ISG15 Monomer Promotes IFNα-mediated Antiviral Activity against Pseudorabies Virus by Facilitating phosphorylation of STAT1/STAT2

Pseudorabies virus (PRV), which presently lacks both an antiviral drug and a viable therapeutic option, is a major viral pathogen that poses a danger to the pig industry worldwide. Interferon-stimulated gene 15 (ISG15) is strongly upregulated during viral infections and has been reported to have proviral or antiviral properties, depending on the virus and host species. Our previous studies demonstrated ISG15 was remarkably upregulated during PRV infection, and the overexpression of ISG15 inhibited PRV replication. Nevertheless, the exact mechanism through which ISG15 influences PRV replication poorly understood unclear. Here, we demonstrate that ISG15 accumulation induced by PRV infection requires viral gene expression and viral growth. Conjugation inhibition assays showed that ISG15 imposes its antiviral effects via unconjugated (free) ISG15 and affects the viral release. In addition, we found ISG15 promoted IFN-mediated antiviral activity against PRV by facilitating the phosphorylation of STAT1 and STAT2, along with an increase of ISGF3-induced ISRE promoter activity. Furthermore, we evaluated the role of ISG15 in host defense to control PRV infection by using ISG15-/- mice model. When challenged with PRV, ISG15-/- mice exhibited increased morbidity and mortality, as well as viral load compared to WT mice. Pathological examination revealed increased lesions, mononuclear cellular infiltration and neuronal death in the brains of ISG15-/- mice, along with the upregulation of the cytokines. Our findings establish the importance of ISG15 in IFN-induced antiviral response and in the control of PRV infection.