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

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• RNA viruses utilize multifunctional proteins to manage limited genomes, impacting viral replication and host interactions.
• The rabies virus P gene produces P1 and P3 isoforms, with P3 exhibiting unique functions like interaction with membrane-less organelles (MLOs) via liquid-liquid phase separation (LLPS).
• Protein isoform multifunctionality may stem from conformational regulation involving intrinsically disordered regions (IDRs) and globular domains.

Purpose of the Study:

• To investigate the structural and functional differences between rabies virus P1 and P3 protein isoforms.
• To elucidate the mechanisms underlying the gain-of-function observed in the P3 isoform, particularly its interaction with MLOs and RNA.
• To understand how conformational regulation contributes to isoform-specific functions in viral replication and host modulation.

Main Methods:

• Comparative analysis of P1 and P3 protein structures and functions.
• Identification of isoform-specific intra-protomer interactions between IDRs and the C-terminal domain.
• Assessment of mutations affecting MLO interactions and their impact on protein conformation and RNA binding.

Main Results:

• Isoform-specific long-range interactions between IDRs and the C-terminal domain were identified, correlating with distinct conformational states, LLPS behavior, and subcellular localization.
• Mutations altering P3's MLO interactions also modulated these long-range intra-protomer interactions.
• While both isoforms interact with MLO-associated proteins and exhibit similar LLPS capacity, only P3 binds RNA, and this binding is linked to gain-/loss-of-function mutations.

Conclusions:

• Conformational differences between P1 and P3 isoforms are critical for regulating LLPS behavior and protein-RNA interactions.
• These conformational dynamics control the accessibility of host LLPS structures, representing a novel strategy for P protein multifunctionality.
• The study uncovers a previously unrecognized mechanism of viral protein function through isoform-specific conformational regulation and interaction with host phase-separated compartments.