Maximal interferon induction by influenza lacking NS1 is infrequent owing to requirements for replication and export

Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. As such, most viral particles cannot complete the life cycle. However, despite this failure, this virus is incredibly successful in the suppression of innate immune detection and the production of interferons, succeeding at remaining undetected in >99% of cells in tissue-culture models of infection. As the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1, what features prevent these events from triggering massive amounts of interferon production? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus. Taken together, the highest level of stimulation we observe (97% of infected cells), requires a high level of replication in the presence of defective viral genomes with NS1 and NS1 alone from the NS segment bearing an inactivating mutation. This is incredibly unlikely to occur within the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection. ### Competing Interest Statement The authors have declared no competing interest.