Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein, a Translational Activator of Influenza Virus

Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5′ untranslated region (5′ UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

流感病毒NS1蛋白是一种RNA结合蛋白，其表达会调控宿主细胞mRNA的多种转录后过程，包括多聚腺苷酸化、剪接以及核质运输。此外，NS1蛋白可增强病毒mRNA的翻译效率，但对宿主细胞mRNA的翻译无此调控作用。为阐明该效应的分子机制，本研究在宿主细胞翻译因子中筛选流感病毒NS1蛋白的互作靶点。研究发现，无论是在流感病毒感染的细胞中，还是在转染NS1 cDNA的细胞中，NS1均可与帽结合复合物eIF4F的大亚基真核翻译起始因子4GI（eukaryotic initiation factor 4GI, eIF4GI）发生免疫共沉淀。采用纯化的His-NS1融合蛋白构建的亲和层析基质开展的实验显示，该融合蛋白可从COS-1细胞中富集内源性eIF4GI，也可结合体外翻译的标记eIF4GI，但无法结合eIF4F复合物中的eIF4E亚基。针对eIF4GI缺失突变体的类似体外结合实验表明，eIF4GI上与NS1结合的结构域位于第157至550位氨基酸残基区间，该区域尚未见翻译机器的其他组分与之结合的报道。此外，通过覆盖结合实验与下拉实验，我们证实NS1与eIF4GI可直接相互作用，且该过程不依赖于RNA。对NS1蛋白上eIF4GI结合结构域的定位研究显示，该蛋白N端前113个氨基酸残基即可介导与eIF4GI的结合，而N端前81个氨基酸残基则无此结合活性。既往研究已证实，前者（N端前113位氨基酸残基）可作为翻译增强子发挥功能，而后者（N端前81位氨基酸残基）则丧失该活性。综合上述实验结果及已发表的相关研究数据，我们提出如下模型：NS1蛋白可将eIF4GI特异性招募至病毒mRNA的5'非翻译区（5′ untranslated region, 5′ UTR），从而使流感病毒mRNA得以优先翻译。