Mechanisms of Genome Segment Assortment in Reovirus

Members of the Reoviridae family of double-stranded (ds)RNA viruses represent a wide range of important human animal and plant pathogens. During replication, each virus must select and package a complete genome, comprised of up to 12 distinct RNA segments. Stoichiometric genome segment selection is achieved through a series of inter-segment RNA-RNA interactions, mediated by virally-encoded proteins with RNA chaperone-like activities. Two of the most well-characterised examples of such proteins are rotavirus NSP2 and avian reovirus sigmaNS. Although both proteins bind multiple single-stranded (ss)RNAs concurrently with near-identical, low-nanomolar affinities, possess helix destabilising activity and can promote RNA duplex formation, it has long remained unclear whether NSP2 and sigmaNS employ similar mechanisms to promote RNA-RNA matchmaking between RNA segments. The work presented in this thesis aims to understand the mechanisms underlying the selective formation of inter-molecular RNA duplexes by the viral RNA chaperone proteins NSP2 and sigmaNS. A comparison of the RNA-binding, helix unwinding and RNA-annealing activities of NSP2 and sigmaNS revealed different modes of RNA chaperone activities. The role of the C-terminal domain of NSP2 in its RNA chaperone activities was subsequently investigated. Single-molecule fluorescence, protein-RNA crosslinking binding kinetics measurements revealed a role for the CTD in RNA dissociation, which was further supported by cryoEM structures of NSP2 alone and in complex with RNA (3.9 Å and 3.2 Å resolution, respectively). Finally, sigmaNS oligomerisation and ribonucleoprotein assembly was explored, revealing that sigmaNS assembles into large, filamentous RNP species. These may serve as a platform for RNA remodelling, as they are sensitive to RNA structure and disassemble upon duplex formation. Together, these data demonstrate the diversity in mechanisms used to facilitate RNA-RNA interactions employed by viral RNA chaperones, and suggest that NSP2 and sigmaNS utilise different auto-regulatory mechanisms to modulate RNA unwinding and annealing.