Structural basis of rotavirus RNA chaperone displacement and RNA annealing.

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling

Unravelling the Mechanism of Rotavirus Viral Factory Formation Using Structural Mass Spectrometry

Upon infection, many viruses form intracellular biomolecular condensates where viral replication occurs and are often referred to as viral factories (VFs). The assembly of these VFs is an obscure process involving the formation of transient, heterogenous protein / RNA assemblies. This thesis demonstrates how structural mass spectrometry (MS) methods can be employed to investigate the structure, dynamics, and interactions of proteins within a biomolecular condensate in vitro and in cell to bridge the gap between high- and low- resolution biophysical and structural techniques. Here, rotavirus (RV) has been utilised as a model system to both study the mechanism of VF formation and demonstrate the use of structural MS to interrogate condensate formation mechanisms. Two non-structural proteins, NSP2 (an RNA chaperone) and NSP5 (an intrinsically disordered protein [IDP] which undergoes hyperphosphorylation in infected mammalian cells), along with viral RNA drive the formation of VFs. In this thesis, data from native MS are presented, which has revealed that NSP2 exists as an octamer and NSP5 as a decamer. Hydrogen-deuterium exchange MS (HDX-MS) has additionally revealed that the N-terminus of NSP5 is least protected from deuterium exchange and therefore likely to be disordered while the C-terminal region of NSP5 is most protected from exchange and thus likely to be more structured. Furthermore, native MS has also implicated the C-terminal region of NSP5 in the formation of higher order structures. This has provided the first glimpses into the structure of this IDP and interactions which mediate oligomerisation. Next, to understand the interaction between NSP2 and NSP5 mediating VF formation, it was necessary to develop a HDX-MS workflow to uncover key interacting regions of NSP2 and NSP5 within a condensate forming environment was conducted. This has revealed NSP5 binding within the C-terminal region of NSP2 (previously identified to promote RNA annealing) possibly induces a conformational change which could contribute to the allosteric regulation of RNA annealing. Finally, using label free quantification (LFQ), comparing infected and uninfected cells, the host proteins / pathways that are modulated upon infection were investigated, revealing possible mechanisms of immune evasion and how VF formation modulates the proteome. The mechanistic insights in VF assembly presented herein are vital for understanding VF function and for developing therapies that interfere with the assembly of mature VFs. Moreover, the work presented in this thesis demonstrates the potential for structural MS methods to interrogate mechanisms of biomolecular condensate formation