RIG-I-mediated antiviral responses to single-stranded RNA bearing 5' phosphates are described. The study shows that influenza A virus infection does not generate double-stranded RNA (dsRNA) and that RIG-I is activated by single-stranded viral genomic RNA with 5' phosphates. This activation is blocked by the influenza NS1 protein, which forms a complex with RIG-I in infected cells. These findings indicate that RIG-I acts as a sensor for single-stranded RNA and is a target for viral immune evasion. The study suggests that RIG-I evolved to sense uncapped RNA for self/nonself discrimination by the innate immune system. The innate immune response to viral infection involves the rapid production of cytokines, particularly type I interferons (IFN-α/β). Specialized plasmacytoid dendritic cells (pDC) produce IFN-α/β upon triggering of toll-like receptors (TLRs) 7, 8, and 9 by viral genomes. Other cell types rely on cytoplasmic sensors like RIG-I and MDA5, which are activated by dsRNA. The influenza NS1 protein suppresses IFN-α/β production by sequestering dsRNA. The study confirms that influenza A virus inhibits IFN-α/β production through NS1. cDC derived from murine bone marrow progenitors infected with a mutant virus lacking NS1 produced 100-fold more IFN-α than wild-type infected cells. No dsRNA was detected in infected cells, suggesting that RIG-I is activated directly by the influenza single-stranded RNA genome. Transfection with influenza viral RNA induced potent activation of the IFN-β reporter and production of IFN-α and IL-6 by BM-DC. This response was RIG-I-dependent and inhibited by NS1. The study also shows that RIG-I recognizes influenza ssRNA genomes bearing 5' phosphates. The presence of 5' phosphates is crucial for RIG-I activation, as treatment with calf intestinal phosphatase abrogated the stimulatory properties of flu vRNA. RIG-I forms stable complexes with RNA containing phosphorylated 5' ends, and NS1 is recruited to such complexes via its RNA binding domain. These findings suggest that RIG-I recognizes viral RNA with 5' phosphates, which serve as a "pathogen-associated molecular pattern" for the innate immune system. This parallels the recognition of cytoplasmic DNA by the innate immune system.RIG-I-mediated antiviral responses to single-stranded RNA bearing 5' phosphates are described. The study shows that influenza A virus infection does not generate double-stranded RNA (dsRNA) and that RIG-I is activated by single-stranded viral genomic RNA with 5' phosphates. This activation is blocked by the influenza NS1 protein, which forms a complex with RIG-I in infected cells. These findings indicate that RIG-I acts as a sensor for single-stranded RNA and is a target for viral immune evasion. The study suggests that RIG-I evolved to sense uncapped RNA for self/nonself discrimination by the innate immune system. The innate immune response to viral infection involves the rapid production of cytokines, particularly type I interferons (IFN-α/β). Specialized plasmacytoid dendritic cells (pDC) produce IFN-α/β upon triggering of toll-like receptors (TLRs) 7, 8, and 9 by viral genomes. Other cell types rely on cytoplasmic sensors like RIG-I and MDA5, which are activated by dsRNA. The influenza NS1 protein suppresses IFN-α/β production by sequestering dsRNA. The study confirms that influenza A virus inhibits IFN-α/β production through NS1. cDC derived from murine bone marrow progenitors infected with a mutant virus lacking NS1 produced 100-fold more IFN-α than wild-type infected cells. No dsRNA was detected in infected cells, suggesting that RIG-I is activated directly by the influenza single-stranded RNA genome. Transfection with influenza viral RNA induced potent activation of the IFN-β reporter and production of IFN-α and IL-6 by BM-DC. This response was RIG-I-dependent and inhibited by NS1. The study also shows that RIG-I recognizes influenza ssRNA genomes bearing 5' phosphates. The presence of 5' phosphates is crucial for RIG-I activation, as treatment with calf intestinal phosphatase abrogated the stimulatory properties of flu vRNA. RIG-I forms stable complexes with RNA containing phosphorylated 5' ends, and NS1 is recruited to such complexes via its RNA binding domain. These findings suggest that RIG-I recognizes viral RNA with 5' phosphates, which serve as a "pathogen-associated molecular pattern" for the innate immune system. This parallels the recognition of cytoplasmic DNA by the innate immune system.