Suppression of the interferon sensitive gene 15 affects the expression of proteins that play a role in HCV replication

Introduction: The interferon stimulated gene 15 (ISG15) has been identified as pro-viral host factor in the pathogenesis of HCV. As an ubiquitin-like protein ISG15 is conjugated to many cellular proteins, but the link between ISG15 and HCV is not well understood. Aim of the present study was to functionally analyse the role of ISG15 and to identify the HCV promoting interactions. Methods: ISG15-specific siRNAs or control siRNAs (NC) were transfected into human and murine hepatoma cell lines, respectively, harboring a subgenomic HCV replicon (human: con1; murine: MH1). For in vivo studies, nanolipid-formulated siRNAs (ISG15, NC) were injected into C57Bl/6 mice to specifically target hepatocytes. Primary murine hepatocytes (PMH) from wild-type C57Bl/6 mice were prepared 48h after siRNA application. Cells were lysed after 24h and 1D-gel electrophoresis, in-gel digestion, RP-HPLC-MS/MS analysis, quantitative analysis and protein identification were performed. Transcriptome analysis was additionally performed. Results: At the time points chosen, transcriptome analysis of ISG15-suppression in replicon cells (con1, MH) and murine liver tissue did not reveal any major changes in gene expression. In contrast, proteome analysis of ISG15-suppression in murine MH1 cells identified different groups of regulated proteins, which have previously been linked to pathways that are associated with HCV replication (p70S6 kinase, mTOR signaling and translational control). Comparative analysis between the MH1 proteome study and the HCV-free PMH study revealed an overlay of seven similar regulated proteins. Among these are two heterogeneous nuclear ribonucleoproteins (Hnrnp) and the Hydroxymethylglutaryl-CoA synthase, all of which have been shown to play a role in HCV replication. Conclusions: These data suggest that ISG15 has multiple effects on cellular processes, including the modulation of signaling pathways that have previously been shown to regulate HCV replication. This is of particular relevance for therapeutic strategies targeting this host factor and may represent a novel therapeutic option for HCV patients, which are non-responders to IFN-based therapies.