Respiratory syncytial virus nonstructural protein 1 inhibits secretion 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. Several vaccines have recently been approved, which are all based on the RSV fusion (F) protein. Among the strategies to induce a broader immune response is the development of live-attenuated RSV vaccines, for example by disabling nonstructural protein 1 (NS1). RSV NS1 has been found to modulate the host immune response by repression of interferon (IFN) production. However, functional characterization of this protein was mainly performed 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 differentiated primary human nasal epithelial cells (HNEC) cultured at air-liquid interface (ALI), which mimic several physiological properties of the human airways. Using a newly developed yeast-based RSV reverse genetics system, inactivating mutations in the NS1 gene were introduced. These mutations did not affect viral replication in Vero and A549 cells. However, in HNEC we observed a delayed replication of the NS1 mutants compared to the WT virus. Bulk RNA sequencing data from early post infection revealed a stronger antiviral signature in HNEC that were infected with NS1 mutants compared to WT, characterized by upregulation of IFN-ß and IFN-?1/2/3 and chemokines CXCL11 and CCL5. This signature was confirmed by analysis of the cytokines that were released into the HNEC basal medium. Finally, the basal medium containing these cytokines was used for an indirect immune cell migration assay. In this set-up, 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 IFNs, but also by reducing early chemokine production and secretion by epithelial cells. Furthermore, our data highlight the suitability of the ALI transwell model for preclinical assessment of live-attenuated vaccine candidates. Overall design: Cell source: well-differentiated primary nasal epithelial cells, cultured at Air-liquid interface 3 different nasal donors were used. Two transwells per condition were infected with the following RSV strains: RSV-X WT (wild-type virus) RSV-X NS1Y125A (Y125A point mutation in the NS1 protein) RSV-X NS1L132A/L133A (L132A/L133A point mutations in the NS1 protein) Mock: no virus Whole cell lysates were made at 20 hours post infection.  RNA was isolated from the cell lysates to perform RNA-seq profiling.