Integrin-FAK signaling dependent non-canonical ISGylation by extracellular ISG15 restricts human respiratory syncytial virus infection in epithelial cells

Interferons (IFN) induce cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to target cellular proteins through ISGylation to confer antiviral activity and can regulate cellular processes associated with infectious, inflammatory and neurodegenerative diseases, and cancer. Apart from ISGylation, unconjugated free ISG15 is also released from cells during various conditions, including virus infection. The role of extracellular ISG15 during virus infection is poorly understood. Additionally, it was unknown whether extracellular ISG15 can activate IFN independent ISGylation via activation of alternative cellular signaling pathway. We have used IFN and IFN signaling incompetent epithelial cells infected with human respiratory syncytial virus (RSV) or treated with purified human ISG15 protein. Additional studies were conducted with IFN competent cells treated with IFN signaling inhibitors, along with extracellular ISG15 blocking antibody and immunofluorescence analysis to demonstrate the role of extracellular ISG15 during virus infection. The involvement of integrin-Focal Adhesion Kinase (FAK) pathway during this event was investigated by using integrin-FAK pathway inhibitors and mutant ISG15 protein (generated based on in silico modeling) that failed to interact with integrin. To demonstrate post-translational regulation, protein chase experiment was performed with protein synthesis inhibitor treated cells. We have identified an alternative non-canonical ISGylation route initiated by extracellular ISG15 and mediated by integrin-FAK signaling, in contrast to the classical IFN-driven ISGylation. We show that extracellular ISG15 acts as a soluble antiviral factor that triggers IFN independent ISGylation and restricts virus infection in epithelial cells. Specifically, extracellular ISG15 engages with cell surface integrins via its RGD-like motif to activate the integrin-FAK pathway, resulting in IFN-independent ISGylation. Furthermore, in the absence of ISG15 transcriptional upregulation, extracellular ISG15 enhances the post-translational stability of basal intracellular ISG15 protein to support ISGylation. Thus, our studies have identified extracellular ISG15 as an antiviral factor that promotes non-canonical IFN independent ISGylation through integrin-FAK signaling and post-translational stabilization of intracellular ISG15 protein. Because ISG15 and ISGylation influence diverse infectious, inflammatory, and degenerative conditions, this alternative ISGylation route has broad implications for antiviral immunity, disease resolution, and development of host-directed therapeutic strategies for various diseases.