Mechanism of influenza A virus NS1 protein interaction with the p85β, but not the p85α, subunit of phosphatidylinositol 3-kinase (PI3K) and up-regulation of PI3K activity
Mechanism of influenza A virus NS1 protein interaction with the p85β, but not the p85α, subunit of phosphatidylinositol 3-kinase (PI3K) and up-regulation of PI3K activity
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DOI：
10.1074/jbc.m802737200
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发表时间：
2008-08-22
影响因子：
4.8
通讯作者：
Zhou, Yan
中科院分区：
文献类型：
--
作者：
Li, Yang;Anderson, Deborah H.;Zhou, Yan
甲型流感病毒感染通过将甲型流感病毒NS1蛋白与PI3K的P85β调节亚基结合，激活磷脂酰肌醇3-激酶(PI3K)/Akt途径。在本研究中，我们报告了NS1与P85β的SH2间(ISH2)结构域结合。对P85βiSH2结构域的突变分析表明，Val-573是介导NS1和P85β相互作用的关键氨基酸。在与P85α的功能互补实验中，我们证明了Met-582位的Val突变导致NS1结合和PI3K活性增加。根据我们的实验结果进行的分子模拟表明，除了NS1SH3结合基序1(AA 164-167)与P85βVal-573之间的相互作用界面外，NS1中的AA 137-142还可能与P85β相互作用。事实上，NS1中的AA 141和142突变破坏了NS1和P85β之间的相互作用。突变型病毒PR8-NS1-141/142不能激活Akt的磷酸化。此外，PI3K检测表明，在野生型病毒感染的细胞中，P85β相关的PI3K活性显著增加。相反，在含有突变NS1而不能与P85β相互作用的突变病毒感染的细胞中，没有看到与P85β相关的PI3K活性上调，这表明PI3K上调依赖于NS1和P85β之间的相互作用。竞争实验和免疫沉淀研究表明，NS1、P85β和P110在细胞内形成复合体。最后，基于预测的NS1-P85-p110复合体的结构模型，讨论了NS1与P85β结合调节PI3K活性的机制。
Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by binding influenza A virus NS1 protein to the p85 beta regulatory subunit of PI3K. In this study, we report that NS1 binds to the inter-SH2 (iSH2) domain of p85 beta. Mutational analyses on p85 beta iSH2 domain defined that Val-573 is the critical amino acid (AA) that mediates NS1 and p85 beta interaction. In reciprocal gain of function experiments with p85 alpha, we demonstrated that mutation to Val at Met-582 leads to NS1 binding and increased PI3K activity. Molecular modeling based on our experimental results suggested that, in addition to the interaction interface between the NS1 SH3 binding motif 1 (AA 164-167) and p85 beta Val-573, AA 137-142 in NS1 might interact with p85 beta. Indeed, mutations of AA 141 and 142 in NS1 disrupted the interaction between NS1 and p85 beta. Mutant virus PR8-NS1-141/142 was not able to activate Akt phosphorylation. Furthermore, PI3K assays demonstrated that, in wild-type virus-infected cells, p85 beta-associated PI3K activity was increased significantly. In contrast, in the mutant virus-infected cells containing mutant NS1 unable to interact with p85 beta, the p85 beta-associated PI3K activity up-regulation was not seen, suggesting that PI3K up-regulation is dependent upon the interaction between NS1 and p85 beta. Competition experiments and the immunoprecipitation studies demonstrated that NS1, p85 beta, and p110 form a complex in cells. Finally, the mechanism by which binding of NS1 to p85 beta regulates PI3K activity was discussed based on a predicted structural model of NS1-p85-p110 complex.