Rapid mapping of functional cis-acting RNA elements by recovery of virus from a degenerate RNA population: application to genome segment 10 of bluetongue virus

The regulatory elements which control the processes of virus replication and gene expression in the Orbivirus genus are uncharacterised both in terms of their locations within genome segments and their specific functions. The reverse genetics system for the type species, Bluetongue virus, has been used in combination with RNA secondary structure prediction to identify and map the positions of cis-acting regions within genome segment 10. Through the simultaneous introduction of variability at multiple nucleotide positions in the rescue RNA population, the functional contribution of these positions was used to map regions containing cis-acting elements essential for virus viability. Nucleotides that were individually lethal when varied mapped within a region of predicted secondary structure involving base pairing between the 5' and 3' end of the transcript. An extended region of predicted perfect base pairing located within the 3' untranslated region (UTR) of the genome segment was also found to be required for virus viability. In contrast to the identification of individually lethal mutations, gross alteration of the composition of this predicted stem region was possible, providing the base pairing potential between the two strands was maintained, identifying a structural feature, predicted to be conserved throughout the Orbivirus genus. The approach of identifying cis-acting sequences through sequencing the recovered virus following the rescue of a degenerate RNA population is broadly applicable to viruses where reverse genetics is available.