Nucleoporins Nup214 and Nup358 as regulators of adenoviral genome import

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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.