Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.
PLoS One. 2013 Aug 23;8(8):e72791. doi: 10.1371/journal.pone.0072791. eCollection 2013.
Although 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.
The 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.
RBV 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 <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.
We 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.
尽管慢性丙型肝炎病毒（HCV）感染已经用干扰素 alpha（IFN-α）和利巴韦林（RBV）联合治疗了十多年，但抗病毒协同作用的机制仍不清楚。我们旨在使用 HCV 细胞培养来确定 IFN-α和 RBV 联合治疗的协同抗病毒机制。
使用 HCV 感染和复制子细胞培养定量测量 IFN-α、RBV 单独和联合治疗的抗病毒疗效。在 Huh-7 细胞培养中研究了这两种药物在 HCV 内部核糖体进入位点（IRES）介导的翻译水平上的直接抗病毒活性。使用 CalcuSyn 软件和 MacSynergy 软件验证了 IFN-α和 RBV 联合治疗的协同抗病毒作用。
RBV 联合 IFN-α可有效抑制 HCV 复制细胞培养。我们的结果表明，IFN-α、干扰素 lambda（IFN-λ）和 RBV 各自抑制 HCV IRES-GFP 的表达，并且它们对 GFP 的表达影响很小，其中翻译不依赖于 IRES。RBV 与 IFN-α或 IFN-λ的联合治疗具有高度协同作用，组合指数<1。我们表明，IFN-α处理诱导 PKR 和 eIF2α 磷酸化的水平，从而阻止 HCV IRES-GFP mRNA 的核糖体加载。在 Huh-7 细胞中沉默 PKR 表达可阻止 IFN-α对 HCV IRES-GFP 表达的抑制作用。RBV 还通过抑制细胞 IMPDH 活性阻断 HCV-IRES mRNA 的多核糖体加载，并诱导 PKR 和 eIF2α 磷酸化。PKR 或 IMPDH 的敲低可阻止 RBV 诱导的 HCV IRES-GFP 翻译。
我们证明了 IFN-α和 RBV 通过防止多核糖体形成来抑制 HCV IRES。IFN-α和 RBV 联合治疗通过两种不同的机制协同抑制 HCV IRES 翻译，涉及 PKR 激活和通过抑制 IMPDH 耗尽细胞内鸟苷池。
