Supplementary Material for: Respiratory syncytial virus nonstructural protein 1 inhibits production of cytokines and chemokines by differentiated primary nasal epithelial cells cultured at air-liquid interface

Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and older adults. Currently approved vaccines target only the viral fusion protein, but live-attenuated vaccines - e.g. through inactivation of nonstructural protein 1 (NS1) - likely induce a broader immune response. NS1 inhibits the immune response by repressing interferon production, but this has mostly been shown in immortalized cell lines, which do not necessarily represent the in vivo situation. Here, we assessed the effect of NS1 mutations on replication and host responses in physiologically relevant differentiated primary human nasal epithelial cells (HNEC) cultured at air-liquid interface (ALI). Using yeast-based reverse genetics, NS1-inactivating mutations were introduced. In differentiated HNECs, NS1 mutants showed delayed replication compared to wild-type virus. Bulk RNA sequencing early after infection revealed stronger antiviral signatures in HNEC infected with NS1 mutants compared to wild-type, characterized by upregulation of interferons and chemokines. Cytokine analysis confirmed these results. Finally, an indirect immune cell migration assay revealed that both WT and NS1-mutant viruses induce migration of mainly neutrophils. In conclusion, this study shows that RSV NS1 supports viral replication not only via inhibition of the production of interferons, but also by reducing early chemokine production and secretion by epithelial cells. Together, our data highlight the suitability of the ALI transwell model for preclinical assessment of live-attenuated vaccine candidates.