A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes

During host cell infection by flaviviruses such as dengue and Zika, acidic pH within the endosome triggers a conformational change in the envelope protein on the outer surface of the virion. This results in exposure of the ∼15 residue fusion peptide (FP) region, freeing it to induce fusion between the viral and endosomal membranes. A better understanding of the conformational dynamics of the FP in the presence of membranes, and the basis for its selectivity for anionic lipid species present within the endosome, would facilitate its therapeutic targeting with antiviral drugs and antibodies. In this work, multiscale modeling, simulations, and free energy calculations (including a total of ∼75 μs of atomic-resolution sampling), combined with imaging total internal reflection fluorescence correlation spectroscopy experiments, were employed to investigate the mechanisms of interaction of FP variants with lipid bilayers. Wild-type FPs (in the presence or absence of a fluorescein isothiocyanate tag) were shown to possess a funneled conformational landscape governing their exit from solvent and penetration into the lipid phase and to exhibit an electrostatically favored >2-fold affinity for membranes containing anionic species over purely zwitterionic ones. Conversely, the landscape was abolished in a nonfunctional point mutant, leading to a 2-fold drop in host membrane affinity. Collectively, our data reveal how the highly conserved flavivirus FP has evolved to funnel its conformational space toward a maximally fusogenic state anchored within the endosomal membrane. Therapeutically targeting the accessible ensemble of FP conformations may represent a new, rational strategy for blocking viral infection.