Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5'UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

中文翻译：

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与细胞mRNA和其他病毒内部核糖体进入位点（IRESs）不同，HCV IRES的翻译活性不需要eIF3亚基e。

病毒依赖于宿主细胞翻译机制进行复制，一种常见的策略是使用不同组的宿主翻译起始因子在病毒RNA中存在内部核糖体进入位点（IRES）。丙型肝炎病毒（HCV）IRES与真核翻译起始因子3（eIF3）结合，但是eIF3复合物及其亚基的确切功能仍待精确定义。为了实现这一目标，我们将重点放在eIF3亚基e上。我们使用了结合了核糖体耗竭的兔网织红细胞裂解物和由对照或eIF3e siRNA转染的HeLa或Huh-7.5细胞制备的核糖体的体外测定方法。eIF3e沉默减少了由各种细胞基因和HCV样IRES的5'UTR介导的翻译。但是，真正的HCV IRES并未观察到这种效果。对eIF3e的沉默降低了eIF3的c，d和l亚基的细胞内水平以及它们与eIF3核心亚基a的联系。对与HCV IRES相关的eIF3亚基的下拉分析显示了相似的作用，并且该亚基对于与HCV IRES的结合至关重要。进行对照和eIF3e沉默的Huh-7.5细胞的HCV感染后，我们发现与体外研究结果一致，eIF3e沉默不会降低HCV复制和病毒蛋白表达。我们得出的结论是，与宿主细胞mRNA不同，HCV RNA翻译不需要完整的eIF3，有利于在eIF3e水平较低的条件下进行病毒表达。对与HCV IRES相关的eIF3亚基的下拉分析显示了相似的效果，并且该亚基对于与HCV IRES结合至关重要。进行对照和eIF3e沉默的Huh-7.5细胞的HCV感染后，我们发现与体外研究结果一致，eIF3e沉默不会降低HCV复制和病毒蛋白表达。我们得出的结论是，与宿主细胞mRNA不同，HCV RNA翻译不需要完整的eIF3，有利于在eIF3e水平较低的条件下进行病毒表达。对与HCV IRES相关的eIF3亚基的下拉分析显示了相似的作用，并且该亚基对于与HCV IRES的结合至关重要。进行对照和eIF3e沉默的Huh-7.5细胞的HCV感染后，我们发现与体外研究结果一致，eIF3e沉默不会降低HCV复制和病毒蛋白表达。我们得出的结论是，与宿主细胞mRNA不同，HCV RNA翻译不需要完整的eIF3，有利于在eIF3e水平较低的条件下进行病毒表达。