Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic dextran polymers. Thus, kinesin-1 uncoats viral DNA and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.

Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential.DNA viruses and retroviruses maintain and replicate their genomes in host cell nuclei by using histone-based nucleosomes, similar to chromatin, or they encode their own DNA binding and DNA-organizing proteins (34,45,47). They assemble and maintain their genomes in different chromatin states by packaging the nucleic acids into proteinaceous capsids and sometimes lipid envelopes and thereby traffic their genome within and transmit it between cells (8, 41). The simian virus 40 (SV40) polyomavirus, for example, packages its virion DNA with cellular core histones and uses histones to replicate in infected nuclei (19). Herpesviruses, on the other hand, condense their double-strand DNA in particles with the help of polyamines and use histones during latent residence within infected nuclei or use irregularly spaced nucleosomes during productive phases of infection (45).Adenoviruses (Ads) replicate and assemble particles in the nucleus. They encode their own histone-like proteins to condense a linear double-strand DNA genome of about 36 kbp into a proteinaceous DNA core. Although it is unknown how the viral DNA is precisely organized in the virion, isolated cores of species C human adenovirus serotypes 2 and 5 (Ad2/5) contain six viral proteins, the basic proteins V (pV), pVII, and pX; the terminal protein covalently attached to the 5Ј ends of the DNA; and small numbe...

Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.

SummaryOrganelles in the endocytic pathway interact and communicate through the crucial mechanisms of fusion and fission. However, any specific link between fusion and fission has not yet been determined. To study the endosomal interactions with high spatial and temporal resolution, we enlarged the endosomes by two mechanistically different methods: by expression of the MHC-class-II-associated chaperone invariant chain (Ii; or CD74) or Rab5, both of which increased the fusion rate of early endosomes and resulted in enlarged endosomes. Fast homotypic fusions were studied, and immediately after the fusion a highly active and specific tubule formation and fission was observed. These explosive tubule formations following fusion seemed to be a direct effect of fusion. The tubule formations were dependent on microtubule interactions, and specifically controlled by Kif16b and dynein. Our results show that fusion of endosomes is a rapid process that destabilizes the membrane and instantly induces molecular-motor-driven tubule formation and fission.

Infections with coxsackievirus B3 (CVB3) are common causes of myocarditis in humans. One detail of CVB3-induced pathogenesis is apoptosis. The interaction between the capsid protein VP2 of the myocardial virus variant CVB3H3 and the proapoptotic host cell protein Siva has recently been observed. In order to characterize the interaction between both proteins more precisely, the binding activity of the CVB3H3 VP2 to Siva was compared to that of the mutant virus CVB3H310A1 VP2. We found that the asparagine at position 165 in VP2 is essential for a stable interaction with Siva influencing also the induction of apoptosis, viral spread, and inflammatory responses in vivo. Furthermore, the specific binding site of Siva to VP2 is located at amino acid positions 118-136. Together, these results show that the interaction between VP2 of CVB3H3 and Siva is a highly specific process involving distinct amino acids on both proteins that most likely influence the outcome of CVB3-caused disease.