Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments

Segmented RNA viruses are ubiquitous pathogens, which include influenza viruses and rotaviruses. A major challenge in understanding their assembly is the combinatorial problem of a non-random selection of a full genomic set of distinct RNAs. This process involves complex protein/RNA interactions, which are often obscured by non-specific binding at concentrations approaching in vvo assembly conditions. Here, we present direct experimental evidence of sequence-specific inter-segment interactions in rotaviruses, taking place in a complex RNA- and protein- rich milieu. We show that binding of the rotaviral protein NSP2 to ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. To identify the sites of these interactions, we have developed an RNA-RNA SELEX approach for mapping the sequences involved in inter-segment base-pairing. Our findings elucidate the molecular basis underlying inter-segment interactions in rotaviruses, paving the way for delineating RNA-RNA interactions that govern assembly of other segmented RNA viruses