Abstract
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.
Highlights
RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication
Previous reports demonstrated that the two viral proteins NSP5 and NSP2 constitute the bulk of viroplasms (Berois et al, 2003; Eichwald et al, 2004; Silvestri et al, 2004; Taraporewala et al, 2006; Criglar et al, 2018)
Together with our recent studies (Papa et al, 2020a), these results suggest that C-terminal tagging of NSP5 impairs its function and RV replication, while not precluding NSP5-EGFP mixing with untagged NSP5/NSP2 condensates that are formed during RV infection
Summary
RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. We show that replication factories of rotaviruses represent proteinRNA 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. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein 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
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