Liquid-liquid phase separation underpins the formation of replication factories in rotaviruses

EMBO J. 2021 Nov 2;40(21):e107711. doi: 10.15252/embj.2021107711. Epub 2021 Sep 15.


RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.

Keywords: RNP granules; biomolecular condensates; microfluidics; viral genome assembly.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Cell Line
  • Cytoplasmic Ribonucleoprotein Granules / drug effects
  • Cytoplasmic Ribonucleoprotein Granules / metabolism*
  • Cytoplasmic Ribonucleoprotein Granules / ultrastructure
  • Cytoplasmic Ribonucleoprotein Granules / virology
  • Gene Expression Regulation, Viral
  • Genes, Reporter
  • Glycols / pharmacology
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Haplorhini
  • Host-Pathogen Interactions / genetics
  • Humans
  • Osmolar Concentration
  • Phosphorylation
  • Propylene Glycol / pharmacology
  • Protein Processing, Post-Translational*
  • RNA-Binding Proteins / antagonists & inhibitors
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Rotavirus / drug effects
  • Rotavirus / genetics*
  • Rotavirus / growth & development
  • Rotavirus / ultrastructure
  • Signal Transduction
  • Viral Nonstructural Proteins / antagonists & inhibitors
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Virus Assembly / drug effects
  • Virus Assembly / genetics
  • Virus Replication / drug effects
  • Virus Replication / genetics


  • Glycols
  • NSP5 protein, rotavirus group A
  • RNA-Binding Proteins
  • Viral Nonstructural Proteins
  • enhanced green fluorescent protein
  • NS35 protein, rotavirus
  • Green Fluorescent Proteins
  • Propylene Glycol
  • hexamethylene glycol