Investigating the dynamic nature of biomolecular condensates that support rotavirus replication

Abstract

Group A rotaviruses (RVAs) induce the formation of viral replication factories, termed viroplasms, where new viral particle packaging and genome replication occur. The formation of such factories is driven by liquid-liquid phase separation (LLPS) of an intrinsically disordered phosphoprotein NSP5, which acts as a scaffold, along with the RNA chaperone NSP2 as the major client biomolecule. While viroplasms have been recognised as ribonucleoprotein condensates that serve as replication sites for RVA, many aspects of their formation, functions, and composition remain poorly understood and require further investigation. Utilising a machine learning-based LLPS prediction tool in combination with reverse genetics, a recombinant RVA expressing an engineered NSP5 variant with amino acid features of low LLPS propensity was rescued. Unexpectedly, this recombinant virus retained the ability to form viroplasms in infected cells. Comparative studies with wild-type RVA suggest that phosphorylation may be crucial in promoting LLPS in NSP5 variants with a low LLPS propensity. Further comparative analysis of host proteome and phosphoproteome following infection with either the low LLPS propensity variant or the wild-type RVA indicates that viroplasms may facilitate immune evasion and modulate various host cellular processes. Therefore, my results suggest additional roles of viroplasms extend beyond simply the sites of viral replication. Additionally, advanced labelling investigations suggest that a diverse range of proteins co-localise with viroplasms during infection, and newly synthesised NSP5 is directed to nascent viroplasms rather than pre-existing ones. In summary, I developed and utilised a variety of tools to investigate the dynamics of RVA viroplasms from three perspectives: their formation, functions, and composition. These findings not only provide new insights into viroplasm but also lay a preliminary foundation for future research.