Recruitment of multi-segment genomic RNAs by Bluetongue virus requires a preformed RNA network.

ABSTRACT

How do segmented RNA viruses correctly recruit their genome has yet to be clarified. Bluetongue virus is a double-stranded RNA virus with 10 segments of different sizes, but it assembles its genome in single-stranded form through a series of specific RNA-RNA interactions prior to packaging. In this study, we determined the structure of each BTV transcript, individually and in different combinations, using 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP identified RNA structural changes during complex formation and putative RNA-RNA interaction sites. Our data also revealed a core RNA-complex of smaller segments which serves as the foundation ('anchor') for the assembly of a complete network composed of ten ssRNA segments. The same order of core RNA complex formation was identified in cells transfected with viral RNAs. No viral protein was required for these assembly reactions. Further, substitution mutations in the interacting bases within the core assemblies, altered subsequent segment addition and affected virus replication. These data identify a wholly RNA driven reaction that may offer novel opportunities for designed attenuation or antiviral therapeutics.