Ribavirin增強干擾素α抗C型肝炎病毒作用之機轉

Abstract

慢性C 型肝炎是一種全球性重要的肝病。目前治療以干擾素 (IFN)-α 加上ribavirin合併治療為主，比起單用IFN-α治療，可將持續性病毒反應 (sustained virologic response,SVR) 由10-20%大幅提高到33-41% (傳統型IFN-α加ribavirin)或54-60% (長效型Pegylated IFN-α加ribavirin)。為何IFN-α與ribavirin合併治療能使治療C 型肝炎的SVR大幅提高，雖抑制的機轉有幾種被提出，但迄今未明。 在IFN-α所誘發表現的抗病毒基因(IFN-stimulated response genes ，ISGs)中---雙股螺旋RNA 活化蛋白酶（double-stranded RNA-activated protein kinase, PKR）在抗C 型肝炎病毒上扮演著重要的角色。當PKR變成一個活化態時，會進一步磷酸化下游基因的表現，如eIF2α。eIF2α被磷酸化後，就會抑制寄主細胞的蛋白質生成，或誘導細胞凋亡（apoptosis），進而使C 型肝炎病毒的複製受到抑制。另外，PKR也會經由免疫調節作用機轉來達到抗病毒的作用。最近有文獻提出，ribavirin也可以誘發IFN-α所刺激表現的基因(ISGs)來抑制病毒的表現。因此，我們的研究假說認為ribavirin或許也可以誘發抗病毒蛋白PKR或一些抗病毒基因的表現來幫助IFN-α抑制hepatitisc C virus (HCV)的複製機轉。所以，本論文分兩個研究方向來探討：(1)Ribavirin是否可以增加抗病毒蛋白PKR的表現，進而抑制HCV的複製機轉﹖(2)Ribavirin是否在細胞內還可以誘發其它未知的抗病毒基因或蛋白質的表現﹖ 第一部份研究ribavirin是否可以增加PKR的活性進而幫助IFN-α來抑制C 型肝炎病毒。我們將human hepatocytes 和HCV replicon cells先用IFN-α、ribavirin或IFN-α合併ribavirin處理；發現ribavirin可以增加PKR和eIF2α的磷酸化，且也會抑制HCV RNA replicons的複製和HCV病毒蛋白質NS3的表現。而且當IFN-α合併ribavirin時，PKR的磷酸化和抑制病毒蛋白質NS3的表現都有明顯的改變。另外，用PKR siRNA的方法，把細胞內的PKR基因的表現量降到最低，發現ribavirin是經由PKR-eIF2α這條路徑來抵抗HCV的增生。再者，從pulse-chase的實驗中也發現ribavirin有減緩PKR降解的速率，但其作用機轉仍未知。另外，我們也發現ribavirin可以不經由PACT (PKR activating protein)的調控來使PKR變成磷酸態的PKR。由以上的實驗結果得知，ribavirin是可以增加PKR的活性進而幫助IFN-α來抑制HCV的複製。 第二部份則是利用蛋白體學和基因微陣列來探討ribavirin是否可以誘發細胞內其它未知的抗病毒基因或蛋白質的表現。在蛋白體的分析實驗中，將human hepatocytes處理ribavirin過後的蛋白質萃取液，經二維凝膠電泳(2D-PAGE)和液態層析偶合串聯式質譜儀(LC-MS/MS)，分析發現有四個差異表現的蛋白質會被ribavirin所誘發。其中三個是已知的蛋白質(Erp29、DCI、GSTO1)，另一個則是未知的蛋白質。這些蛋白質初步鑑定為與thyroglobulin（Tg）的摺疊(folding)，不飽合脂肪酸中的氧化作用酶的代謝，硫醇類轉化酶和monomethylarsonate還原酶的活性反應有關。另外，在基因微陣列分析實驗中發現，經過ribavirin處理的Huh-7細胞，有56個基因的表現是增加的，32個基因是降低表現的。而這些有差異表現的基因，按照其參與的生物功能性和具有的分子功能進行分析，包含了與細胞的分裂與增殖，轉錄和轉譯因子、離子通道因子、糖類的代謝酶、訊息傳遞因子、能量代謝酶、免疫反應以及蛋白質降解酶(ubiquitin protease)等多方面有關。因此，經由這兩種技術發現了許多的基因或蛋白質可被ribavirin所調控表現。而這些蛋白質和基因的表現與抗病毒間的相關性是我們感興趣且要進一步研究探討。 綜合我們的實驗結果，ribavirin不僅可以增加PKR的活性表現，也可以誘發一些細胞性的蛋白質和基因的表現。所以，在未來的實驗中，我們將進一步暸解PKR的降解過程和這些蛋白質和基因與抗病毒間的相關性。

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.