Untangling the processes of replication and encapsidation in Picornavirales

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Despite their importance as pathogens of both animals and plants, and their relatively simple, non-enveloped capsids, remarkably little is known about the mechanism of RNA packaging in members of the family Picornavirales. This project will make use of recent developments in plant transient expression at the John Innes Centre (JIC) and cryo-electron microscopy (cryo-EM) at the University of Leeds (UoL) to determine the mechanism of RNA encapsidation in cowpea mosaic virus (CPMV), a bipartite member of this large virus family. In particular, it will build on the recent demonstration that co-expression of the CPMV coat protein precursor (VP60) and the proteinase necessary for its processing (24K), leads to the assembly of RNA-free capsids into which replicating RNA molecules can be packaged (JIC) and the ability to observe RNA within CPMV particles by cryo-EM (UoL). Specifically, the requirement of replication for packaging will be determined and interactions between particles and replicase- associated proteins will be analysed. Further, the ability of RNAs of differing sizes and sequences to be efficiently packaged will be assessed and the structures adopted by different RNAs within particles will be determined. Finally, the roles of specific amino acids, particularly those at the C-terminus of the small (S) coat protein in the encapsidation process will be determined. As well as being important for our understanding of an important part of the replication cycle of the Picornavirales, the results of this study will also be of relevance to the general field of RNA-protein interactions. Furthermore, they will be highly significant for the development of CPMV particles as a means of delivering specific RNA molecules.

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