ISG15 regulates Zika Virus Replication through Jak/STAT Signaling pathway and autophagy pathway
Yancui Wang, Xinzhong Liao, Hang Zhao, Chunhui Yang, Yujia Li, Min Xu, Xiaoqiong Duan, Shilin Li and Limin Chen
Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, Sichuan, China
Background＆aims: ZIKV infection has been shown to up-regulate ISG15 expression. In this study, we sought to explore whether ISG15 regulates ZIKV replication and its underlying mechanism.
Methods: Overexpression of ISG15 was achieved by transfection of ISG15 plasmid into A549 cells and the cells were infected with ZIKV. Silencing of ISG15 was achieved by siRNA transfection into A549 cells and the silencing efficiency was confirmed by western blot. The expression level of ISG15, type I Interferons (IFNα/β) and ZIKV RNA were quantified by Real-Time PCR, the ability of ZIKV replication and the Jak/STAT signaling pathway was examined by NS1,p-STAT1 and p-STAT2,(western blot), Interferon-sensitive response element (ISRE) activity (dual-luciferase reporter assay) , and ISG expression (real-time PCR). The autophagy signaling pathway was examined by beclin-1 and LC3Ⅰ/Ⅱ(western blot).
Results: ZIKV infection led to the increased expression of ISG15.Overexpression of ISG15 increased ZIKV replication in an ISGylation-dependent manner. Furthermore, ISG15 attenuated IRF3 to inhibit IFN production thus attenuated the IFN-mediated Jak/STAT signaling pathway as shown by the reduced expression of p-STAT1, p- STAT2, ISRE activity, and expression levels of ISGs in the presence of IFNα stimulation. SiRNA specifically targeted against ISG15 in A549 cells promote IFN (IFNα and IFNβ) production, 40% lower level of ZIKV RNA replication was observed in ISG15 silenced cells compared to the irrelevant siRNA control group. Additionally, ISG15 could activate autophagy pathway as shown by the increased expression of beclin-1 and LC3II to increase ZIKV replication.
Conclusions: ISG15 induced by ZIKV infection is a critical host factor that is utilized by ZIKV to confer resistance to type I interferon and to promote autophagy. Therefore, ISG15 could be a potential target for developing novel therapeutic agents against ZIKV, and potentially other viruses' infections.