MacCallum Paul R, Jack Samantha C, Egan Philip A, McDermott Benjamin T, Elliott Richard M, Chan Shiu-Wan
Faculty of Life Sciences, The University of Manchester, Jackson's Mill, PO Box 88, Sackville Street, Manchester M60 1QD, UK.
Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK.
J Gen Virol. 2006 Nov;87(Pt 11):3251-3262. doi: 10.1099/vir.0.82051-0.
Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2alpha phosphorylation. A phosphomimetic of eIF2alpha, which mimics the structure of the phosphorylated eIF2alpha, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2alpha. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2alpha-independent mechanism, whereas the simultaneous phosphorylation of eIF2alpha repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2alpha-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.
慢性丙型肝炎常与氧化应激相关。丙型肝炎病毒（HCV）利用内部核糖体进入位点（IRES）元件进行翻译，这与大多数细胞蛋白的帽依赖性翻译不同。为了解在氧化应激下病毒翻译是如何被调节的，使用双顺反子报告基因构建体和过氧化氢（H2O2）作为应激诱导剂，将HCV IRES介导的翻译与帽依赖性翻译进行了比较。在对H2O2敏感的HeLa细胞中，H2O2以时间和剂量依赖性方式抑制翻译，这与eIF2α磷酸化的动力学过程相伴。一种模拟磷酸化eIF2α结构的eIF2α磷酸模拟物，在没有H2O2的情况下足以抑制翻译。在对H2O2耐药的HepG2细胞中，H2O2激活了HCV IRES介导的翻译和帽依赖性翻译，这与磷酸化eIF2α水平的升高有关。据推测，H2O2可能通过一种不依赖eIF2α的机制刺激HepG2细胞中的翻译，而eIF2α的同时磷酸化则抑制了部分翻译活性。事实上，在存在不可磷酸化突变体eIF2α-SA的情况下，翻译抑制被解除，导致在暴露于H2O2后两种翻译活性进一步增强。在对H2O2表现出中等敏感性的HuH7细胞中，用不同剂量的H2O2处理后，HCV IRES介导的翻译和帽依赖性翻译活性均上调，但在低水平H2O2时诱导水平最高，这可能代表了H2O2的生理水平。在此水平下，与帽依赖性翻译相比，HCV IRES介导的翻译优先上调。
