Mapping Binding Sites of Nucleoprotein Within the Influenza Virus RNA Genome.

With the advent of next-generation sequencing, a plethora of techniques have been developed to uncover nucleic acid interactions with unprecedented resolution. For example, UV-crosslinking and immunoprecipitation (CLIP) assays have been coupled to deep-sequencing to identify RNA-protein interactions and precisely map the RNA footprint regions. Here, we describe a CLIP-based technique that allows the genome-wide mapping of influenza nucleoprotein (NP)-binding sites to genomic viral RNA (vRNA). This method has been applied to show that the influenza viral genome is not uniformly coated with nucleoprotein, but instead enriched in certain regions and depleted in others. As subtle changes in genome sequence have been shown to globally alter NP-binding sites, this technique will be useful to deduce how different strains adjust their genome organization and what parameters govern NP-vRNA interactions.