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Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
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  11. Properties Of Rabies Virus Phosphoprotein And Nucleoprotein Biocondensates Formed In Vitro And In Cellulo

Properties of rabies virus phosphoprotein and nucleoprotein biocondensates formed in vitro and in cellulo

Quentin Nevers1, Nathalie Scrima1, Damien Glon1

  • 1Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France.

Plos Pathogens
|December 8, 2022

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View abstract on PubMed

Summary
This summary is machine-generated.

Rabies virus (RABV) nucleoprotein (N) and phosphoprotein (P) form liquid-like viral factories called Negri bodies (NBs) via liquid-liquid phase separation (LLPS). This study reveals P

Area of Science:

  • Virology
  • Cell Biology
  • Biophysics

Background:

  • Rabies virus (RABV) transcription and replication occur in Negri bodies (NBs), which are liquid-like viral factories formed by liquid-liquid phase separation (LLPS).
  • The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in cells can recapitulate NB formation, serving as a minimal system to study their properties.

Purpose of the Study:

  • To investigate the dynamics and formation mechanisms of NBs using fluorescently tagged RABV proteins.
  • To identify the specific protein domains and residues involved in the phase separation and condensate formation of N and P.
  • To elucidate the physicochemical properties of these viral condensates and their relevance to the rabies virus life cycle.

Main Methods:

  • Fluorescence Recovery After Photobleaching (FRAP) to analyze protein dynamics in minimal cell systems and infected cells.

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  • Construction and analysis of fluorescently labeled N protein mutants.
  • In vitro phase separation assays using purified P protein and N0-P complexes in crowded environments.
  • Liposome association and RNA binding studies of P condensates.

    • Main Results:

    • RABV nucleoprotein (N) dynamics differ from phosphoprotein (P) within condensates, with no observed fluorescence recovery for N.
    • Specific arginine residues and exposed loops in N were identified as crucial for condensate formation.
    • N mutants showed varied effects in infected cells, including exclusion from NBs and dominant-negative impacts on infection.
    • Purified P and N0-P complexes formed liquid condensates in vitro, with P condensates associating with liposomes and concentrating RNA.
    • P condensates underwent a liquid-to-gel transition upon ageing, while N0-P droplets were destabilized by RNA.

    Conclusions:

    • Phosphoprotein (P) plays a central role in the formation of Negri bodies (NBs).
    • Physicochemical properties of P and N0-P condensates, such as RNA interaction and liquid-gel transition, are relevant to NB formation, membrane association, and viral particle release.