Gondelaud Frank, Bignon Christophe, Ptchelkine Denis, Carrière Frédéric, Longhi Sonia
Laboratoire Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257, Aix Marseille University and Centre National de la Recherche Scientifique (CNRS), Marseille, France.
Aix Marseille Univ, CNRS, UMR7281 Bioénergétique et Ingénierie des Protéines, Marseille, France.
Protein Sci. 2025 Apr;34(4):e70085. doi: 10.1002/pro.70085.
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) 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 compared to PNT3, suggesting that another W 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 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 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.
亨德拉病毒（HeV）和尼帕病毒（NiV）是高优先级的生物安全4级病原体，可导致致命的神经和呼吸系统疾病。它们的P基因不仅编码P蛋白（一种必需的聚合酶辅助因子），还编码毒力因子V和W。我们之前表明，HeV的W蛋白（W）形成淀粉样纤维，并且其一个亚结构域PNT3可单独形成纤维。然而，与PNT3相比，全长W的纤维化动力学要快得多，这表明W的另一个区域有助于纤维化过程。在这项研究中，我们确定了跨越第2至110位残基的区域（PNT1）是与W纤维化相关的关键区域。通过定点诱变，结合硫黄素T结合实验和负染色透射电子显微镜，我们表明PNT1内一个预测的隐匿性淀粉样生成区域（CAR）是纤维化的主要驱动因素，并破译了潜在的分子机制。使用傅里叶变换红外光谱，我们表明PNT1纤维富含交叉β-折叠。序列比对显示CAR在亨尼帕病毒属中具有保守性，并能够鉴定出一个迄今从未报道过的促淀粉样生成基序。实验表明，形成纤维的能力是亨尼帕病毒PNT1蛋白共有的特性。总体而言，这项研究揭示了W纤维化的分子机制，并呼吁未来开展研究，探索新鉴定的促淀粉样生成基序作为抗病毒方法的有价值靶点的相关性。
