinin trimers in close proximity. If removing cholesterol increases the lateral density of hemagglutinin, this should result in an increase in the rate of fusion

Viral factors in influenza pandemic risk assessment

The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk

Atomic-Resolution Simulations Predict a Transition State for Vesicle Fusion Defined by Contact of a Few Lipid Tails

Membrane fusion is essential to both cellular vesicle trafficking and infection by enveloped viruses. While the fusion protein assemblies that catalyze fusion are readily identifiable, the specific activities of the proteins involved and nature of the membrane changes they induce remain unknown. Here, we use many atomic-resolution simulations of vesicle fusion to examine the molecular mechanisms for fusion in detail. We employ committor analysis for these million-atom vesicle fusion simulations to identify a transition state for fusion stalk formation. In our simulations, this transition state occurs when the bulk properties of each lipid bilayer remain in a lamellar state but a few hydrophobic tails bulge into the hydrophilic interface layer and make contact to nucleate a stalk. Additional simulations of influenza fusion peptides in lipid bilayers show that the peptides promote similar local protrusion of lipid tails. Comparing these two sets of simulations, we obtain a common set of structural changes between the transition state for stalk formation and the local environment of peptides known to catalyze fusion. Our results thus suggest that the specific molecular properties of individual lipids are highly important to vesicle fusion and yield an explicit structural model that could help explain the mechanism of catalysis by fusion proteins