Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

PurposeAlthough chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture. MethodsThe antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software. ResultsRBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes ConclusionsWe demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.研究目的：尽管慢性丙型肝炎病毒（hepatitis C virus, HCV）感染的治疗已采用干扰素α（interferon alpha, IFN-α）与利巴韦林（ribavirin, RBV）联合方案逾十年，但其抗病毒协同作用的分子机制仍未得到充分阐释。本研究旨在借助HCV细胞培养模型，探明IFN-α与RBV联合治疗的协同抗病毒机制。 方法：本研究采用HCV感染及复制子细胞培养体系，定量检测IFN-α、RBV单药及二者联合给药的抗病毒活性。同时在Huh-7细胞培养体系中，针对HCV内部核糖体进入位点（internal ribosome entry site, IRES）介导的翻译过程，探究两种药物的直接抗病毒效应。此外，分别使用CalcuSyn软件与MacSynergy软件验证IFN-α与RBV联合治疗的协同抗病毒效果。 结果：RBV与IFN-α联合给药可有效抑制细胞培养体系中的HCV复制。本研究结果显示，IFN-α、λ干扰素（interferon lambda, IFN-λ）及RBV均可抑制HCV IRES介导的绿色荧光蛋白（green fluorescent protein, GFP）表达，而对非IRES依赖翻译的GFP表达影响极微。RBV分别与IFN-α或IFN-λ联合给药时，其联合指数证实二者具有高度协同效应。 结论：本研究证实，IFN-α与RBV均可通过阻止多聚核糖体形成，抑制HCV IRES介导的翻译过程。IFN-α与RBV联合治疗可通过两种不同机制协同抑制HCV IRES翻译：一是激活蛋白激酶R（protein kinase R, PKR），二是通过抑制次黄嘌呤核苷酸脱氢酶（inosine monophosphate dehydrogenase, IMPDH）耗竭细胞内鸟苷池。