Elina Gerber-Tichet, Université de Montréal

Elina Gerber-Tichet1,2 (presenter) Marta Acchioni3 Lydie Martin1,2 Chiara Acchioni3 Dacquin Kasumba1,4 Marco Sgarbanti3 Nathalie Grandvaux1,4 ; 1 Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada 2 Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada 3 Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy 4 Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada

Building & Escaping

Background: Respiratory syncytial virus NS2 is a multifunctional antagonist of type I interferon responses, but its effects are often partial and context-dependent, complicating quantitative cell-based assays. We developed a stable, virus-free reporter system in human lung epithelial A549 cells to measure NS2-mediated inhibition of RIG-I–dependent activation of the IFNB promoter. Methods: A549 cells stably expressing N-terminally Flag-tagged NS2 and the corresponding control cells were engineered by retroviral integration of a cassette co-expressing a non-targeting short hairpin RNA that constitutively activates RIG-I and a firefly luciferase gene driven by the human IFNB promoter. Results: Constitutive expression of the non-targeting shRNA induced robust IRF3 phosphorylation at Ser396, confirming engagement of the RIG-I pathway, and this signal was reduced in NS2-expressing reporter cells. Across five paired experiments, NS2 reduced IFNB promoter–driven luciferase activity by 58.9% ± 5% (SEM) versus control (paired t test p = 0.004; mean paired Δ = −50,479; 95% CI −74,643 to −26,315). Conclusions: These stable A549 reporter lines provide a sensitive and reproducible readout of NS2-mediated antagonism of interferon beta induction in a controlled, infection-free setting. The luminescence-based format is compatible with scaling for early-stage discovery of small molecules or biologics that counteract NS2 function and can be adapted to other viral immune antagonists.