Abstract
Hepatitis C virus (HCV) infection is primarily treated with regimens that contain pegylated interferon alpha (PEG-IFN-alpha). IFN induces antiviral effects through the up-regulation of many interferon-stimulated genes (ISGs). While many ISGs have previously been identified, limitations of screening approaches employed to date raise the possibility that other anti-HCV ISGs are yet to be discovered. In the present study, a novel screening strategy combined suppression subtractive hybridisation (SSH) and recombinant Dicer generated siRNA pools to screen for anti-HCV ISGs - utilising a replicon model of HCV which is sensitive to IFN-alpha, and in which the impact on HCV replication can be readily assessed through the incorporation of a bicistronic luciferase reporter gene. This approach does not require a priori gene sequence data, and thereby opens up the possibility of detecting the anti-HCV activity of novel genes, functional polymorphisms and splice variants. SSH and its related technique mirror orientation selection (MOS) were employed to isolate differentially expressed genes from IFN-alpha treated Huh-7 cells. The isolated SSH and MOS genes were cross-referenced with microarray data to identify likely mediators of the anti-HCV replicon effects of IFN-alpha treatment. Subsequently, SSH/MOS cDNA clones were used to produce complementary dsRNA, which was digested with recombinant Dicer to generate target-specific siRNA pools, that were then screened for their ability to suppress the effects of IFN (using luciferase activity as a measure of replicon RNA copy number) following transfection into the stable HCV-replicon cell line (Huh-7 Luc). The list of positive screen hits included ZC3HAV1 and IFIT1. Further validation experiments showed the long isoform of ZC3HAV1 to be a new bona fide anti-replicon ISG. Thus, this study demonstrates the utility of unbiased screening approaches in better understanding how IFN-alpha limits HCV replication.