Unraveling the molecular grammar and the structural transitions underlying the fibrillation of a viral fibrillogenic domain.

Hendra virus (HeV) is a biosafety level 4 human pathogen belonging to the Henipavirus genus within the Paramyxoviridae family. In HeV, the phosphoprotein-encoding gene also drives the synthesis of the V and W proteins that are two major players in the host innate immune response evasion. These three proteins share a common intrinsically disordered N-terminal domain (NTD) and have distinct C-terminal domains. We recently reported the ability of a short region (i.e., PNT3), located within the shared NTD, to form fibrils. We subsequently identified a PNT3 motif (EYYY) critically involved in fibrillation and deciphered the contribution of each tyrosine to the process. Herein, we combined mutational studies with various biochemical and biophysical approaches to further investigate the molecular mechanisms underlying PNT3 fibrillation. The results show that (i) lysine residues play a critical role in driving fibrillation, (ii) hydrophobic residues affect the nucleation step, and (iii) charge distribution strongly affects the fibrillation propensities. Vibrational Raman spectroscopy data further validated the role of lysine residues in promoting fibrillation and enabled documenting the formation of cross-β amyloid structures. Altogether, these results illuminate the molecular mechanisms involved in fibril formation and pave the way towards the rational design of inhibitors.