Nonstructural Protein 1 Mediates HMGB1 Release by Targeting Histone H1.0 After Respiratory Syncytial Virus Infection In Vivo and In Vitro

High mobility group box-1 (HMGB1) is implicated in airway inflammation during the late phase of respiratory syncytial virus (RSV) infection. Despite its recognized role, the specific mechanism underlying its release post-RSV infection remains ambiguous. The nonstructural protein 1 (NS1) has been associated with interactions with numerous host proteins, affecting diverse physiological processes, and it is speculated to be involved in the release of HMGB1. We utilized an in vivo model of RSV-infected mice and an in vitro model of RSV-infected A549 and 16HBE cells to investigate the role of NS1 in promoting HMGB1 release. Small interfering RNA was employed to deplete NS1, while lentiviral vectors were used for NS1 overexpression. The interaction between NS1 and H1.0 was confirmed by immunofluorescence analysis, immunoprecipitation, GST pull-down assays, surface plasmon resonance analysis and in silico study. Our study revealed that silencing the NS1 gene reduced the levels of HMGB1 protein and suppressed airway inflammation during the late stage of RSV infection. Depletion of NS1 led to decreased levels of intracellular and extracellular HMGB1 in A549 and 16HBE cells, while over-expression of NS1 increased HMGB1 expression. Furthermore, NS1 and HMGB1 directly interacted with histone H1.0, as confirmed by GST pull-down, surface plasmon resonance and in silico analyses. Overexpression of NS1 disrupted the binding of HMGB1 to H1.0, while silencing of NS1 enhanced their interaction. The research findings indicate that NS1 interacts with H1.0, thereby inhibiting the binding of HMGB1 to H1.0. Consequently, this interaction results in the release of HMGB1 into both the cytoplasm and the extracellular space.