Interaction of influenza virus NS1 protein and the human homologue of Staufen in vivo and in vitro
Interaction of influenza virus NS1 protein and the human homologue of Staufen in vivo and in vitro
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DOI：
10.1093/nar/27.11.2241
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发表时间：
1999-06-01
影响因子：
14.9
通讯作者：
Ortín, J
中科院分区：
文献类型：
--
作者：
Falcón, AM;Fortes, P;Ortín, J
利用酵母中的双杂交遗传陷阱，对能够与流感病毒NS1相互作用的人类蛋白进行了筛选。分离到与果蝇Staufen蛋白（hStaufen）人类同源基因相对应的cDNA，满足双杂交方案的所有遗传控制。利用从lambda人类文库中分离的hStaufen cDNA，在体内和体外鉴定了hStaufen与NS1蛋白的相互作用。同时转染NS1 cDNA和hStaufen的部分cDNA，可使重组hStaufen蛋白从其在细胞质中的正常积累位点重新定位到NS1蛋白的细胞核位置。NS1和hStaufen蛋白可以从共转染细胞的提取物、含有两种蛋白的提取物的混合物以及流感病毒感染细胞的提取物中共免疫沉淀，此外，这两种蛋白在流感病毒感染细胞的核糖体和多体部分中共定位。用含有纯化的hStaufen和体外翻译的NS1蛋白的树脂进行下拉实验，也检测了相互作用。NS1基因的缺失定位表明，含有n端81个氨基酸的突变蛋白不能与hStaufen相互作用，尽管保留了完整的rna结合能力。这些结果讨论了hStaufen的可能作用机制及其与流感病毒感染的相关性。
A screening for human proteins capable of interacting with influenza virus NS1 has been carried out using the two-hybrid genetic trap in yeast. A cDNA corresponding to the human homologue of Drosophila melanogaster Staufen protein (hStaufen) was isolated that fulfilled all genetic controls of the two-hybrid protocol. Using a hStaufen cDNA isolated from a lambda human library, the interaction of hStaufen and NS1 proteins was characterised in vivo and in vitro. Co-transfection of NS1 cDNA and a partial cDNA of hStaufen led to the relocalisation of recombinant hStaufen protein from its normal accumulation site in the cytoplasm to the nuclear location of NS1 protein. NS1 and hStaufen proteins could be co-immunoprecipitated from extracts of co-transfected cells and from mixtures of extracts containing either protein, as well as from extracts of influenza virus-infected cells, Furthermore, both proteins co-localised in the ribosomal and polysomal fractions of influenza virus-infected cells. The interaction was also detected in pull-down experiments using a resin containing purified hStaufen and NS1 protein translated in vitro, Deletion mapping of the NS1 gene indicated that a mutant protein containing the N-terminal 81 amino acids is unable to interact with hStaufen, in spite of retaining full RNA-binding capacity. These results are discussed in relation to the possible mechanisms of action of hStaufen and its relevance for influenza virus infection.