Effect of pH on the influenza fusion peptide properties unveiled by constant-pH molecular dynamics simulations combined with experiment

Diana Lousa,Ana S Veiga,Cláudio M Soares,Miguel A R B Castanho,António M Baptista,Antónia R T Pinto,Sara R R Campos

doi:10.1038/s41598-020-77040-y

Diana Lousa, Ana S Veiga + Show 5 more

Open Access

https://doi.org/10.1038/s41598-020-77040-y

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Abstract

The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide’s effect on the membrane.

Highlights

The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment
The first 23 amino acid residues of HA2 are important in the fusion process, since this region inserts into the host membrane, promoting fusion and is, known as the fusion peptide[4]
Fusogenic activity of the influenza fusion peptide analyzed by a Förster Resonance Energy Transfer (FRET)‐based assay

Summary

Introduction

The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. The first 23 amino acid residues of HA2 are important in the fusion process, since this region inserts into the host membrane, promoting fusion and is, known as the fusion peptide[4]. This region is very conserved across different hemagglutinin subtypes (18 out of 23 residues are strictly conserved among all influenza A strains) and several mutations have been shown to abolish or impair its function[4]. Subsequent simulation studies by Worch et al using temperature replica exchange molecular dynamics found that the influenza fusion peptide can adopt these two configurations and indicate that he membranespanning configuration corresponds to the lowest free energy minimum for the 23-residue long fusion peptide with a charged N-terminus[15,16]

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