Rabies Virus Phosphoprotein Exhibits Thermoresponsive Phase Separation with a Lower Critical Solution Temperature.

Area of Science:

• Virology
• Biophysics
• Molecular Biology

Background:

• Rabies virus (RABV) forms Negri bodies, essential sites for viral RNA synthesis and replication.
• The mechanisms governing the assembly and maturation of these membrane-less organelles are not fully understood.
• RABV phosphoprotein (P) is a key component of the viral RNA synthesis machinery and acts as a scaffold for Negri body assembly.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying the self-assembly and phase separation of RABV phosphoprotein (P).
• To investigate the role of P's intrinsically disordered regions and globular domains in condensate formation.
• To develop a molecular model explaining P-driven liquid phase separation.

Main Methods:

• In vitro self-association assays of RABV P.
• Analysis of thermoresponsive phase separation behavior.
• Investigation of salt concentration effects on liquid phase separation.
• Conformational analysis of P dimers and their interactions.

Main Results:

• RABV P self-association drives thermoresponsive phase separation with a lower critical solution temperature.
• Protein dimers assemble below saturation concentration, driven by conformation-specific interactions.
• Reentrant liquid phase separation occurs within a narrow salt concentration range.
• A minimal molecular model involving three conformational states of P explains the observed phenomena.

Conclusions:

• RABV P's intrinsically disordered N-terminal arms and C-terminal globular domains mediate condensate formation.
• Attractive, conformation-specific interactions drive liquid phase separation.
• The study proposes a model where disordered arms control interactions between giant dipoles, consistent with experimental observations.
• Understanding P's self-assembly provides insights into Negri body formation and viral replication.