Respiratory Syncytial Virus (RSV) is the first cause of viral bronchiolitis and pneumonia in young children and the elderly. Airway mucosal immunity is the first barrier against RSV, having interferons (IFN) as central actors of the antiviral response. However, RSV and other respiratory viruses have evolved various evasion strategies to counteract such IFN responses. This study aims at characterizing the role of RSV Non-structural Protein 2 (NS2) as a putative modulator of innate response to infection.
We infected reconstituted human airway epitheliums (HAE) with wild-type (WT) or NS2 gene-deleted (ΔNS2) RSV strains. We then combined longitudinal kinome and RNAseq analyses to identify host factors and pathways modulated in response to both strains and also specific of ΔNS2. We finally used knock-out (KO) cell lines and specific inhibitors to validate our hypotheses.
RSV-ΔNS2 was attenuated in HAE model, with lower viral titers and an earlier induction of IFN than the WT. ΔNS2 infection resulted in significantly increased activity of STK38L, a kinase involved in IRF3 phosphorylation, suggesting a role of NS2 in the negative modulation of IRF3-based antiviral response. This hypothesis was confirmed by the loss of the ΔNS2 attenuated phenotype compared to the WT in an IRF3-KO cells. Total or partial loss of ΔNS2 attenuation was also observed in IFNAR1 and IFNLR1-KO cells, respectively. Longitudinal functional enrichment and weighted co-expression network analysis enabled the identification of functional and co-expression gene modules specifically linked to the innate immune response, which are significantly associated with infection by the ΔNS2 but not the WT virus. Protein-protein interaction (PPI) network analysis further revealed dozens of interaction partners of activated kinases within these modules.
Our results highlight RSV NS2 protein as a major negative modulator of IFN antiviral responses through, although not exclusively, interference with STK38L-IRF3 signaling. Ongoing regulomics and functional perturbation studies should identify and validate specific factors and paths to further map key NS2-associated PPIs.