Conformational dynamics, RNA binding, and phase separation regulate the multifunctionality of rabies virus P protein

Rabies virus (RABV) phosphoprotein (P) is a multifunctional viral protein that plays essential roles in viral replication, transcription, and immune evasion. Although encoded by a single gene, P is expressed as multiple isoforms with distinct cellular functions, particularly the full-length P1 and the N-terminally truncated P3. The molecular basis for this functional diversification has remained unclear. This study investigates how conformational dynamics, RNA binding, and liquid–liquid phase separation (LLPS) regulate the multifunctionality of 　P. They show that P1 and P3 adopt distinct conformational states. P1 predominantly exhibits a compact intramolecular structure, whereas P3 displays greater conformational flexibility. Both isoforms are capable of undergoing LLPS in vitro, indicating that phase separation alone does not account for their functional differences. Instead, RNA binding emerges as a key determinant of isoform-specific activity. P3, but not P1, exhibits strong RNA-binding ability. RNA binding by P3 is shown to be critical for its selective localization to membrane-less organelles such as nucleoli and PML nuclear bodies, as well as for interactions with microtubules and immune-related cellular structures. Disruption of RNA binding impairs these P3-specific functions without abolishing phase separation. 
(MN)