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Phase Separation Mediated Compartmentalisation of Rotavirus Replication https://doi.org/10.17863/CAM.125247 Acker, Julia Borodavka, Alexander Liquid-liquid phase separation (LLPS) of biopolymers is a fundamental mechanism underlying the formation of cytoplasmic inclusions that function as sites of genome replication and viral particle assembly in rotaviruses. Intrinsically disordered proteins are key drivers of LLPS. In rotaviruses, the disordered protein NSP5 binds the RNA chaperone NSP2, viral RNAs and other viral proteins to form replication factories known as viroplasms. Early in infection, these conden- sates are liquid-like and dynamic, whereas at later stages they undergo maturation that coincides with hyperphosphorylation of NSP5. In this thesis, a minimal system comprising recombinant NSP2 and NSP5 was established, in order to study the formation of viroplasms and the effects of NSP5 phosphorylation on viral replication. This system revealed a phosphorylation-dependent allosteric switch in NSP5 that regulates its interaction with NSP2. Comparative analyses demon- strated that NSP5 variants differ in their intrinsic propensity to phase separate: high-propensity variants phase separate spontaneously, while low-propensity variants require phosphorylation to nucleate condensates. This establishes phosphorylation as a context-dependent regulator of viroplasm assembly across diverse rotavirus strains. Phosphorylation further coincides with selective recruitment of viral RNA into viroplasms. Using an in cellulo system, it was shown here that recruitment occurs only when intact 3’ untranslated regions are present and when the viral polymerase VP1, together with NSP2, localises to viral condensates. Regulation of strain-specific RNA partitioning into viroplasms may furthermore act as a mechanism to coordi- nate rotavirus strain reassortment, as co-infection of different strains does not lead to mixing of different RNAs, as shown using in situ hybridisation techniques. Together, these findings establish LLPS as the organising principle of rotavirus replication factories and reveal how NSP5 phosphorylation, sequence diversity, and RNA features converge to regulate condensate behaviour, genome assembly, and ultimately viral replication. University of Cambridge 2025-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng EPSRC project reference: 2597129 SBS-DTP https://www.repository.cam.ac.uk/handle/1810/395885 https://www.repository.cam.ac.uk/bitstreams/fd034bac-4675-4bb9-a2d1-c0bf601c6783/download fdf9d523183930c388193a6a6ab60f7c https://www.repository.cam.ac.uk/bitstreams/8870fe38-9392-4c05-9f65-b52bdeeda080/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved LLPS Phase separation Virus