Fusion Peptide Insertion Configuration Determines the Mechanism of Membrane Curvature Induction in Human Parainfluenza Virus Type 3

Viral entry requires remodeling the host membrane with a short fusion peptide (FP) that anchors and deforms the target lipid bilayer. However, the contribution of the FP throughout the full fusion pathway remains unknown. Here, all-atom molecular dynamics simulations define the membrane- deformation mechanisms associated with two distinct insertion configurations of membrane-bound human parainfluenza virus type 3 (HPIV3) FP. Molecular simulations quantifying the linear response of curvature to the peptide are complemented with analyses of lipid organization and FP-lipid interactions to define the underlying membrane-deformation mechanisms. The surface-parallel FP configuration generates positive membrane curvature through deformation of the proximal leaflet, consistent with the classical hydrophobic insertion (wedging) mechanism. In contrast, the transmembrane FP configuration generates curvature through localized deformation of the distal leaflet, a distinct mechanism that would strongly influence later stages of fusion. Cryo-electron tomography shows that target membrane curvature at virus-host contact sites is consistent with the molecular dynamics prediction. In the absence of F-protein activation and FP insertion, the cryo-ET-determined curvature distribution shifts toward negative values, thereby isolating the role of the fusion protein in inducing early-stage curvature that enables the host and virus membranes to approach one another. Modeling key mutational changes isolates how aromatic and electrostatic interactions act together to promote curvature generation. Taken together, these findings establish FP insertion configuration as a determinant of productive membrane curvature across the full fusion pathway.