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

Abstract

Rotaviruses transcribe eleven distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all eleven types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.

Rotavirus Particle Disassembly and Assembly In Vivo and In Vitro

“…It is thought that virus assembly begins with the specific interaction of VP1, the viral polymerase, with conserved bases at the 3′ ends of ssRNA(+) that serve as precursors of the dsRNA segments, forming a pre-core complex in which VP1 is inactive [ 70 , 71 , 72 , 73 ]. These 11 different cytoplasmic complexes are specifically recruited to the viroplasms, which are cytoplasmic electron-dense inclusions where viral ssRNAs and viral proteins accumulate [ 74 , 75 ]. It is in the viroplasms that most of the events of rotavirus morphogenesis are compartmentalized.…”

“…Recently, viroplasms have been shown to act as biomolecular condensates [ 74 , 75 , 78 , 89 , 90 ] formed by the liquid–liquid phase separation (LLPS) of the proteins NSP5 and NSP2, where NSP5 acts as the main driver of LLPS (scaffold), of which NSP2 is a client protein [ 89 ]. Immediately after infection, viroplasms show liquid-like behavior that matures to solid-like condensates via the accumulation of other viral RNA and proteins that participate in these condensates as clients and by post-translational modifications, particularly the phosphorylation of NSP5.…”

Structural Studies of Bacteriophage Φ6 and Its Transformations during Its Life Cycle

“…While the cystoviruses package their three ssRNA segments into preformed procapsids, the eucaryotic dsRNA viruses form their capsids around preassembled particles containing the 9-12 ssRNA segments within organized cellular structures called viral factories or viroplasms [3]. How these particles come together is complex and still requires intensive study [71]. The demonstration that only one P4 hexamer per procapsid is necessary for packaging followed by minus-strand synthesis (replication) led to the proposal that there is a special vertex different from the others [67].…”

“…While the cystoviruses package their three ssRNA segments into preformed procapsids, the eucaryotic dsRNA viruses form their capsids around preassembled particles containing the 9-12 ssRNA segments within organized cellular structures called viral factories or viroplasms [3]. How these particles come together is complex and still requires intensive study [71].…”

Seeing Biomolecular Condensates Through the Lens of Viruses

“…Prior to RNA encapsidation, viral factories accumulate a vast number of transcripts destined for packaging (58). This aspect of some viral factories, including those formed during rotavirus infection, shares several common features with other ribonucleoprotein (RNP) granules that contain multiple RNAs.…”