Mechanism of interferon unresponsiveness in hepatitis C virus infection

Abstract

Hepatitis C virus (HCV) is a positive-stranded RNA virus of the genus Hepacivirus in the family Flaviviridae that infects approximately 170 million people worldwide. Acute HCV infection is spontaneously cleared in 20 to 30% of patients

however, the majority of patients fail to clear HCV and develop chronic persistent infection, which tends to progress to life-threatening liver diseases such as liver cirrhosis and hepatocellular carcinoma. In 2005, the first HCV infection system in cell culture was established using clone Japanese Fulminant Hepatitis-1 (JFH-1), and the development of cell culture-derived HCV (HCVcc) systems has allowed us to understand how hosts respond to HCV infection and how HCV evades the host responses. Although the mechanisms underlying the different outcomes of HCV infection are not fully understood, innate immune responses seem to have a critical impact on the outcome of HCV infection, as demonstrated by the prognostic value of interferon (IFN)-λ gene polymorphisms among patients with chronic HCV infection. After HCV infection, innate sensing of the virus in infected hepatocytes occurs through the pattern recognition receptors, leading to downstream signaling that results in the induction of type III and I IFNs. These secreted IFNs have been demonstrated as inducers of antiviral interferon-stimulated genes (ISGs) in HCV-infected cells. Upregulation of these ISGs is sustained in HCV-infected livers. However, paradoxically, HCV-infected patients with high levels of ISGs in their liver at baseline respond poorly to pegylated $IFN- \alpha-based$ treatment. Previous reports have identified ubiquitin specific peptidase 18 (USP18) as a critical factor conferring unresponsiveness to exogenous IFN-α treatment by blocking the generation of phosphorylated signal transducer and activator of transcription (STAT) proteins, although the mechanism underlying the maintenance of increased level of USP18 protein in HCV-infected liver was not clearly elucidated. In this dissertation, I investigated and clarified the mechanism of prolonged ISG expression and its role in IFN responsiveness during HCV infection in relation to unphosphorylated interferon-stimulated gene factor 3 (U-ISGF3), recently identified as a tripartite transcription factor formed by high levels of interferon-regulatory factor 9 (IRF9), STAT1, and STAT2 without tyrosine phosphorylation of the STATs. First, I established high-titer HCVcc infection system and HCVcc-permissive, immune-competent cell culture model. I confirmed the robust production of IFN-λs and IFN-β in primary human hepatocytes and cell lines after high-titer HCVcc infection. Second, I found that the protein level of USP18 is increased in HCV-infected liver and IFN-λ-treated hepatoma cells, blocking the phosphorylation of STAT1 in response to exogenous IFN-α. Recently, intracellular free interferon-stimulated gene 15 (ISG15) was reported to regulate the stability of USP18 protein. I checked the level of ISG15 in HCV-infected liver and IFN-λ-treated hepatoma cells, and it was upregulated at both mRNA and protein levels. Silencing ISG15 decreased the protein level of USP18 in these cells and restored the USP18-mediated attenuation of STAT1 phosphorylation after treatment with IFN-α. Moreover, forced expression of IFNL4 also conferred unresponsiveness to IFN-α via ISG15/USP18 axis. Therefore, I concluded that ISG15 sustains the abundance of USP18, resulting in defective STAT1 phosphorylation in response to exogenous IFN-α in HCV-infected liver. Next, I noticed that the level of U-ISGF3, but not PY-STAT1, was significantly elevated in HCV-infected patients’ livers. U-ISGF3 was also significantly elevated in response to IFN-λ and IFN-β in HCV-infected primary human hepatocytes and Huh-7-TLR3 cells. Moreover, the U-ISGF3 components were detected in the nucleus of HCV-infected cells and livers. Chromatin immunoprecipitation revealed that U-ISGF3 is bound to the promoters of a subset of ISGs (U-ISGs) and prolongs their expression in HCV-infected cells and livers. Forced expression of IRF9, phosphorylation-defective STAT1, and STAT2 upregulated U-ISGs and restricted HCV chronic replication without exogenous IFN treatment. ISG15 is one of the U-ISGs, and high levels of U-ISGF3 and ISG15 paradoxically conferred unresponsiveness to IFN-α therapy by regulating USP18 protein level. Taken together, I suggest that prolonged activation of ISGs in HCV-infected liver is mediated by U-ISGF3 which is induced by continuous IFN-λ stimulation including IFN- λ4, and upregulation of ISG15 by U-ISGF3 is critical in maintaing the protein level of USP18, leading to unresponsiveness to IFN-α. This novel mechanism of U-ISGF3-ISG15-USP18 axis in HCV-infected liver provides new insights into the treatment of chronic HCV infection.