Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA.
J Mol Biol. 2010 Jul 16;400(3):393-412. doi: 10.1016/j.jmb.2010.04.059. Epub 2010 May 4.
Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2alpha, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5'-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of approximately 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.
蛋白激酶 R (PKR) 是先天免疫反应的重要组成部分。在双链 RNA (dsRNA) 的存在下,PKR 自身磷酸化,使其能够磷酸化其底物真核起始因子 2alpha,导致翻译停止。PKR 的典型激活剂是病毒感染过程中产生的长 dsRNA,尽管某些其他 RNA 也可以激活 PKR。最近的一项研究表明,丙型肝炎病毒 (HCV) RNA 5'非翻译区中存在的全长内部核糖体进入位点 (IRES) 抑制 PKR,而另一项研究表明它可以激活 PKR。我们在这里表明,全长 IRES 既可以激活也可以抑制 PKR。HCV IRES 具有复杂的二级结构,由四个结构域组成。虽然已经证明结构域 III-IV 可以激活 PKR,但我们在这里报告结构域 II 也可以强烈激活 PKR。结构域 II 的结构映射和突变分析表明,尽管 RNA 的双链区域对于激活很重要,但环区也有贡献。结构比较表明,结构域 II 具有多个非 Watson-Crick 特征,模拟 A 型 dsRNA。结构域 II 的典型和非典型特征累计约为 33 个无分支碱基对,这是 PKR 激活所需的最短 dsRNA 长度。这些结果为 PKR 被各种偏离传统 PKR 激活剂中长螺旋延伸的 RNA 结构基序激活提供了进一步的结构基础。当 IRES 的其他结构域无法接近时,HCV IRES 的结构域 II 对 PKR 的激活对先天免疫反应具有重要意义。我们还研究了 HCV 非结构蛋白 5A (NS5A) 结合 IRES 各种结构域并改变激活的能力。提出了一个模型,用于解释 IRES 的结构域 II 和 NS5A 如何在 HCV 感染过程中控制宿主和病毒翻译。