Unravelling the role of key amino acid residues of the parainfluenza fusion peptide in membrane fusion.

Parainfluenza viruses enter host cells by fusing their envelope with the cell membrane. In this process mediated by the fusion glycoprotein, the fusion peptide plays an essential role in membrane binding and triggering fusion. Previously, we demonstrated that the parainfluenza fusion peptide (PIFP) oligomerizes into porelike structures within the membrane, leading to membrane perturbations, fusion, and leakage. Additionally, we identified two key amino acid residues in the PIFP, F103 and Q120, which are important in inducing lipid tail protrusion and maintaining peptide-peptide interactions, respectively. Here, we seek to elucidate the role of these two residues in the PIFP function by studying the impact of F103A and Q120A substitutions on peptide activity. We compared the substituted peptides with the native peptide using biophysical experiments and molecular dynamics (MD) simulations. Our results show that the F103A substitution significantly impairs PIFP's interaction with the membrane and its ability to induce lipid mixing and membrane leakage in experimental assays. Moreover, a decrease in lipid perturbation and water flux through the membrane was observed in the MD simulations. In contrast, the Q120A substitution appears to have minimal impact on membrane interaction and PIFP-induced membrane leakage. Interestingly, a pronounced change in the interpeptide interactions within the membrane of the substituted peptides was observed in the MD simulations. These findings provide crucial insights into the potential role of F103 and Q120 in PIFP activity: the N-terminal phenylalanine (F103) is pivotal for membrane insertion and fusion, while the Q120 is crucial for regulating peptide oligomerization and pore formation.