Adenoviruses are nonenveloped viruses with an ϳ36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin ␣, importin ␤, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.Adenoviruses (Ads) are nonenveloped, double-stranded DNA viruses with a diameter of ϳ90 nm. They contain an outer capsid shell with icosahedral symmetry composed of 12 vertices and 20 facets surrounding the viral core with the genomic DNA. Extending from each of the 12 vertices of the capsid is the spike-like fiber protein, which is anchored to the vertices by the penton protein. Ad infection of cells is initiated by attachment of the fiber to the Coxsackie adenovirus receptors of cells, followed by association of the penton with ␣V integrins. This secondary penton interaction is required for fiber release and viral uptake into clathrin-coated pits of the early endosomal pathway (10, 43, 51; reviewed in references 9 and 34). In the early endosome, a poorly understood mechanism involving a drop in pH induces conformational changes in the capsid, which appears to release proteins of the vertex region, including protein VI and penton (17). An amphipathic helix at the N terminus of protein VI is thought to mediate disruption of the endosomal membrane and aid in the release of the remaining partially uncoated capsid into the cytoplasm (52). The nucleocapsid then moves toward the nucleus in a microtubule-and dynein-dependent mechanism (26) and docks at the nuclear pore complex (NPC), the proteinaceous channel that mediates transport across the nuclear envelope. Finally, the viral genome is imported into the nucleus prior to initiation of viral replication (16,44).In contrast to the Ad capsid, the core does not display a well-ordered symmetry or the coaxial coiling of DNA previously observed with most bacteriopha...

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 ( A denoviruses (AdVs) are nonenveloped DNA viruses consisting of an icosahedral capsid of ϳ90-nm diameter and an inner nucleoprotein core containing a linear double-stranded DNA genome of ϳ36 kbp (1-3). The major structural component of the capsid is the hexon trimer that is present in 240 copies. On the outer surface of the capsid at each of the 12 vertices, fiber proteins are anchored to the penton base. A number of minor capsid proteins on the outer and inner surfaces of the virus particle help to stabilize the capsid (4). The DNA is directly associated with the core proteins, including protein X, the "terminal protein," which is covalently linked to the 5= DNA termini, protein VII, and protein V, which connects the core to the outer capsid.AdV enters the cells by receptor-mediated endocytosis during which the virion becomes partially uncoated (3). Uncoating involves a series of events, culminating with endosomal membrane lysis by protein VI, which allows access of the particle to the cytosol (5). The partially disassembled capsid is then translocated along microtubules to the nucleus using the dynein/dynactin motor complex (6, 7). AdV then interacts with the nuclear envelope (NE) at nuclear pore complexes (NPCs) (8), and the viral genome is translocated into the nucleus by means of nuclear import receptors and/or histone H1 (8-10).NPCs are evolutionarily conserved large protein complexes of ϳ100 MDa spanning the NE that mediate trafficking into and out of the nucleus. Although small molecules passively diffuse through the NPC, macromolecules larger than ϳ20 to 40 kDa are transported in an active manner. This pathway is mediated by cellular transport receptors, including the karyopherin beta family that facilitates the translocation of most proteins and certain RNAs (11,12). NPCs are formed by ϳ30 proteins, which are thought to be present in multiples of 8 copies (13). A third of all nucleoporins (Nups) contain intrinsically disordered regions enriched in Phe-Gly (FG) repeats. The FG repeat domains directly bind karyopherins (12) and play an essential role in trafficking of

The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin–cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin α, importin β, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.