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• W2129333552 abstract "Article1 October 1997free access The role of the nuclear pore complex in adenovirus DNA entry Urs F. Greber Urs F. Greber University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Maarit Suomalainen Maarit Suomalainen University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Robert P. Stidwill Robert P. Stidwill University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Karin Boucke Karin Boucke University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Melanie W. Ebersold Melanie W. Ebersold Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT, 06510 USA Search for more papers by this author Ari Helenius Ari Helenius Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT, 06510 USA Search for more papers by this author Urs F. Greber Urs F. Greber University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Maarit Suomalainen Maarit Suomalainen University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Robert P. Stidwill Robert P. Stidwill University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Karin Boucke Karin Boucke University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland Search for more papers by this author Melanie W. Ebersold Melanie W. Ebersold Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT, 06510 USA Search for more papers by this author Ari Helenius Ari Helenius Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT, 06510 USA Search for more papers by this author Author Information Urs F. Greber1, Maarit Suomalainen1, Robert P. Stidwill1, Karin Boucke1, Melanie W. Ebersold2 and Ari Helenius2 1University of Zurich, Department of Zoology, Winterthurerstrasse 190, 8057 Zurich, Switzerland 2Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, CT, 06510 USA The EMBO Journal (1997)16:5998-6007https://doi.org/10.1093/emboj/16.19.5998 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA-associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium-depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O-linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus-binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus. Introduction Viruses carry genetic information from one cell to the other. Viruses, which replicate in the nucleus, import their genome into the nucleus at early stages of infection. They either access the nuclear interior after mitotic breakdown of the nuclear envelope, as in the case of oncoretroviruses, or transport their genome across the nuclear envelope into the interphase nucleus (for review, see Fields et al., 1996). To reach the interphase nucleus, the viral genome must cross three barriers, the plasma membrane, the cytosol and the nuclear envelope. After passing the plasma membrane and the cytosol, nuclear RNA viruses, such as human immunodeficiency virus (HIV) or influenza virus, import a high molecular weight nucleoprotein complex into the nucleus. This process requires signals, cellular factors, functional nuclear pore complexes (NPCs) and a special configuration of the nucleoprotein complex (Gallay et al., 1996; Stevenson, 1996; Whittaker et al., 1996). The small DNA virus SV40 accesses the nucleus through the nuclear pores by some kind of a conformational change in its capsid and uncoats the DNA for transcription and replication within the nucleus (for review, see Greber and Kasamatsu, 1996). Larger DNA viruses, such as adenoviruses, herpesviruses, baculoviruses or hepadnaviruses, uncoat their genome to a variable extent before they reach the nuclear membrane (Miller, 1996; Roizman and Sears, 1996; Shenk, 1996; Kann et al., 1997; Sodeik et al., 1997). When they dock at the NPC, they are still enwrapped by a protein capsid, ready to undergo a final disassembly reaction and genome injection into the nucleoplasm. To analyze the mechanisms of DNA import into the nucleus, we are using adenovirus type 2 in cultured human epitheloid cells. Adenoviruses naturally enter human airway cells and produce progeny virions within the nucleus of the infected cell (for review, see Horwitz, 1990). Adenovirus particles have a diameter of ∼90 nm and contain at least 11 different structural polypeptides and a linear double-stranded DNA molecule. The DNA is condensed with proteins V, VII and μ, and covalently associated with the terminal protein and non-covalently with the cysteine protease L3/p23 (Mangel et al., 1993; Stewart et al., 1993). The DNA is connected to the inside wall of the capsid via protein VI. The icosahedral capsid consists of six or seven different proteins. More than 75% of the capsid mass is contributed by the hexon protein. Hexon is held together by the cementing protein IX, which accounts for ∼4% of the capsid mass (van Oostrum and Burnett, 1985). Protein IIIa, another capsid-stabilizing component, links adjacent facets of the icosahedron. The vertices of the capsid are made up of pentameric penton base and protruding trimeric fiber proteins. Fibers make a primary contact with a cell surface receptor of the immunoglobulin gene family (Bergelson et al., 1997; Hong et al., 1997; Tomko et al., 1997). Adenovirus enters the cells by a stepwise uncoating program (Greber et al., 1994). First, the fibers are released, followed by other coat proteins. Interactions between the viral penton base and a secondary cell surface receptor, αvβ5 integrins, mediate virus uptake into endosomes (Wickham et al., 1993). Integrins and the penton base are thought to assist the acid-stimulated virus release from early endosomes, ∼15 min after internalization into cold synchronized cells (Greber et al., 1993; Wickham et al., 1994; Prchla et al., 1995). By lysing the endosome, virus passes to the cytosol and then to the nuclear membrane. Penton base–integrin interactions together with reducing agents in endosomes or the cytosol also reactivate the viral cysteine protease L3/p23 inside the capsid. L3/p23 then degrades the internal protein VI (Cotten and Weber, 1995; Greber et al., 1996). This step thus weakens the capsid for final dissociation and DNA import into the nucleus. The nucleus is separated from the cytoplasm by the nuclear envelope, a double membrane with an intermediate filament network, the lamina (for review, see Goldberg and Allen, 1995). The nuclear envelope membranes are contiguous with the endoplasmic reticulum (ER) membrane. Their cisternae are major intracellular calcium stores (for review, see Pozzan et al., 1994). Embedded in both nuclear membranes are large macromolecular structures, the NPCs. NPCs control transport of proteins and nucleic acids in and out of the nucleus. They are anchored in the membranes via the pore membrane domain, a specialized subdomain of the nuclear envelope which lines the pore and links the outer and inner nuclear membranes (for reviews, see Davis, 1995; Panté and Aebi, 1996b). In higher eukaryotic cells, the pore membrane domain is an integral part of the scaffolding framework and contains transmembrane proteins, such as the major NPC glycoprotein gp210 or Pom121 (for review, see Gerace and Foisner, 1994). Small diffusional channels with a diameter of ∼5–9 nm are situated in the scaffolding framework (Hinshaw et al., 1992). These sites are thought to allow passive diffusion of small ions and molecules smaller than 30–50 kDa in and out of the nucleus (for review, see Davis, 1995). Nuclear import of most cellular proteins depends on nuclear localization signals (NLSs) and signal decoding machineries (for different models, see Duverger et al., 1995; Melchior and Gerace, 1995; Rexach and Blobel, 1995; Görlich and Mattaj, 1996; Panté and Aebi, 1996a; Pollard et al., 1996). Nuclear import can be operationally separated into two steps, energy-independent attachment of the transport ligand to the cytoplasmic face of the NPC and energy-dependent translocation. Stable attachment of transport ligands to NPCs in higher eukaryotic cells involves cytosolic factors and O-linked N-acetylglucosamine-containing NPC glycoproteins (Finlay et al., 1987; Adam and Adam, 1994). Translocation of NLS-bearing proteins occurs via the central transporter region of the NPC in cooperation with additional cytosolic factors (for review, see Davis, 1995; Panté and Aebi, 1996a). In addition, lumenal factors of the nuclear envelope have a role in maintaining the functionality of the NPC. Binding of a monoclonal antibody to a lumenal epitope of the transmembrane glycoprotein gp210 reduces the nuclear envelope permeability to small macromolecules and signal-bearing proteins in living cells (Greber et al., 1990; Greber and Gerace, 1992). Recent experiments have identified an additional lumenal factor, calcium ions, affecting NPC function. Depletion of calcium from the nuclear envelope by calcium ionophores or the calcium ATPase inhibitor thapsigargin blocks passive diffusion and signal-mediated transport across the nuclear envelope in somatic mammalian cells and frog oocytes (Greber and Gerace, 1995; Stehno-Bittel et al., 1995; Sweitzer and Hanover, 1996). The inhibition is most likely due to a steric block of the central and peripheral NPC Wtransport channels, as suggested by atomic force microscopy in Xenopus nuclear envelopes (Perez-Terzic et al., 1996). In this study, we addressed the mechanisms of adenovirus DNA import into the nucleus in living cells. Our results demonstrate that viral DNA is imported through the NPCs. Depletion of ER/nuclear envelope calcium inhibited DNA and protein VII translocation into the nucleus, but not virus targeting to the nuclear pores or virus disassembly. Wheat germ agglutinin (WGA) or antibodies against O-linked NPC glycoproteins blocked protein VII import, virus attachment to the nuclear envelope and virus disassembly. The data indicate that pore complexes have multiple roles in adenovirus entry into the nucleus. They provide a docking site for a cytoplasmic virus at the nuclear envelope, they contain a virus disassembly activity and they are a gateway for the incoming genome into the nucleus. Results Calcium requirements during adenovirus infection To investigate the mechanisms by which incoming adenovirus DNA is imported into the cell nucleus, we tested whether calcium depletion from intracellular stores of HeLa cells by thapsigargin would affect the efficiency of adenovirus infection. Thapsigargin is an irreversible high affinity inhibitor of the ER-associated calcium ATPase (Thastrup et al., 1990). It rapidly depletes calcium from internal ER and nuclear envelope cisternae in many permanent cell lines, including epidermal carcinoma cells (Chao et al., 1992). Calcium depletion from the ER and nuclear envelope cisternae with thapsigargin or calcium ionophores previously has been shown to inhibit nuclear transport of classical NLS-bearing proteins and small dextrans in mammalian cells (Greber and Gerace, 1995; Stehno-Bittel et al., 1995). However, low calcium concentrations (∼100 nM free calcium, buffered with EGTA) in the extracellular medium for up to 1 h did not entirely deplete internal calcium stores and did not inhibit nuclear protein import. Virus was allowed to bind at a multiplicity of infection (m.o.i.) of 5–10 to the surface of HeLa cells in the cold using a normal calcium-containing medium. Infection was started by adding medium at 37°C. At various times thereafter, extracellular and lumenal calcium stores were depleted by adding 37°C medium containing ∼100 nM free calcium buffered with EGTA (low calcium medium), in the presence or absence of thapsigargin. The extracellular calcium concentration was kept low to prevent influx of calcium across the plasma membrane in response to depleting the internal stores (Fasolato et al., 1994; Clapham, 1995). After 3 h, the inhibitors (EGTA and thapsigargin) were washed out and the calcium concentration returned to normal. Infection was scored by counting the number of cells positive for the immediate early transcription factors E1A at 14 h using indirect immunofluorescence microscopy. About 90% of the cells, which were not depleted of calcium, were E1A positive (Figure 1A). This corresponded well with the expected efficiency of virus binding to cells in cold medium, which is ∼10% of the input virus (Greber et al., 1993). When thapsigargin/EGTA was added to the cells at 0, 10, 20 or 50 min after warming, the number of E1A-positive cells was reduced 5- to 10-fold as compared with control cells kept in regular calcium medium. Depleting calcium at 100 min or later post-warming still had a marginal effect on E1A expression, yielding ∼60% positive cells. It is possible that translation of E1A was inhibited due to calcium store depletion. In separate experiments, we showed that store depletion reduced the incorporation of [35S]methionine into cellular proteins by 30–50% (data not shown; see also Brostrom and Brostrom, 1990). Adding low calcium medium without thapsigargin at 0, 10 or 20 min, but not at 50 or 100 min after warming, also delayed E1A expression. We concluded that internal calcium stores and perhaps extracellular calcium were important for one or several early step(s) in cell infection with adenovirus. Figure 1.Calcium depletion from the ER/nuclear envelope inhibits adenovirus infection. (A) Purified adenovirus (10 p.f.u./cell) was bound in the cold to HeLa cells grown on glass coverslips. Samples were warmed in either normal calcium-containing medium, EGTA/calcium (E/Ca) medium or E/Ca medium containing 0.5 μM thapsigargin (Tg). After 3 h, medium was replaced with DMEM containing fetal bovine serum. Infection was scored by immunostaining against E1A 14 h after warming. (B) Purified [35S]methionine-labeled adenovirus (50 000 c.p.m. per dish) was bound to HeLa cells in the cold. Samples were warmed for 60 min as described in (A) and virus internalization measured by trypsin digestion of cell surface-bound hexon protein as described (Greber et al., 1993). Download figure Download PowerPoint Earlier studies using anti-virus antibody accessibility measurements had suggested that virus uptake into cells requires divalent cations (Svensson and Persson, 1984). To test whether extracellular calcium was required for virus endocytosis, we determined the efficiency of virus uptake using a trypsin digestion assay and [35S]methionine-labeled virus pre-bound in the cold (Figure 1B). Either with or without thapsigargin in low concentrations of extracellular calcium, no trypsin-resistant hexon was detected up to 60 min post-warming, indicating that virus was unable to enter these cells. In contrast, in the presence of normal amounts of extracellular calcium, viruses were internalized rapidly and efficiently (Figure 1B). Approximately 80% of the pre-bound viruses became trypsin resistant 20 min post-warming, in agreement with earlier results in unperturbed cells (Greber et al., 1993). Nuclear envelope/ER calcium is not required for virus transport to the nuclear envelope To investigate the nature of the intracellular calcium-dependent step(s), extracellular calcium and internal calcium stores were depleted after the virus had been internalized into cells, but before it had reached the nucleus, i.e. 20–25 min post-warming. Depletion of calcium was performed with the reversible calcium ionophore ionomycin (Greber and Gerace, 1995). Thapsigargin and A23187 were also tested and gave essentially the same results as ionomycin. We first analyzed whether virus transport to the nucleus was affected in ionomycin-treated cells using fluorescein isothiocyanate (FITC)-labeled adenovirus. Virus labeling was performed without inhibiting viral infectivity, as determined by plaque titration (data not shown). Approximately 58% of the FITC was incorporated into the major capsid protein hexon and ∼27% into the minor hexon-associated protein IX. Penton base and fiber each contained ∼7.5% of the total FITC. Entry of FITC-labeled adenovirus into HeLa cells was analyzed by fluorescence microscopy at different times after warming (Figure 2). Cell surface-bound viruses gave a typical plasma membrane staining pattern, with a prominent signal at cell borders (Figure 2a). After 20 min of warming in calcium-containing medium, a discretely punctate cytoplasmic pattern was observed, and at 60 min a predominant nuclear envelope rim staining was seen (Figure 2b and c). The majority of the punctate fluorescence signals were most likely individual virus particles, as concluded from serial confocal microscopy sections across whole cells (data not shown). In addition, we found no sign of virus aggregation in transmission electron micrographs (see also Figure 3). At 60 min after warming, a large fraction of viruses was distributed around the nuclear envelope. The same result was obtained with cells that were treated with ionomycin in EGTA/calcium medium at 20 min post-virus internalization (Figure 2d). Figure 2.Adenovirus transport to the nuclear envelope in calcium-depleted cells. Purified FITC-labeled adenovirus was bound to the cell surface in the cold at 20 μg/ml and internalized in warm calcium medium for 0, 20 or 60 min (a–c). Cells were also warmed for 20 min in calcium medium, followed by incubation in EGTA/calcium medium containing 5 μg/ml ionomycin up to 60 min post-warming (d). Samples were fixed in paraformaldehyde and analyzed on a Reichert-Jung Polyvar fluorescence microscope equipped with a CCD camera. Download figure Download PowerPoint Figure 3.Adenovirus docking to nuclear pore complexes in calcium-depleted cells. Purified adenovirus was bound to HeLa cells in the cold at 120 μg/ml and internalized into cells for 60 min in calcium medium (a) or EGTA/calcium medium (E/Ca) containing ionomycin (b) as described in Figure 2. Samples were fixed in 2% glutaraldehyde, embedded in Epon and processed for thin section electron microscopy as described in Materials and methods. Download figure Download PowerPoint Transmission electron microscopy indicated that adenovirus particles were associated with NPCs in ionomycin-treated cells, indistinguishably from mock-treated cells (Figure 3). Identical results were obtained with FITC-labeled virus (data not shown). We thus concluded that depletion of lumenal calcium had no effect on adenovirus transport across the cytoplasm to the nuclear membrane and attachment to NPCs. Nuclear envelope/ER calcium is needed for DNA transport into the nucleus We next investigated whether nuclear import of incoming viral DNA and DNA-bound protein VII was disturbed in calcium-depleted cells. We determined the subcellular localization of viral DNA by a fluorescence in situ hybridization (FISH) assay at different times after warming cells containing filled or ionomycin-depleted internal calcium stores (Figures 4 and 5). DNA fragments of 200–500 bp in length from isolated adenovirus were labeled by nick translation in the presence of dUTP–Texas Red according to standard protocols (Sambrook et al., 1989). Hybridization of probe DNA to target nucleic acids was performed by denaturation at 92°C and subsequent hybridization at 37°C as described (Ishov and Maul, 1996). Figure 4.Nuclear transport of viral capsid and DNA. FITC-labeled adenovirus bound to HeLa cells at 20 μg/ml was internalized in calcium medium (a–c), calcium medium containing 5 μM actinomycin D (d–f) for 180 min or in calcium medium for 25 min followed by EGTA/calcium medium (E/Ca) containing 5 μg/ml ionomycin for up to 180 min (g–i). Samples were fixed in methanol and paraformaldehyde, and processed for in situ hybridization using Texas Red-labeled adenovirus type 2 DNA as a probe. Non-infected cells were processed identically (k–m). Double fluorescence images and Nomarsky differential interference optics pictures were recorded with a CCD camera on a Reichert-Jung Polyvar microscope. Download figure Download PowerPoint Figure 5.Nuclear import of viral DNA and associated protein VII is blocked at the nuclear envelope in calcium-depleted cells. Purified adenovirus bound to HeLa cells at 20 μg/ml was internalized in calcium medium in the presence of 0.5 mM cycloheximide (CHX) for 25 min (A and B, a) or 180 min (A and B, b), respectively. Other cells were incubated in calcium medium for 25 min followed by EGTA/calcium medium (E/Ca) containing 5 μg/ml ionomycin for up to 180 min (A and B, c). Samples were either fixed directly or washed in warm calcium medium and incubated in regular medium for up to 360 min post-warming (A and B, d). Non-infected cells were processed identically (A and B, e). Samples were either processed for in situ hybridization with Texas Red-labeled virus DNA (A) or processed for immunostaining of protein VII (B). Confocal fluorescence micrographs were recorded across the centers of the cells using the FITC and Texas Red filter sets, respectively. Results are displayed as single optical slices with an estimated thickness of 0.2 μm each (A) or as a stack of four slices, each ∼0.2 μm thick and 0.5 μm apart from the next slice (B). Download figure Download PowerPoint We first analyzed the DNA localization of FITC-labeled adenovirus in control cells under normal calcium conditions at 180 min post-warming, either in the presence or absence of 5 μg/ml actinomycin D (Figure 4a–f). At this concentration, actinomycin D is a potent inhibitor of DNA polymerase I and II transcription (Perry and Kelley, 1970). When images across the centers of the cells were recorded with a sensitive charge coupled device (CCD) camera, FITC-labeled viral capsid proteins could be seen at the nuclear envelope. The Texas Red fluorescent DNA probe was found throughout the nucleus and occasionally in the cytoplasm. The Texas Red signal represented incoming viral genomes, since first, the distribution was the same whether actinomycin D was present or absent. Second, neither FITC, nor Texas Red staining was found in uninfected cells (Figure 4k–m). Third, the Texas Red signal was quantitatively quenched by incubation of the infected cells with DNase, but not with RNase post-fixation in methanol (data not shown). When internal calcium stores were depleted of calcium with ionomycin from 25 to 180 min post-internalization, capsid proteins were still found at the nuclear envelope, but viral DNA was now concentrated around the nuclear envelope (Figure 4g–i). DNA was not seen across the nucleus. Taken together, these results indicate that in cells depleted of internal calcium stores, DNA remained in apparently close association with the capsid, excluded from the nucleus. Under normal calcium conditions, DNA effectively separated from the capsid and was apparently imported into the nucleus. To confirm that viral DNA was indeed transported into the nucleus under normal calcium conditions, we analyzed the FISH assays by confocal microscopy. The nuclear envelope was visualized with anti-lamin A, B and C antibodies by indirect immunofluorescence (Figure 5A, green signal). After 25 min of warming cells containing unlabeled adenovirus, punctate signals of viral DNA (in red) were found almost exclusively in the cytoplasm (Figure 5A, a). Most probably, the majority of the fluorescent dots in these confocal sections represented single DNA molecules. A few particles appeared already at the nuclear envelope, as indicated by the yellow color of the merged lamin and DNA stainings. No DNA signal was detected in mock-infected cells (Figure 5A, e). After 180 min of warming in normal calcium medium, the majority of adenovirus DNA was found inside the nucleus, enclosed by the lamina rim (Figure 5A, b). Some DNA was located near the nuclear envelope, suggesting that this DNA was transported rather slowly into the nucleus. DNA staining could be detected inside the nucleus as early as 60 min post-warming (data not shown). In cells depleted of lumenal calcium by ionomycin from 25 to 180 min post-internalization, viral DNA was excluded from the nucleus and localized in the vicinity of the cytoplasmic face of the nuclear envelope (Figure 5A, c). When ionomycin/EGTA was removed and cells incubated in normal calcium-containing medium, DNA was imported into the nuclei, as shown at 360 min post-internalization (Figure 5A, d). Similar results were also obtained after 270 min (data not shown). These results confirmed that the cells were not poisoned irreversibly by the ionomycin/EGTA treatment. No significant inhibition of DNA import was observed when cells were treated with EGTA/calcium alone from 25 to 180 min post-internalization, suggesting that calcium was not depleted sufficiently from internal stores under these conditions (data not shown). When internal calcium was depleted by ionomycin/EGTA at 180 min post-warming, viral DNA was still found inside the nucleus, indicating that calcium depletion did not extract previously imported DNA from the nucleus (data not shown). In a parallel experiment, the localization of incoming DNA-associated protein VII was analyzed by confocal microscopy and indirect immunofluorescence labeling in the presence of cycloheximide, which blocks viral and cellular protein synthesis (Figure 5B). Simultaneous staining of protein VII and viral DNA was unfortunately not possible under the conditions used for in situ hybridization (data not shown). As expected, protein VII was not detected after 25 min of warming, since viral DNA and associated proteins were still enwrapped by an essentially intact coat (Figure 5A and B, panels a, see also Greber et al., 1993). No protein VII was detected in uninfected cells (Figure 5B, e). In the presence of calcium, protein VII was found inside the nucleus and also at the nuclear envelope after 180 min, similarly to the viral DNA (Figure 5B, b). In calcium store-depleted cells, protein VII was enriched at the nuclear envelope, but not detected inside the nucleus, suggesting that virus disassembly could occur, but not nuclear import (Figure 5B, c). When calcium was restored, protein VII was found inside the nucleus, in agreement with the in situ hybridization results (Figure 5B, d). The data demonstrated that depletion of calcium from internal cisternae had no effect on intracellular virus targeting to the nuclear envelope, but inhibited translocation of DNA and associated protein VII across the nuclear envelope. Attachment to nuclear pore complexes is needed for capsid disassembly It had been shown earlier by biochemical and genetic data that capsid disassembly was required for the transport of the DNA-associated protein VII into the nucleus (Miles et al., 1980; Greber et al., 1996). In order to test if the inhibition of DNA and protein VII import into the nucleus of calcium-depleted cells was due to reduced virus dissociation, we quantitated capsid disassembly using two different biochemical assays. Confocal microscopy together with the Bio-Rad area software was used to determine the abundance of hexon epitopes for a polyclonal anti-hexon antibody (Baum et al., 1972; Greber et al., 1995, 1996). We also determined the amount of hexon-associated DNA by co-immunoprecipitation experiments of [3H]thymidine-labeled virus DNA with an anti-hexon antibody exactly as described before (Greber et al., 1996). The antigens of the major coat protein picked up by the first assay are of low abundance within the native particle, but are more abundant in stripped down or disassembled particles. The second assay measures the dissociation of virus DNA from hexon, provided that the degradation of the linker protein between the capsid and the viral chromosome, protein VI, has occurred. In both assays, we detected no significant differences in virus uncoating between calcium-depleted cells and control cells (Figure 6). Cells treated with thapsigargin in low calcium medium at 20 min of warming showed the typical increase in hexon fluorescence with time, reaching, at 80 min, nearly the same levels as the control cells or the cells treated with low calcium-containing medium alone (Figure 6A). Likewise, in the inhibitor-treated and the control cells, ∼50 and 65%, respectively, of the viral DNA was dissociated from hexon after 150 min of internalization (Figure 6B" @default.
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• W2129333552 title "The role of the nuclear pore complex in adenovirus DNA entry" @default.
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