Comprehensive in virio structure probing analysis of the influenza A virus identifies a functional RNA structure involved in replication and segment interactions

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Intersegment interactions are necessary for segment bundling, and secondary structures on vRNA are assumed to be involved in the process. However, the RNA structure required for segment bundling remains unidentified because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins. Here, we revealed the global intersegment interactions and the secondary structure of the vRNA in virion. We demonstrated that a pseudoknot structure was formed on a segment in the virion and the impairment of replication and packaging of the other specific segment was observed in cells infected with recombinant virus which had mutations in the pseudoknot structure. Moreover, we showed that the intersegment interactions were reconstituted in the recombinant virus. Our data provides the first evidence that the functional RNA structure on the influenza A virus genome affects segment bundling.