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. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? 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 the 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, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given 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. Author summaryThe production of interferons in response to viral threat is a potent barrier to successful infection. Viruses like influenza A virus encode potent interferon antagonists, suppressing detection of viral replication such that only an incredibly small number of cells successfully produce interferons. Strikingly, even in the absence of its major interferon antagonist, NS1, influenza still only induces an interferon response in a minority of infected cells. Using a combination of single-cell RNAseq, flow cytometry, and classical bulk methods, we explored how heterogeneity, both genetic and phenotypic, in a virus lacking NS1, explains the observed rarity of the interferon response. In doing so, we find that, in the absence of NS1, viral replication is absolutely required to induce an interferon response, and that both active export and the presence of defective viral genomes can greatly enhance, but are not strictly required, for the detection of this virus. The confluence of events which we find maximizes the host response is incredibly unlikely to occur alongside the absence of the NS1 protein via the most common forms of genetic variation in the viral population, perhaps explaining, in part, how a virus with such a high error rate nevertheless has such a high rate of success at evading cell intrinsic innate immunity.