Mechanism of Ribavirin in Enhancement of the Action of IFN-α against Hepatitis C Virus
Date Issued
2007
Date
2007
Author(s)
Liu, Wei-Liang
DOI
zh-TW
Abstract
Hepatitis C virus (HCV) infection is a major health problem worldwide, which may lead to chronic hepatitis, liver cirrhosis, hepatocellular carcinoma or liver failure. Therapy for chronic hepatitis C has improved markedly by the combination of ribavirin with conventional interferon-α (IFN-α) or peginterferon-α compared with IFN-α monotherapy, however, the mechanism of action of ribavirin in the treatment of chronic hepatitis C remains to be clarified.
Among the IFN-α induced antiviral mechanisms, activation of PKR (double-stranded RNA-activated protein kinase) is an essential component which plays an important role in innate immunity against viral infection. The anti-HCV effect of PKR is achieved by blocking viral proteins synthesis as a consequence of PKR-mediated phosphorylation and activation of alpha subunit of eukaryotic initiation factor 2 (eIF2α). Moreover, in the recent studies suggest that ribavirin can directly upregulate the IFN-stimulated response genes (ISGs) expression, and HCV replication could be inhibited through PKR pathway. Therefore, the PKR plays a role in cell defense against virus infection. Besides the PKR, whether the ribavirin could induce the expression of some other genes or proteins as an antiviral mechanism is also worthy to be investigated. Therefore, in this thesis we explore two parts for characterization the biochemical and biological properties of ribavirin.
The first part is to study whether the ribavirin upregulates the PKR activity and enhance IFN-α action against hepatitis C virus. Primary human hepatocytes and HCV replicon cells were treated with ribavirin and IFN-α. PKR activities were assayed by immunoblots. A pulse-chase experiment of half-life of PKR protein was performed to study whether ribavirin decreases PKR degradation. We used small interference RNA (siRNA) to knockdown PKR to assess its importance in suppressing HCV replication in the replicon system. Ribavirin could upregulate the phosphorylation levels of PKR and eIF2α, leading to suppress HCV replication. The effects of ribavirin plus IFN-α on PKR activity were greater than observed with either ribavirin or IFN-α alone. Knockdown PKR increased the HCV replication, supporting the importance of PKR in controlling HCV replication. Pulse-chase experiment showed that ribavirin could reduce the degradation rate of PKR protein. These results suggest that the anti-HCV action of ribavirin is partly attributable to its ability to upregulate PKR activity.
The second part of our work is using the proteomic and microarray technology to investigate whether the ribavirin could induce differentially expressed genes or proteins associated with antivirus. In the proteomic analysis, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins in human hepatocytes after ribavirin treatment. Compared the treated or untreated with ribavirin, 4 differential expressed spots were identified and cut off from silver staining gel, in gel digested with typsin, analyzed with LC-MS/MS and searched in the Swiss-Prot and NCBI nonredundant database, 3 known proteins and 1 unknown protein were preliminarily identified. The 3 known proteins were functions related to the folding of thyroglobulin (Erp29), metabolism of unsaturated fatty acid (DCI), glutathione-dependent thioltransferase, dehydroascorbate reductase, and monomethylarsonate reductase (GSTO1). In the microarray study, the total RNA was extracted from Huh-7 cells isolated from untreated and treated with ribavirin. After hybridization the membrane format of microarray containing 9600 spots of cDNAs with the prepared probes, the membrane was scanned for blue intensity to screen the differently expressed genes. Compared with the control group, the ribavirin exposed group has 56 up-regulated known genes and 32 down-regulated known genes. The differently expressed genes were functionally related with ion channels and transport proteins, cyclins, cell receptors, cell growth and proliferation, cell signal transduction, ubiquitin protease, metabolism, and immunity. These differentially expressed genes and proteins may provide valuable clues for further study of the new anti-HCV mechanisms of ribavirin to anti-HCV.
Taken together, our results show that ribavirin not only could upregulate PKR activity to anti-HCV, but also would regulate the expression of some genes and proteins. In the future, we will investigate the modification of these genes and proteins in the process of ribavirin to antivirus.
Subjects
Ribavirin
PKR
HCV
IFN-α
PKR-siRNA
SDGs
Type
other
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