A Stable Reporter Cell Line to Study Respiratory Syncytial Virus NS2-Mediated Inhibition of IFNB Promoter Activation

Human respiratory syncytial virus (RSV) is an enveloped, non-segmented, negative-sense RNA virus and a major cause of severe lower respiratory tract disease in infants, older adults, and immunocompromised individuals. A hallmark of RSV pathogenesis is early evasion of innate immunity mediated by the non-structural proteins NS1 and NS2. NS2 is a multifunctional interferon (IFN) antagonist that dampens both IFN{beta} induction and type I IFN responsiveness by targeting multiple nodes of the RIG-I/IFN signaling axis. Because NS2 inhibition is typically partial and context dependent, its quantitative assessment in conventional cell-based assays can be challenging. Here, we report the generation and characterization of a stable, virus-free reporter system in human lung epithelial A549 cells to measure NS2-mediated inhibition of RIG-I-dependent activation of the human IFNB promoter. The platform combines stable NS2 expression with genomic integration of a dual cassette encoding (i) a non-targeting short hairpin RNA that constitutively activates RIG-I signaling and (ii) a firefly luciferase reporter driven by the IFNB promoter. This configuration provides a sensitive and reproducible luminescence readout of IFNB promoter activity under controlled, infection-free conditions, enabling robust quantification of NS2 antagonism with reduced experimental variability. The luminescence-based format is readily scalable for early-stage discovery and prioritization of small molecules or biologics that counteract NS2 function. More broadly, the modular design can be adapted to other viral immune antagonists, signaling pathways, or cell types, supporting comparative studies where pathway modulation must be evaluated under highly standardized and reproducible conditions.