Phase separation and viral factories: unveiling the physical processes supporting RNA packaging in dsRNA viruses

Abstract

Understanding of the physicochemical properties and functions of biomolecular condensates has rapidly advanced over the past decade. More recently, many RNA viruses have been shown to form cytoplasmic replication factories, or viroplasms, via phase separation of their components, akin to numerous cellular membraneless organelles. Notably, diverse viruses from the Reoviridae family containing 10-12 segmented double-stranded RNA genomes induce the formation of viroplasms in infected cells. Little is known about the inner workings of these membraneless cytoplasmic inclusions and how they may support stoichiometric RNA assembly in viruses with segmented RNA genomes, raising questions about the roles of phase separation in coordinating viral genome packaging. Here, we discuss how the molecular composition of viroplasms determines their properties, highlighting the interplay between RNA structure, RNA remodelling, and condensate self-organisation. Advancements in RNA structural probing and theoretical modelling of condensates can reveal the mechanisms through which these ribonucleoprotein complexes support the selective enrichment and stoichiometric assembly of distinct viral RNAs.

Keywords: RNA structure; RNA:RNA interactions; RNP granules; X-ray RNA footprinting; viral factories.

Figure 1.. Rotavirus replication takes place in viroplasms formed via phase separation.

(A) A proposed model of the rotavirus viroplasm and its key contributions to the viral replication cycle. Viral transcription following the uncoating of endocytosed triple-layered particles (TLPs) results in the extrusion of viral mRNAs followed by their translation. This creates a cytoplasmic pool of viral proteins, including viroplasm-forming NSP5 and NSP2. These proteins undergo LLPS to form early viroplasms, which recruit additional clients required for viral assembly. RNA recruitment is selective, favouring viral transcripts over cellular ones, ensuring the enrichment of eleven distinct types of viral RNAs (step 1). RNA relocalisation to the viroplasm is associated with RNA remodelling, promoting interactions with other RNAs and viral proteins (step 2). Viral inner capsid protein VP2 shells assemble around growing clusters of up to eleven distinct single-stranded RNAs in distinct micro-domains (step 3). The remaining unpackaged regions of these RNA complexes fill pre-assembled VP2 shells (step 4). Packaged RNA undergoes replication before or during assembly of double-layered particles (DLPs, step 5). The endoplasmic reticulum (ER)-resident viral protein NSP4 recruits DLPs assembled in viroplasms into the ER (step 6), where TLPs are assembled (step 7). (B) Electron microscopy image of rotavirus viroplasm with NSP2 octamers dispersed throughout the viroplasm and particle intermediates visible only at the surface of the condensate [16].

Figure 2.. Current understanding of RNA structure in condensates.

(A) The environment inside RNP condensates can remodel ssRNA structure. Inside condensates, large folded ssRNAs can undergo partial unfolding reforming extensive inter-molecular base-pairing with adjacent ssRNAs. (B) A model of RNA enrichment controlled by the RNA structure that may affect condensate's selectivity and composition by presenting distinct binding sites that recruit additional clients (ssRNAs and proteins). Further partitioning of ssRNAs (orange) or RNA-binding proteins (RBP, green) through base-pairing or binding site display, respectively, allow dynamic control of the RNP granules’ composition.

Figure 3.. RNA structure probing in condensates.

Organic solvents traditionally used for RNA structure probing, such as DMSO, can disrupt liquid-like condensates both in vitro and in cellulo. (A) Top: in vitro condensates formed by mixing of recombinant rotavirus proteins NSP5 and an Atto-488-tagged NSP2 are partially dissolved in the presence of 5% dimethylsulfoxide (DMSO) and fully dissolved after the addition of 10% DMSO as used in SHAPE-MaP. Bottom: Replication factories formed upon infection with RV are fluorescently labelled via partitioning of transiently expressed NSP5-eGFP into viroplasms. Viroplasms disappear in the presence of 5% and 10% DMSO. Scale bar = 10 µm. (B) XFP can be used to probe RNA structure inside condensates. Frozen biological or reconstituted samples are exposed to high-energy X-ray radiation, which generates ·OH radicals from water radiolysis. Radical-mediated cleavage of the RNA at solvent-exposed sites is recorded as reverse transcriptase (RT) stops during next-generation sequencing and is used to model RNA structure.