Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.
Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.
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DOI：
10.1016/j.jmb.2010.04.059
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发表时间：
2010-07-16
影响因子：
5.6
通讯作者：
Bevilacqua PC
中科院分区：
文献类型：
--
作者：
Toroney R;Nallagatla SR;Boyer JA;Cameron CE;Bevilacqua PC
蛋白激酶PKR是先天免疫反应的重要组成部分。在dsRNA存在的情况下，PKR被自动磷酸化，这使得它能够磷酸化其底物eIF2α，导致翻译停止。典型的PKR激活剂是病毒感染期间产生的长dsRNA，尽管某些其他RNA也可以激活。最近的一项研究表明，存在于丙型肝炎病毒(HCV)RNA 5‘端非编码区的全长内部核糖体进入位点(IRES)抑制PKR，而另一种研究表明它激活PKR。我们在这里表明，全长IRES的激活和抑制都是可能的。丙型肝炎病毒IRES具有复杂的二级结构，包括四个结构域。虽然已经证明结构域III-IV激活PKR，但我们在这里报告IRES的结构域II也有效地激活。结构图谱和结构域II的突变分析表明，虽然RNA的双链区对激活是重要的，但环区也起到了重要作用。结构比较表明，结构域II具有多个模仿A-型dsRNA的非Watson-Crick特征。结构域II的典型和非典型特征累积到∼33个未分支碱基对，这是PKR激活所需的最小长度。这些结果为进一步了解PKR激活的结构基础提供了进一步的见解，这些结构基序通过不同的RNA结构基序来激活，这些结构基序偏离了传统PKR激活剂中发现的长螺旋延伸。当IRES的其他结构域可能无法访问时，由丙型肝炎病毒IRES的II结构域激活PKR对先天免疫反应有影响。我们还研究了丙型肝炎病毒非结构蛋白NS5A与IRES的不同结构域结合并改变激活的能力。提出了一个模型，说明IRES和NS5A的结构域II如何在丙型肝炎病毒感染过程中控制宿主和病毒的翻译。
The protein kinase PKR is an essential component of the innate immune response. In the presence of dsRNA, PKR is autophosphorylated, which enables it to phosphorylate its substrate, eIF2α, 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′-UTR 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 non-canonical features of domain II cumulate to a total of ∼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 non-structural protein 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.
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