Influenza A viruses (IAVs) are segmented single-stranded negative sense RNA viruses that constitute a major threat to human health. The IAV genome consists of eight RNA segments contained in separate viral ribonucleoprotein complexes (vRNPs) that are packaged together into a single virus particle. While IAVs are generally considered to have an unstructured single-stranded genome, it has also been suggested that secondary RNA structures are required for selective packaging of the eight vRNPs into each virus particle. Here, we employ high-throughput sequencing approaches to map both the intra and intersegment RNA interactions inside influenza virions. Our data demonstrate that a redundant network of RNA-RNA interactions is required for vRNP packaging and virus growth. Furthermore, the data demonstrate that IAVs have a much more structured genome than previously thought and the redundancy of RNA interactions between the different vRNPs explains how IAVs maintain the potential for reassortment between different strains, while also retaining packaging selectivity. Our study establishes a framework towards further work into IAV RNA structure and vRNP packaging, which will lead to better models for predicting the emergence of new pandemic influenza strains and will facilitate the development of antivirals specifically targeting genome assembly.
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