Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

Abstract

RNA viruses induce 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. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus

“…Rotavirus replication factories, or viroplasms, have long been known as membrane-less organelles that emerge in the cytoplasm shortly after infection 12 . Our recent findings have revealed that these viral organelles are formed via liquid-liquid phase separation (LLPS) of the RV RNA-binding proteins NSP2 and NSP5 17 . Similar liquidlike membrane-less RNP condensates are ubiquitous in cells 42,[44][45][46][47] ; however, each specific type of an RNP condensate being unique in their protein and RNA composition that can be dynamically modulated by various processes.…”

“…In order to monitor rotavirus transcription during the course of infection, we took advantage of the MA104 cell line stably expressing an EGFP-tagged NSP5 that readily partitions into the viroplasmic condensates 18,31,32 . At a multiplicity of infection (MOI) of 10, NSP5-EGFP-tagged granules were readily detected as soon as 2-3 hours post infection (hpi) ( Fig.…”

“…Recently, we have discovered that early replication stage (2-6 hours post infection) viroplasms represent liquid condensates that are formed via phase-separation of the RV RNA chaperone NSP2 and a non-structural phosphoprotein NSP5 17 . These condensates could be rapidly and reversibly dissolved by treating RV-infected cells with small aliphatic diols 17 . A fraction of the RV transcripts was released from viroplasms when they were briefly treated with 4.5% propylene glycol or 4% 1,6-hexanediol, resulting in the reversible recruitment of the RV transcripts into these condensates when these compounds were removed 17 .…”

“…These condensates could be rapidly and reversibly dissolved by treating RV-infected cells with small aliphatic diols 17 . A fraction of the RV transcripts was released from viroplasms when they were briefly treated with 4.5% propylene glycol or 4% 1,6-hexanediol, resulting in the reversible recruitment of the RV transcripts into these condensates when these compounds were removed 17 . In principle, the formation of dynamic ribonucleoprotein condensates in RV-infected cells could facilitate selective enrichment of cognate viral transcripts required for a robust and stoichiometric RNA assembly and packaging.…”

“…Recently, we have discovered that early replication stage (2-6 hours post infection) viroplasms represent liquid condensates that are formed via phase-separation of the RV RNA chaperone NSP2 and a non-structural phosphoprotein NSP5 17 . These condensates could be rapidly and reversibly dissolved by treating RV-infected cells with small aliphatic diols 17 .…”