Nucleoporins Nup214 and Nup358 as regulators of adenoviral genome import

核孔蛋白是构成核孔复合物（NPC）的蛋白质，限制和控制细胞核和细胞质之间的分子流动。单个核孔蛋白在NPC的胞质和核质侧的不对称分布为转运复合物的组装和定向提供了结构框架，一些核复制病毒将NPC作为将其基因组递送到靶细胞的核质中的通道。两种主要的细胞质核孔蛋白Nup 214和Nup 358与人类病原体如腺病毒、疱疹病毒和人类免疫缺陷病毒的病毒基因组输入有关。腺病毒由于其高效的基因组递送策略而被认为是非常有效的基因治疗工具和疫苗接种载体。对于腺病毒，我们最近表明，在Nup 214的N-末端的片段作为主要的NPC对接站点的基因组包含病毒粒子和确定的主要衣壳蛋白六邻体作为病毒对接决定簇。我们的初步数据进一步表明，Nup 358在病毒体解体以释放病毒基因组的后对接步骤中和/或在释放的基因组本身的核输入中起重要作用。因此，了解NPC在病毒基因组递送中的作用可以开辟新的抗病毒策略的道路，可能针对常见的病毒机制，同时有助于产生更有效的基于载体的tools.In本项目中，我们将确定Nup 214上腺病毒衣壳的最小对接区域，并确定Nup 358中参与基因组释放和导入的功能域。我们将结合联合收割机Nup片段和衣壳/六邻体之间的生物化学结合研究与细胞中的消耗/重建研究。使用半透化细胞系统，我们将能够将我们的分析范围缩小到Nup 214和Nup 358的必需区域。我们将通过携带标记基因的病毒和直接检测病毒基因组来补充我们在感染研究中的数据。最终，我们将使用Nup 214和/或Nup 358中鉴定的区域/结构域来产生具有修饰的核孔蛋白的细胞，并研究靶向NPC是否可以使细胞对病毒感染具有抗性。

Nucleoporins, the proteins constituting the nuclear pore complex (NPC), restrict and control the flow of molecules between the nucleus and the cytoplasm. The asymmetric distribution of individual nucleoporins at the cytoplasmic and the nucleoplasmic side of the NPC provides the structural framework for transport complex (dis)assembly and directionality.Several nuclear replicating viruses use the NPC as gateway to deliver their genome into the nucleoplasm of target cells. Two major cytoplasmic nucleoporins, Nup214 and Nup358, have been implicated in viral genome import for human pathogens such as adenoviruses, herpesviruses and human immunodeficiency virus. Adenoviruses are considered as very effective tools for gene therapy and as vaccination vector because of their efficient genome delivery strategy. For adenoviruses, we recently showed that a fragment at the N-terminus of Nup214 serves as primary NPC docking site for the genome containing virion and identified the major capsid protein hexon as the viral docking determinant. Our preliminary data further suggest that Nup358 plays an important role in post docking steps of virion disassembly to release the viral genome and/or in the nuclear import of the released genome itself. Thus, understanding the role of the NPC in viral genome delivery could open the way to novel antiviral strategies, potentially targeting common viral mechanisms while at the same time helping to generate more efficient vector based tools.In this project, we will identify the minimal docking region for the adenovirus capsid on Nup214 and identify the functional domains in Nup358 involved in genome liberation and import. We will combine biochemical binding studies between Nup fragments and capsids/hexon with depletion/reconstitution studies in cells. Using a semi-permeabilized cell system, we will be able to narrow our analysis to the essential regions of Nup214 and Nup358. We will complement our data in infection studies with viruses that harbour marker genes and by directly detecting viral genomes. Ultimately, we will use the identified regions/domains in Nup214 and/or Nup358 to generate cells with modified nucleoporins and to investigate whether targeting the NPC can make cells refractory to viral infection.