title: Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214.
abstract: In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.
  
  
  OBJECTIVE
  AdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA University of Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France aurelia.cassany@u-bordeaux.fr.
authors.forename: Aurélia
authors.initials: A
authors.lastname: Cassany
authors.affiliation: University of Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ragues
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Tinglu
authors.initials: T
authors.lastname: Guan
authors.affiliation: University of Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Bégu
authors.affiliation: University of Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Harald
authors.initials: H
authors.lastname: Wodrich
authors.affiliation: University of Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Service de Virologie, Bordeaux, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Kann
authors.affiliation: Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Glen R
authors.initials: GR
authors.lastname: Nemerow
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Larry
authors.initials: L
authors.lastname: Gerace
mesh_terms: D021581:Active Transport, Cell Nucleus; D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D036022:Capsid Proteins / Q000378:metabolism*; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D054884:Host-Pathogen Interactions; D006801:Humans; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D034622:RNA Interference; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C085914:NUP214 protein, human; D028861:Nuclear Pore Complex Proteins; C018315:hexon capsid protein, Adenovirus