Segment specific inverted repeat sequences in bluetongue virus mRNA are required for interaction with the virus non structural protein NS2

Computational secondary structure prediction of all 10 Bluetongue virus (BTV-10) RNA transcripts and mutant inverted repeat transcripts were performed. Transcripts with intact 5′ and 3′ inverted repeat sequences, all indicated base-pairing between the 5′ and 3′ ends when optimal folding parameters were applied. Secondary structure analysis of the mutant transcripts lacking the inverted repeat sequences indicated alterations in the secondary structures resulting from altered base-pairing. The importance of the inverted repeat sequences in RNA–protein binding was subsequently investigated. Deletion mutant clones lacking the 5′ and/or 3′ inverted repeat sequences have been constructed. A baculovirus recombinant expressing the BTV NS2 protein and radioactively synthesized RNA transcripts were subjected to nitrocellulose RNA–protein binding assays. The cumulative results suggested that the inverted repeat deletion mutants display weaker binding compared to BTV-10 segment 8 with intact 5′ and 3′ inverted repeat sequences. Inverted repeats may influence RNA–protein binding by altering the secondary structure of the RNA and consequently the specific NS2 protein-binding sites may no longer be available.