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Nucleoporins Nup214 and Nup358 as regulators of adenoviral genome import

核孔蛋白 Nup214 和 Nup358 作为腺病毒基因组输入的调节剂

基本信息

批准号：
286487595
负责人：
Professor Dr. Ralph Kehlenbach
金额：
--
依托单位：
Institut für Molekularbiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/286487595?language=en
关键词：
Nucleoporins Nup214 Nup358 regulators adenoviral

项目摘要

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.

核孔蛋白是构成核孔复合物（NPC）的蛋白质，限制和控制分子在细胞核和细胞质之间的流动。单个核孔蛋白在NPC细胞质和核质侧的不对称分布为运输复合物（反）组装和方向性提供了结构框架。几种核复制病毒利用NPC作为门户，将其基因组传递到靶细胞的核质中。两种主要的细胞质核孔蛋白Nup214和Nup358与腺病毒、疱疹病毒和人类免疫缺陷病毒等人类病原体的病毒基因组导入有关。腺病毒因其高效的基因组传递策略而被认为是基因治疗和疫苗接种载体的有效工具。对于腺病毒，我们最近发现Nup214 n端的一个片段作为含有病毒粒子的基因组的主要NPC对接位点，并鉴定出主要的衣壳蛋白六邻体是病毒的对接决定因素。我们的初步数据进一步表明，Nup358在病毒粒子分解后的对接步骤中释放病毒基因组和/或释放的基因组本身的核输入中发挥重要作用。因此，了解NPC在病毒基因组传递中的作用可以为新的抗病毒策略开辟道路，潜在地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工具。在本项目中，我们将确定Nup214上腺病毒衣壳的最小对接区域，并确定Nup358中参与基因组释放和导入的功能域。我们将结合Nup片段与衣壳/己体之间的生化结合研究和细胞中的耗尽/重构研究。使用半渗透细胞系统，我们将能够将分析范围缩小到Nup214和Nup358的基本区域。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。最后，我们将利用Nup214和/或Nup358中已鉴定的区域/结构域来生成具有修饰核孔蛋白的细胞，并研究靶向NPC是否可以使细胞对病毒感染产生抗性。