RNA viruses trigger innate immune responses through distinct signaling pathways involving RIG-I and MDA5 helicases. This study compares the roles of RIG-I, MDA5, and IPS-1 in innate immune signaling against various RNA viruses, including Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae. RIG-I and MDA5 recognize viral RNA and initiate signaling through the IPS-1 adaptor protein, leading to interferon production and antiviral responses. RIG-I is essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus, while MDA5 is involved in picornavirus infections. IPS-1 is critical for innate immune signaling, as its absence impairs interferon production and gene expression. Functional genomics analyses reveal that RIG-I is essential for the expression of genes involved in innate and adaptive immunity, apoptosis, and cytokine signaling during influenza infection. These findings highlight the distinct and redundant roles of RIG-I and MDA5 in RNA virus-triggered innate immune responses, with implications for viral pathogenesis and host defense. The study also identifies a RIG-I-responsive gene bioset linked to immunity and disease during influenza A virus infection. Overall, the results emphasize the importance of IPS-1 in host immunity to RNA viruses and the differential roles of RIG-I and MDA5 in pathogen recognition and signaling.RNA viruses trigger innate immune responses through distinct signaling pathways involving RIG-I and MDA5 helicases. This study compares the roles of RIG-I, MDA5, and IPS-1 in innate immune signaling against various RNA viruses, including Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae. RIG-I and MDA5 recognize viral RNA and initiate signaling through the IPS-1 adaptor protein, leading to interferon production and antiviral responses. RIG-I is essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus, while MDA5 is involved in picornavirus infections. IPS-1 is critical for innate immune signaling, as its absence impairs interferon production and gene expression. Functional genomics analyses reveal that RIG-I is essential for the expression of genes involved in innate and adaptive immunity, apoptosis, and cytokine signaling during influenza infection. These findings highlight the distinct and redundant roles of RIG-I and MDA5 in RNA virus-triggered innate immune responses, with implications for viral pathogenesis and host defense. The study also identifies a RIG-I-responsive gene bioset linked to immunity and disease during influenza A virus infection. Overall, the results emphasize the importance of IPS-1 in host immunity to RNA viruses and the differential roles of RIG-I and MDA5 in pathogen recognition and signaling.