From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and the Department of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania 15261.
From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and.
J Biol Chem. 2014 May 30;289(22):15718-28. doi: 10.1074/jbc.M114.572099. Epub 2014 Apr 10.
The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.
HIV-1 Nef 毒力因子与多种宿主细胞信号蛋白相互作用。Nef 与Src 家族激酶的 SH3 结构域结合，导致激酶激活，这对于病毒感染性、复制和 MHC-I 下调至关重要。Itk 和其他 Tec 家族激酶也存在于 HIV 靶细胞中，并且 Itk 与 HIV-1 感染性和复制有关。然而，将 Itk 与 HIV-1 联系起来的分子机制尚不清楚。在这项研究中，我们使用基于细胞的双分子荧光互补测定法探索了 Nef 与 Tec 家族激酶的相互作用。在这种方法中，Nef 与伴侣激酶的相互作用使融合到每个蛋白质 C 端的非荧光 YFP 片段相互接近，导致 YFP 互补和明亮的荧光信号。使用双分子荧光互补，我们观察到 Nef 与 Tec 家族成员 Bmx、Btk 和 Itk 相互作用，但不与 Tec 或 Txk 相互作用。与 Nef 的相互作用通过激酶 SH3 结构域发生，并定位于质膜。该测定法中，所有主要 HIV-1 亚型的 Nef 等位变体与 Itk 强烈相互作用，表明这种相互作用具有高度保守性。Itk 激酶活性的选择性小分子抑制剂（BMS-509744）强烈抑制野生型 HIV-1 的感染性和复制，但不抑制 Nef 缺陷突变体。Nef 在转染细胞中诱导组成型 Itk 激活，该激活对抑制剂处理敏感。总之，这些结果首次提供了证据表明 Nef 与 Tec 家族的细胞质酪氨酸激酶相互作用，并表明 Nef 在病毒生命周期中为 HIV-1 和 Itk 信号提供了机制联系。
