Effects of Wild Type and Mutant HA Fusion Peptides on Kinetics and Activation Thermodynamics of Stalk and Pore Formation: Mechanistic Implications

Abstract

Viral fusion peptides are 20-25 residue N-terminal regions of type 1 viral fusion proteins that are known to be critical to viral infection and to promote fusion between model membrane vesicles brought into close contact by poly(ethylene glycol) (PRG). However, almost nothing is known about how they function. We report the effects of wild-type (WT) hemagglutinin fusion peptide and its G1S, G1V and W14A mutants on the kinetics of PEG-mediated fusion of 23 nm vesicles of lipid composition dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin and cholesterol in a 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 according to a 2-step or 3-step (Biophys. J., 2007, 92; 4012) sequential model, in order to obtain activation thermodynamics for each step of the fusion process (Biophys J., 2012, 103: 2751). We also used fluorescence probes to monitor the influence of peptides on bilayer interfacial and acyl chain order, bilayer free volume and water penetration. These data were all considered in terms of recently published mechanistic models for the transition states for each of the steps of fusion (Biophys J., 2012, 103: 2751). WT peptide is proposed to catalyze step 1 due to its ability to occupy membrane free volume and stabilize TS1; it eliminates step 2 by destabilizing the second intermediate; and catalyzes step 3 by favoring correlated fluctuations of lipids from cis and trans leaflets of an unstable second intermediate. Supported by NIGMS grant 32707 to BRL.