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

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants, older adults, and immunocompromised individuals. A hallmark of RSV pathogenesis is early innate immune evasion by the nonstructural proteins NS1 and NS2. NS2 is a multifunctional interferon (IFN) antagonist that dampens both IFNβ induction and type I IFN responsiveness by targeting multiple nodes of the RIG-I/IFN signaling axis. Because NS2-mediated inhibition is often partial and context dependent, quantifying its effects in conventional cell-based assays is challenging. Here, we report the generation and characterization of a stable, virus-free reporter system in human A549 cells to measure NS2-dependent inhibition of dsRNA-driven IFNB induction. The platform combines stable NS2 expression with genomic integration of a dual cassette encoding a non-targeting short hairpin RNA that constitutively activates IRF-3 to induce a luciferase reporter under IFNB promoter control. This configuration yields a sensitive, reproducible luminescence readout of IFNB promoter activity in controlled, infection-free conditions, enabling robust quantification of NS2 antagonism with reduced variability. The scalable luminescence-based format supports early-stage discovery and prioritization of inhibitors that counteract NS2 function, and the modular design is adaptable to other viral antagonists, pathways, and cell types for standardized comparative studies.