Functional Domains and the Antiviral Effect of the Double-Stranded RNA-Dependent Protein Kinase PKR from Paralichthys olivaceus
Functional Domains and the Antiviral Effect of the Double-Stranded RNA-Dependent Protein Kinase PKR from Paralichthys olivaceus
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DOI：
10.1128/jvi.02385-07
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发表时间：
2008-04
影响因子：
5.4
通讯作者：
Rong Zhu;Yi-Bing Zhang;Qi-ya Zhang;J. Gui
中科院分区：
文献类型：
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作者：
Rong Zhu;Yi-Bing Zhang;Qi-ya Zhang;J. Gui
双链RNA（dsRNA）依赖性蛋白激酶PKR通过磷酸化真核起始因子2 α（eIF 2 α）抑制病毒蛋白合成，介导保守的抗病毒途径。然而，人们对包括鱼类在内的低等脊椎动物的相关数据知之甚少。最近，PKR样或PKZ的鉴定解决了鱼类中是否存在正向PKR的问题。在这里，我们确定了第一个鱼PKR基因从日本比目鱼（POPKR）。PoPKR编码一种蛋白质，该蛋白质显示出哺乳动物PKR特征的保守结构，具有用于双链RNA结合的N末端区域和用于抑制蛋白质翻译的C末端区域。PoPKR的催化活性进一步证明了它是体外蛋白质翻译抑制所必需的。PoPKR以低水平组成型转录，并且在病毒感染后高度诱导。值得注意的是，PoPKR过表达增加eIF 2 α磷酸化并抑制比目鱼胚胎细胞中大菱鲆弹状病毒（SMRV）的复制，而在表达无催化活性的PKR-K421 R变体的转染细胞中磷酸化和抗病毒作用受损，表明PoPKR通过磷酸化底物eIF 2 α抑制病毒复制。免疫共沉淀实验证实了PoPKR与eIF 2 α之间的相互作用，PoPKR特异性短干扰RNA的转染进一步证实了SMRV感染过程中PoPKR催化eIF 2 α磷酸化的增强。目前的数据提供了重要的证据PKR介导的抗病毒途径在鱼类的存在，并揭示相当大的保护功能域和鱼类和哺乳动物之间的PKR蛋白的抗病毒作用。
ABSTRACT The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2α phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2α. The interaction between PoPKR and eIF2α is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2α phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.