Sequence-encoded determinants drive distinct early aggregation pathways and different structural outcomes in PNT1 fibrils across Henipavirus members.

Protein aggregation is increasingly recognized as a biologically relevant process in viral proteins, yet the molecular determinants governing such phenomena remain poorly understood. Intrinsically disordered proteins (IDPs) are ubiquitous in viral proteomes, where conformational plasticity not only enables functional diversity but also permits aberrant self-assembly. Members of the Paramyxoviridae family, including henipaviruses such as the Nipah and Hendra viruses (two biosafety level-4 pathogens), encode the P, V, and W proteins that share a common intrinsically disordered N-terminal domain (NTD). V and W are virulence factors that undergo fibrillation. Here, we explored the aggregation landscape of seven homologous PNT1 subdomains from the NTD that harbors a conserved cryptic amyloidogenic region (CAR). By integrating Taylor dispersion analysis (TDA), Raman spectroscopy, and all-atom molecular dynamics (MD) simulations, we unraveled both early assembly kinetics and structural outcomes. Despite the conserved CAR motif, the PNT1 homologs exhibit strikingly divergent aggregation behaviors. TDA revealed distinct oligomerization pathways with variations in nucleation, growth kinetics, and monomer recruitment, indicating virus-specific pathways. Raman spectroscopy unveiled substantial variations in β-sheet content, side-chain packing, and aromatic residue environments, while MD simulations showed that conformational heterogeneity in the CAR flanking regions modulates hydrogen-bond networks and structural stability. These findings indicate that the sequence context beyond a conserved amyloidogenic core governs the aggregation landscape of Henipavirus P, V, and W proteins. Evolutionary diversification modulates conformational heterogeneity and assembly pathways, giving rise to distinct structural outcomes. This work sheds light on the molecular grammar underlying viral protein fibrillation with potential implications for viral pathogenicity.