| BackgroundThere are about 150 million people infected with Hepatitis C Virus (HCV) worldwide. HCV typically causes a chronic infection that is often associated with liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). As such, HCV-related liver disease is a serious public health problem in many countries. Pegylated interferon-a and ribavirin (PEG-IFN/RBV) is widely used to treat chronic hepatitis C virus infection with notorious adverse reactions due to the broad expression of IFN-a receptors on all nucleated cells. Accordingly, a type-Ⅲ IFN with restricted receptors distribution is much safer as an alternative for HCV therapy.Interferon Stimulated Genes (ISGs) are not only the ultimate weapon to exert the antiviral activity in IFN pathway but also a double-edged sword. Some ISGs are negative regulators (suppressors) to inhibit IFN signal transduction, including USP18 and SOCS1, which is a possible reason leading to HCV refractory state. USP18 (ubiquitin specific protease 18) is an ubiquitin-specific protease that interacts with the subunit of type Ⅰ IFN receptor (IFNAR2) to inhibit type-Ⅰ IFN signal pathway. However, whether USP18 suppresses type-Ⅲ IFN signal pathway is still elusive. Similarly, SOCS1 (suppressor of cytokine signaling 1) has long been thought to block type Ⅰ interferon signaling through interaction with Tyk2 as an endogenous suppressor. Nevertheless, the role of SOCS1 in HCV endocytosis and secretion remains unknown.Numerous studies, including studies from our lab, indicated that intrahepatic ISGs expression before therapy was associated with treatment outcomes in patients chronically infected with HCV. Furthermore, PBMC (peripheral blood mononuclear cell) might be a perfect alternative to liver biopsy as an easier non-invasive predictive test considering the limitation of difficulty in obtaining liver biopsy samples. In consideration of the apparent limitations of current HCV therapy, especially high failure rate and universal side effects, prediction of treatment outcomes prior to the initiation of therapy and understanding the molecular mechanisms of HCV resistance to IFN-based therapy are two important goals in HCV research. This current research is aiming to explore the role of the two negative regulators of IFN signaling pathway (SOCS1 and USP18) in HCV infection and IFN resistance.Objectives:1 To study the different induction kinetics of ISGs by type I and typeⅢ IFNs;2 To determine the role of USP18/SOCS1 over-expression in type Ⅰ (IFN-α) and type Ⅲ (IFN-λ) signaling pathways as well as in HCV life cycle (endocytosis, replication and secretion);3 To explore the possible relationship between differential mRNA expression levels of related genes in PBMCs from chronic HCV patients and treatment outcomes.Methods:1 Huh7.5.1 cells were transfected with pCR3.1/USP18 orpCR3.1/SOCS1 to confirm target gene expression at mRNA level (Realtime PCR) and protein level (Western blot) and the optimal plasmid DNA dose, transfection reagent (PEI) dose and IFN concentration were further optimized. The induction kinetics of ISGs after stimulation with IFN-α or IFN-λ in the absence and presence of USP18/SOCS1 over-expression was studies by Realtime PCR.2 HCV Replicon cells were transfected with pCR3.1/USP18 or pCR3.1/SOCS1 to study the role of increased expression of USP18 or SOCS1 in both type Ⅰ and type Ⅲ IFN signaling pathways. STAT1 phosphorylation state, interferon stimulated response element (ISRE) activity and ISG expressions levels were analyzed. Also, pCR3.1/USP18 or pCR3.1/SOCS1 was transfected into HCVcc system to determine the effect of over-expression of USP18 or SOCS1 on HCV endocytosis, replication and secretion by Realtime PCR and virus tittering (TCID50).3 PBMCs were obtained from forty-eight patients with chronic HCV after treatment and cultured in vitro with/without IFN-a for 8h. Total RNAs were extracted and mRNA levels of related genes were determined by Realtime PCR. The possible correlation between expression levels of related genes in PBMCs and treatment outcomes was explored.Results:1 IFN-α and IFN-λ, activated antiviral ISGs (ISG15 and MxA) expression differently, with delayed but prolonged activation by IFN-λ stimulation compared to IFN-a; the differential induction patterns of USP18 and SOCS1 by IFN-α and IFN-λ may contribute to this difference in ISG activation kinetics although both SOCS1 and USP18 inhibit type 1 and type Ⅲ IFN signaling.2 USP18/SOCS1 over-expression abrogates anti-HCV effect of both IFN-α and IFN-λ, leading to increased HCV RNA replication in both HCV replicon cells and JFH1 HCV culture system. In line with this, USP18/SOCS1 over-expression inhibited STAT1 phosphorylation, attenuated IFN-stimulated response elements (ISRE) reporter activity, and blocked ISG expression. Furthermore, USP18 over-expression promoted HCV endocytosis by up-regulating the mRNA levels of HCV receptors (SR-BI and LDLR) although HCV secretion was not affected following over-expression of either USP18 or SOCS1.3 There is a possible correlation between differential expression levels of some host genes in PBMCs from chronic HCV patients and treatment outcomes. We found that the baseline (without in viro IFN-a stimulation) expression levels of MxA, SOCS1, IFNAR1 and IFNλR are higher in treatment non-responders than in responders although the baseline expression levels of ISG 15 and USP18 showed no difference.Conclusions:1 Differential induction patterns of negative regulator (USP18 and SOCS1) of IFN signaling pathway by IFN-α and IFN-λ. may contribute to distinct activation kinetics of antiviral ISGs (ISG15 and MxA) by IFN-α and IFN-λ, which was a possible reason for IFN refractory state.2 USP18/SOCS1 over-expression abrogated anti-HCV effect of both IFN-α and EFN-λ to increase RNA replication. USP18 over-expression up-regulated mRNA levels of HCV entry receptors to facilitate HCV entry.3 There is a possible correlation between baseline expression levels of MxA, SOCS1, IFNAR1 and IFNλR in PBMCs from HCV patients and treatment outcomes, which may be further developed as an easy-access prognostic test. |
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