Influenza A virus defective viral genomes are inefficiently packaged into virions relative to wild-type genomic RNAs.

Defective interfering particles (DIPs) are commonly produced by diverse families of RNA viruses and have directly implicated in modulating clinical infection outcomes. Based on this, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs that carry large internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain very poorly understood. Here, we used a specialized next generation sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This dataset provides a rich, unbiased profile of the deletion-forming preferences of the influenza virus replicase, revealing the enrichment of specific sequence features surrounding DelVG deletions. Additionally, by comparing the early intracellular DelVGs with those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.