Wild-Type and Mutant Hemagglutinin Fusion Peptides Alter Bilayer Structure as Well as Kinetics and Activation Thermodynamics of Stalk and Pore Formation Differently: Mechanistic Implications

Abstract

Viral fusion peptides are short N-terminal regions of type-1 viral fusion proteins that are critical for virus entry. Although the importance of viral fusion peptides in virus-cell membrane fusion is established, little is known about how they function. We report the effects of wild-type (WT) hemagglutinin (HA) fusion peptide and its G1S, G1V, and W14A mutants on the kinetics of poly(ethylene glycol)(PEG)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, and cholesterol (molar ratio of 35:30:15:20). Time courses of lipid mixing, content mixing, and content leakage were obtained using fluorescence assays at multiple temperatures and analyzed globally using either a two-step or three-step sequential ensemble model of the fusion process to obtain the rate constant and activation thermodynamics of each step. We also monitored the influence of peptides on bilayer interfacial order, acyl chain order, bilayer free volume, and water penetration. All these data were considered in terms of a recently published mechanistic model for the thermodynamic transition states for each step of the fusion process. We propose that WT peptide catalyzes Step 1 by occupying bilayer regions vacated by acyl chains that protrude into interbilayer space to form the Step 1 transition state. It also uniquely contributes a positive intrinsic curvature to hemi-fused leaflets to eliminate Step 2 and catalyzes Step 3 by destabilizing the highly stressed edges of the hemi-fused microstructures that dominate the ensemble of the intermediate state directly preceding fusion pore formation. Similar arguments explain the catalytic and inhibitory properties of the mutant peptides and support the hypothesis that the membrane-contacting fusion peptide of HA fusion protein is key to its catalytic activity.

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

“…To obtain kinetics from single-event studies, one must record many traces of events that follow initiation of fusion between docked membranes (vesicles attached to other vesicles or to membranes) and then analyze the time probability distributions (dwell-time distributions) to distinguish distinct events and obtain their characteristic times (rates). Elsewhere (27), we describe in detail the relationship between the ensemble approach and a well-documented single-event study of fusion of docked vesicles (28), and show that pictures of fusion obtained by the two methods are consistent. Although both approaches have advantages and disadvantages, ensemble measurements have the advantage of requiring much shorter experiment times.…”

SARS-CoV fusion peptides induce membrane surface ordering and curvature

“…That is, the more fusogenic SARS FP peptide can also support membrane fusion by stabilization of porous structures. Experimental and computational studies have also indicated stabilization of the pore state by fusion peptides from influenza HA 37,92 and parainfluenza virus (PIV5) F protein 93 . Thus, our findings imply that both SARS fusion peptides can act at different stages of the fusion process to facilitate membrane fusion.…”

Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms

“…It has been suggested spectroscopically that this such activity may correlate with semi-closed fusion peptide conformations (25). Experimental studies on PEG-mediated vesicle fusion with exogenous fusion peptides offer indirect support for this model (26). Separately, spectroscopic experiments and simulations on isolated influenza fusion peptides suggest that they are capable of inducing membrane curvature (27)(28)(29)(30), which could alter the free-energy barriers to fusion either by changing the starting reference state or by stabilizing highly curved intermediates (31,32).…”