A conserved motif in Henipavirus 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.