Rabies virus (RABV) replication occurs in cytoplasmic, membrane-less compartments known as Negri bodies (NBs), formed through liquid-liquid phase separation (LLPS) of viral components. The phosphoprotein (RABV P) is a central, intrinsically disordered scaf fold of the viral replication machinery. This thesis investigates the structural, biophysical, and dynamic properties of RABV P, with emphasis on its phase separation behavior and interactions with molecular partners. To enable this, recombinant expression and purification protocols were optimized to produce stable, high-quality protein samples for reproducible analyses.

We first characterized the intrinsic phase behavior of RABV P in vitro. The protein undergoes thermoresponsive LLPS with a lower critical solution temperature (LCST), forming reversible condensates within a narrow range of protein and salt concentrations. This process is driven by multivalent interactions within a heterogeneous ensemble of conformations, where dimers assemble into higher-order oligomers prior to phase separation. The resulting phase diagram reveals a complex, reentrant system governed by a balance between electrostatic repulsion and attractive dipole-dipole interactions.

The role of ionic conditions was further examined. While NaCl induced reentrant phase separation, LLPS strongly depended on ion identity rather than ionic strength alone. Chloride salts promoted condensate formation, whereas bromide salts did not, indicating ion-specific (Hofmeister-type) effects. Systematic trends showed that fluoride enhances phase separation, while cation effects are weaker. Divalent ions also promoted LLPS, highlighting valency contributions. Chemical perturbations confirmed that condensates are stabilized by weak interactions: 1,6-hexanediol partially disrupted droplets, whereas ATP fully dissolved them. Notably, RABV P intrinsically phase separates even in water, modulated by pH, protein concentration, and ionic conditions.

Time-resolved small-angle X-ray scattering (SAXS) revealed the structural evolution underlying LLPS. Following a temperature jump, RABV P undergoes a hierarchical assembly process, transitioning from dispersed species to larger structures. Early conformational rearrangements precede the formation of intermediate clusters, followed by growth into larger assemblies. These structures remain disordered and liquid-like, supporting a multistep nucleation-and-growth mechanism.

The host protein LC8 was investigated as a regulator of RABV P condensation. LC8 binds a conserved motif in RABV P with high affinity, forming a defined complex and partitioning into condensates. Functionally, LC8 enhances phase separation by increasing condensate size, enriching RABV P in the dense phase, and broadening the phase-separation window. It shifts phase boundaries toward lower concentrations and temperatures while preserving liquid-like properties. These results indicate that LC8 actively promotes condensation by stabilizing interaction-competent conformations and enhancing intermolecular connectivity.

To assess whether LC8 can compensate for intrinsic multivalency, a truncated RABV P lacking the dimerization domain was analyzed. Although LC8 bound this construct, the interaction was weaker and failed to restore robust phase separation. Only weak condensation was observed under crowding conditions, demonstrating that LC8 cannot substitute for the native dimerization-driven multivalency.
Overall, this work establishes RABV P as a finely tuned multivalent scaffold whose phase behavior arises from the interplay of intrinsic disorder, ion-specific effects, and hierarchical assembly. LLPS emerges as a multistep, non-ideal process rather than a simple binary transition. LC8 acts as a key host regulator that enhances phase separation without altering condensate dynamics, while intrinsic multivalency remains essential. These findings provide a mechanistic framework for understanding viral condensate formation and highlight potential avenues for antiviral intervention. .
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