Kwaschik, F.; Pichler, I.; Ruiz, J. L.; Kufner, V.; Huber, M.; Hale, B. G.

RNA:DNA hybrids, and their aberrant accumulation, are key regulators of human genome integrity and innate immunity. Given the functional parallels and extensive interplay between the human genome and viral genetic material, RNA:DNA hybrids also have established roles during infections with DNA viruses and retroviruses. Nevertheless, the presence and consequences of RNA:DNA hybrids in RNA virus infections, which lack a DNA phase, remain largely uncharacterized. Here, we show that infection of human cells with influenza A virus (IAV), but not several other respiratory negative-sense RNA viruses, induces the unexpected accumulation of RNA:DNA hybrids in perinuclear regions. Sequencing of infection-induced cytoplasmic RNA:DNA hybrids revealed that they derive predominantly from the human host genome, with apparent preference for intronic and intergenic sequences, although an intriguing fraction also originates from the IAV genome, with some enrichment for specific viral segments. Notably, we identify the IAV non-structural protein, NS1, as a key determinant of RNA:DNA hybrid localization and stability: NS1 is required for, and co-localizes with, perinuclear RNA:DNA hybrids, and its absence appears to sensitize RNA:DNA hybrids to metabolism by the cellular ribonuclease, RNase H1. Furthermore, experimental loss of RNase H1 attenuates IAV-induced antiviral responses, including type I interferon and inflammatory gene expression programs, indicating that RNA:DNA hybrid metabolism likely contributes to host defense. Overall, our findings uncover host- and viral-origin RNA:DNA hybrids as a previously unrecognized feature of a human pathogenic RNA virus infection, and suggest a mechanism by which a viral product antagonizes host responses mediated via these unusual nucleic acids by spatial sequestration.