Free ISG15 Inhibits the Replication of Peste des Petits Ruminants Virus by Breaking the Interaction of Nucleoprotein and Phosphoprotein

Peste des petits ruminants virus (PPRV) causes a highly contagious disease in small ruminants and severe economic losses in developing countries. PPRV infection can stimulate high levels of interferon (IFN) and many IFN-stimulated genes (ISGs), such as ISG15, which may play a key role in the process of viral infection. However, the role of ISG15 in PPRV infection and replication has not yet been reported. In this study, we found ISG15 expression to be significantly upregulated after PPRV infection of caprine endometrial epithelial cells (EECs), and ISG15 inhibits the proliferation of PPRV. Further analysis showed that free ISG15 could inhibit PPRV proliferation. Moreover, ISG15 does not affect the binding, entry, and transcription but does suppress the replication of PPRV. A detailed analysis revealed that ISG15 interacts and colocalizes with both viral N and P proteins and that its interactive regions are all located in the N-terminal domain. Further studies showed that ISG15 can competitively interact with N and P proteins and significantly interfere with their binding. Finally, through the construction of the C-terminal mutants of ISG15 with different lengths, it was found that amino acids (aa) 77 to 101 play a key role in inhibiting the binding of N and P proteins and that interaction with the P protein disappears after the deletion of 77 to 101 aa. The present study revealed a novel mechanism of ISG15 in disrupting the activity of the N(0)-P complex to inhibit viral replication. IMPORTANCE PPRV, a widespread and fatal disease of small ruminants, is one of the most devastating animal diseases in Africa, the Middle East, and Asia, causing severe economic losses. IFNs play an important role as a component of natural immunity against pathogens, yet the role of ISG15, an IFN-stimulated gene, in protecting against PPRV infection is currently unknown. We demonstrated, for the first time, that free ISG15 inhibits PPRV proliferation by disrupting the activity of the N(0)-P complex, a finding that has not been reported in other viruses. Our results provide important insights that can further understand the pathogenesis and innate immune mechanisms of PPRV.