Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
College of Animal Science and Technol, China Agricultural University, Beijing 100193, China.
Int J Biol Macromol. 2023 Mar 1;230:123191. doi: 10.1016/j.ijbiomac.2023.123191. Epub 2023 Jan 9.
Viral mRNA of coronavirus translates in an eIF4E-dependent manner, and the phosphorylation of eIF4E can modulate this process, but the role of p-eIF4E in coronavirus infection is not yet entirely evident. p-eIF4E favors the translation of selected mRNAs, specifically the mRNAs that encode proteins associated with cell proliferation, inflammation, the extracellular matrix, and tumor formation and metastasis. In the present work, two rounds of TMT relative quantitative proteomics were used to screen 77 cellular factors that are upregulated upon infection by coronavirus PEDV and are potentially susceptible to a high level of p-eIF4E. PEDV infection increased the translation level of ribosomal protein lateral stalk subunit RPLp2 (but not subunit RPLp0/1) in a p-eIF4E-dependent manner. The bicistronic dual-reporter assay and polysome profile showed that RPLp2 is essential for translating the viral mRNA of PEDV. RNA binding protein and immunoprecipitation assay showed that RPLp2 interacted with PEDV 5'UTR via association with eIF4E. Moreover, the cap pull-down assay showed that the viral nucleocapsid protein is recruited in mGTP-precipitated complexes with the assistance of RPLp2. The heterogeneous ribosomes, which are different in composition, regulate the selective translation of specific mRNAs. Our study proves that viral mRNA and protein utilize translation factors and heterogeneous ribosomes for preferential translation initiation. This previously uncharacterized process may be involved in the selective translation of coronavirus.
冠状病毒的病毒 mRNA 以依赖 eIF4E 的方式进行翻译，并且 eIF4E 的磷酸化可以调节这个过程，但 p-eIF4E 在冠状病毒感染中的作用尚不完全清楚。p-eIF4E 有利于特定 mRNA 的翻译，特别是编码与细胞增殖、炎症、细胞外基质以及肿瘤形成和转移相关的蛋白质的 mRNA。在本工作中，使用两轮 TMT 相对定量蛋白质组学筛选了 77 种细胞因子，这些因子在冠状病毒 PEDV 感染时上调，并且可能易受高水平 p-eIF4E 的影响。PEDV 感染以依赖 p-eIF4E 的方式增加核糖体蛋白侧向茎部亚基 RPLp2（而不是亚基 RPLp0/1）的翻译水平。双顺反子双报告基因测定和多核糖体图谱显示，RPLp2 对于翻译 PEDV 的病毒 mRNA 是必需的。RNA 结合蛋白和免疫沉淀测定表明，RPLp2 通过与 eIF4E 结合与 PEDV 5'UTR 相互作用。此外，帽结合下拉测定显示，病毒核衣壳蛋白在 RPLp2 的协助下，与 mGTP 沉淀复合物结合。组成不同的异核糖体调节特定 mRNA 的选择性翻译。我们的研究证明病毒 mRNA 和蛋白质利用翻译因子和异核糖体进行优先翻译起始。这个以前未被描述的过程可能参与了冠状病毒的选择性翻译。
