Genome scale ordered RNA structure (GORS) is found throughout the genome of many single stranded positive sense RNA viruses, including plant viruses. It was hypothesised that GORS may function to help evade RNAi either by preventing the generation of siRNAs or by stopping RNAi-mediated cleavage of the target. This project used Potato Leafroll Virus (PLRV) to investigate the function of GORS in plant viruses. The RNA structure of a 750nt region of the genome was modified to have less, more or the same energy as the WT sequence. The physical structure of these sequences was shown to be different using two distinct methods. Viral infectivity was tested and although all four viruses were able to replicate and spread to distal leaves, the WT virus was always able to outcompete the variant viruses in competition assays. This suggests GORS provides a distinct selective advantage to the WT virus. The effect on the siRNA response was tested using a dedicated siRNA assay. In plants, this showed that the WT sequence was more resistant to degradation by siRNAs than the variant sequences in the presence of their specific inducers. The WT inducer was also not able to cause suppression of the other targets indicating that this inducer failed to produce siRNAs or that they were not effective. The siRNA populations generated during infections were sequenced and the profiles compared. This showed that all four viruses stimulated the production of siRNAs but the location of siRNA hotspots differed. It is therefore hypothesised that GORS may function to evade the RNAi response by directing the generation of less effective siRNAs. The data presented in this thesis not only informs current work on GORS and RNA structure in viral genomes but also has wider implications for research on siRNAs and food biosecurity.

The rotavirus replication cycle has not been fully characterised, one vital stage of virus replication involves large cytoplasmic occlusion bodies termed viroplasms. These are the sites of synthesis and replication of dsRNA, packaging of viral RNA into newly synthesized cores and the formation of double-shelled previrions. The detailed mechanism by which these events occur is poorly understood but is thought to be mediated by the non-structural proteins localised to these structures. Rotavirus gene segment 11 expresses two proteins NSP5 and NSP6 which are found in alternate open reading frames. NSP5 exists is several isoforms which differ on their level of phosphorylation. It has been shown to be essential for virus replication and localises to the viroplasms. The smaller NSP6 protein is the most uncharacterised of all of the rotavirus proteins. It has however been shown to interact with NSP5 and has been tentatively suggested to be localised to the viroplasms. To further investigate these two proteins the pET expression system was utilised to obtain purified protein which was subsequently used to generate mono specific polyclonal antisera. Studies into the function and localisation of these proteins found that both localised to the viroplasms and their relative distributions within these structures were defined. NSP6 was found to be expressed at a low level throughout the course of a rotavirus infection and in contrast to other non-structural proteins, to have a high rate of turnover. The RNA binding ability of both NSP5 and NSP6 was investigated using quantitative filter binding assays and these showed both have sequence independent nucleic acid binding ability. Studies were also conducted into the mechanism of NSP6 expression from the second open reading frame of gene 11, the results obtained being consistent with a leaky scanning mechanism of expression.