A conserved motif in <i>Henipavirus</i> P/V/W proteins drives the fibrillation of the W protein from Hendra virus

The Hendra (HeV) and Nipah (NiV) viruses are high-priority, biosafety level-4 pathogens that cause fatal neurological and respiratory disease. Their P gene encodes not only the P protein, an essential polymerase cofactor, but also the virulence factors V and W. We previously showed that the W protein of HeV (W^HeV) forms amyloid-like fibrils and that one of its subdomains, PNT3, fibrillates in isolation. However, the fibrillation kinetics is much faster in the case of the full-length W^HeV compared to PNT3, suggesting that another W^HeV region contributes to the fibrillation process. In this work, we identified the region spanning residues 2-110 (PNT1) as the crucial region implicated in W^HeV fibrillation. Through site-directed mutagenesis, combined with thioflavin T binding experiments and negative-staining transmission electron microscopy, we showed that a predicted cryptic amyloidogenic region (CAR) within PNT1 is the main driver of fibrillation and deciphered the underlying molecular mechanism. Using FTIR, we showed that PNT1 fibrils are enriched in cross β-sheets. Sequence alignment revealed conservation of the CAR across the Henipavirus genus and enabled the identification of a hitherto never reported pro-amyloidogenic motif. The ability to form fibrils was experimentally shown to be a common property shared by Henipavirus PNT1 proteins. Overall, this study sheds light on the molecular mechanisms underlying W^HeV fibrillation and calls for future studies aimed at exploring the relevance of the newly identified pro-amyloidogenic motif as a valuable target for antiviral approaches.