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.
Acchioni et al. (2026) studied this question.