Biophysical Elucidation of the Distinct Roles the Nipah Virus Attachment and Fusion Glycoproteins Play in the Entry Process

Nipah virus (NiV), a World Health Organization priority pathogen, is a lethal enveloped RNA virus within the Paramyxoviridae family. NiV attachment G and fusion F glycoproteins execute viral-cell or cell–cell membrane fusion during viral entry and syncytia formation, respectively. Despite past studies, several aspects of the molecular-level orchestration underlying this process, such as how F, G, and their interactions regulate fusion, remain poorly understood, obscured by challenges in decoupling fusion determinants in complex cellular environments. Here, we report a reconstitution-based approach for mimicking the NiV-host interface, employing virus-like particles with NiV M, F, and/or G proteins and plasma membrane vesicles derived from NiV-permissive host cells. We found this minimal system can accurately recapitulate NiV-host cell fusion, and used this platform for biophysical dissection of NiV fusion using wild-type or mutant F and G proteins affecting fusion at distinct intermediate steps. We show that F and G affect hemifusion and pore formation stages of NiV fusion in distinct ways, revealing a tightly regulated fusion landscape. Our approach also delivers one of the first direct measurements of temperature thresholds for NiV fusion, providing novel insights into energetic requirements underlying viral fusogenicity. These findings demonstrate the power of this approach in dissecting aspects of NiV fusion not accessible via cell-based assays. Thus, this study serves as a blueprint for comprehensive fusion analysis of other viruses with multicomponent fusion machinery and host protein receptors, and could be repurposed into therapeutic screening platforms targeting their fusion mechanisms, necessary for developing novel antivirals.