The Clone Expression Of Interferon-stimulated Gene 16(ISG16) And Its Role In Hepatitis C Virus Replication And Type Ⅰ Interferon Signaling

Background:Hepatitis C virus (HCV) is the pathogen that cause non-A non-B hepatitis, with about 150 million people chronically infected world-wide. After infected with HCV, only 20% of patients can effectively clear the virus, and chronic HCV infection has a close association with liver fibrosis, cirrhosis and hepatocellular carcinoma. As such HCV infection remains a huge medical burden in most countries. The current standard of care (SOC) for the treatment of chronic HCV infection in most developing countries is the combination therapy with pegylated interferon alpha (PEG-IFNa) and ribavirin (RBV), but this therapy has many limitation such as long duration of treatment, high costs and various side-effects. Previous studies from our group indicated that higher expression levels of ISG16 were detected in the pre-treatment liver tissues of patients chronically infected with HCV who do not respond to interferon-based therapy. Interferon-stimulated gene 16 (ISG16) is one of the interferon-stimulated genes (ISGs) whose expression levels can be induced by type I interferons. However, its roles on HCV production and type I interferon signaling pathway are not well defined.Objective:This study tested the effect of over-expression of ISG16 on HCV RNA replication and IFN anti-HCV activity in both J6/JFH-1 HCV culture system and HCV replicon cells. In order to explore the potential mechanism, we also tested its effect on type I interferon signaling pathway. ISG16 expression levels from peripheral blood mononuclear cells(PBMCs) of treatment response and non-response HCV patients in the presence and ansence of IFN-a stimulation were also examined to correlate ISG16 expression with treatment outcomes.Methods:We used gene cloning method to construct high expression ISG16 plasmid- pcDNA3. 1-3×tag-ISG16, and then transfected it into Huh7.5.1 and Conlb cells. Realtime-PCR was used to detect the expression levels of ISG16, HCV and other ISGs, and western blot was utilized to detect ISG16 and P-STAT1 protein expression. Double fluorescent reporter gene assay was used to detect the interferon-stimulated response element (ISRE) activity. Realtime-PCR was used to detect the expression levels of ISG16 in PBMCs of treatment response and non-response patients of HCV.Results:Expression plasmid of ISG16 was successfully constructed and transfected into J6/JFH-1 HCV culture system and HCV replicon cells. Over-expression of ISG16 promoted HCV RNA replication and inhibited the anti-HCV activity of interferon-a. ISG16 affected type I interferon signaling by prolonging the phosphorylation of P-STAT1 and reducing the ISRE activity although the down-stream ISGs expression was not affected. Although the baseline ISG16 expression showed no statistically significant difference in PBMCs of patients with chronic HCV infection between treatment responders and non-responders, ISG16 expression increased more significantly in treatment response patients following IFN-a treatment in vitro.Conclusion:Over-expression of ISG16 promoted HCV RNA replication and blunted the anti-HCV activity of interferon-a.