Influenza A Virus NS1 Limits Recognition of Double-Stranded Transposable Elements by Cytosolic RNA Sensors

Influenza A virus (IAV) infection triggers de-repression of host transposable elements (TEs), which have the potential to form double-stranded (ds)RNAs and stimulate innate antiviral immunity. However, as wild-type IAV is generally a poor inducer of innate immunity, it remains unclear whether de-repressed TEs actually form dsRNAs recognizable by host cytosolic RNA sensors, or whether IAV might antagonize such sensing. Here, we performed strand-specific total RNA-Seq on nuclear and cytosolic fractions from cells infected with wild-type IAV or a recombinant IAV lacking NS1, a viral dsRNA-binding protein. Both infections led to global increases in host TE RNAs with bioinformatic and experimental evidence for double-strandedness. However, only NS1-deficient IAV infection led to significant amounts of TE-dsRNAs translocating to the cytosol, and co-precipitations identified that wild-type NS1 associates with TE-dsRNAs. Furthermore, a functional screen indicated that TE-dsRNAs can be engaged by various host cytosolic RNA sensors, including RIG-I, MDA5, ZBP1, and PKR. Our data reveal the double-stranded nature of infection-triggered host TEs and suggest an NS1-mediated sequestration mechanism to limit their cytosolic abundance and broad activation of diverse sensors.