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

Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, an in vivo DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the in vivo visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study.