Viroplasms: Assembly and Functions of Rotavirus Replication Factories

Abstract

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein-RNA condensates that may be formed via liquid-liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

Keywords: CRISPR-Csy4 genome editing; NSP2; NSP5; liquid-liquid phase separation; protein-RNA condensates; replication cycle; reverse genetics; rotavirus; viroplasm.

Figure 1

Rotavirus structure, replication cycle. (A,B) Schematic representation of triple-layered and double-layered rotavirus particles (TLPs and DLPs, respectively). Outer layer: VP7, VP4 (spikes); middle layer: VP6; inner layer (core): VP2. The core contains 11 segments of dsRNA (diagrammatically shown as concentric rings) and the viral transcription–replication complex, consisting of the RNA-dependent RNA polymerase (RdRp), VP1, and the capping enzyme, VP3. Rotavirus DLPs are transcription active, producing (+)ssRNAs from each genomic RNA segment. Adapted from: ViralZone, SIB Swiss Institute of Bioinformatics; (C) Rotavirus replication cycle (diagram) including formation of viroplasms. For details, see text. Adapted from: ViralZone, SIB Swiss Institute of Bioinformatics.

Figure 2

Rotavirus viroplasm formation. (A) Formation of rotavirus viroplasm. Upper panel: Immunofluorescent image of a rotavirus-infected cell at 10 hpi, stained with anti-NSP5 antibody (red) and DAPI (blue). Scale bar, 10 μm. Lower panel: Electron micrographs of MA104 cells infected with wildtype (WT) rotavirus (MOI, 75 FFU/cell) at 10 hpi. V, viroplasm. The white open box denotes an area shown at higher magnification in the right-hand EM photograph. White arrows indicate endoplasmic membranes surrounding viroplasms; black arrowheads indicate viral particles with an envelope. Scale bar, 500 nm. Adapted with permission from Papa et al. [25]; (B) Confocal imaging of rotavirus transcripts via fluorescence in situ hybridisation (FISH). FISH probes specific to the rotavirus transcripts (segment 1–11) were used to visualise RV transcripts in EGFP-NSP5-tagged viroplasms (green). RNA signal is shown in yellow (Quasar 560-labelled probes). Nuclei were stained with DAPI (blue), Scale bar, 10 μm. Adapted from Strauss et al. [26]; (C) Rotavirus infection of cells constitutively expressing fluorescently labelled NSP2 and NSP5 proteins. Recruitment of NSP5-EGFP in green (left) or NSP2-mCherry in red (right) proteins to viroplasms in rotavirus-infected MA104 cells constitutively expressing the two fluorescent fusion proteins. DAPI (blue) detects nuclei. Scale bar: 10 μm. From Papa [27].

Figure 3

Replication of rRV-NSP5/S67A mutant: Pivotal role of phosphorylation in assembling round-shaped and fully functional viroplasms. (A) Immunofluorescent images of MA104 cells infected with rRV-wt and rRV-NSP5/S67A (MOI of 15 FFU/cell) at 5 hpi (upper panels) or 10 hpi (lower panels). Cells were stained with anti-NSP5 (red) and DAPI (blue). Scale bar, 15 μm. From Papa et al. [25]; (B) Electron microscopy image of MA104 cells infected with rRV-NSP5/S67A (MOI, 75 FFU/cell) at 10 hpi. V, viroplasm. White arrows indicate the endoplasmic reticulum surrounding viroplasms; black arrowheads indicate putative viral particles with an envelope. Scale bar, 500 nm. From Papa et al. [25]; (C) RNA Fluorescence In Situ Hybridisation (RNA-FISH) on MA104-NSP5-EGFP cells infected with rRV-NSP5/S67A. Viroplasms detected with NSP5-EGFP (green) and viral RNA (probe specific for genome segment 6-specific probe was with Cy3 (red). Co-localization of viroplasms and RNA is indicated by white arrows. Scale bar, 15 μm. From Papa et al. [25].

Figure 4

(diagram). Assembly of viroplasms during rotavirus infection. NSP5 (pink) acts as a scaffold that recruits RNA chaperone NSP2 (cyan doughnut-shaped octamers), and other RNA-binding clients (e.g., VP1, VP2 and VP3). NSP5 and NSP2 undergo condensation at low micromolar concentrations, forming protein droplets known as ‘viroplasm-like structures’ [21]. Viral transcripts undergo enrichment in these condensates, likely through protein–RNA interactions between NSP2, or VP1 and the scaffolding protein NSP5. VP1 recognizes 3′terminal sequences of all eleven distinct (+)ssRNA transcripts, thus allowing them to enter the dense NSP5/NSP2 phase of a viroplasm. Other multivalent RNA-binding proteins, i.e., the viral capping enzyme VP3, form the ribonucleoprotein (RNP) complexes that can be absorbed into the NSP2/NSP5 condensates. The unique molecular environment (concentrated viral (+)ssRNA transcripts, RNA chaperone NSP2, the scaffolding protein NSP5, and the inner core protein VP2) are expected to be conducive to the multi-RNA genome assembly and packaging steps. Adapted from Borodavka et al. [15] and Geiger et al. [35].