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• W2029784758 abstract "Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process. Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process. nuclear pore complexes nuclear localization sequence bovine serum albumin phosphate-buffered saline 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol wheat germ agglutinin guanosine 5′-O-(3-thiotriphosphate) Proteins are imported into the nucleus through aqueous channels that span the nuclear envelope called nuclear pore complexes (NPCs).1 Whereas ions and molecules less than ∼20–40 kDa can diffuse passively through the NPC, larger proteins are transported by saturable pathways that are energy- and signal-dependent. The classical signals that specify nuclear protein import (nuclear localization sequences (NLSs)) are short stretches of amino acids rich in basic residues, although other classes of NLSs have been described recently (1.Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 2.Standiford D.M. Richter J.D. J. Cell Biol. 1992; 118: 991-1002Crossref PubMed Scopus (33) Google Scholar). The transport of NLS-bearing proteins into the nucleus requires the participation of soluble cytosolic factors (3.Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (766) Google Scholar). The advent of a cytosol-dependent in vitro assay for nuclear import (3.Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (766) Google Scholar) has led to the identification of a number of these factors (4.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (911) Google Scholar), namely importin-α (also called the NLS receptor and karyopherin-α), importin-β (also called p97 and karyopherin-β), Ran (TC4), and NTF2 (also called p10 and pp15). An initial step in the import of proteins containing classical NLSs occurs in the cytoplasm, where the substrate binds to the receptor importin-α. Subsequently, this complex docks at the cytoplasmic face of the NPC together with importin-β. The complex is then translocated through a central gated channel of the NPC into the nuclear interior. Movement of the import complex through the NPC requires Ran and NTF2 (5.Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 6.Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (638) Google Scholar, 7.Paschal B.M. Delphin C. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7679-7683Crossref PubMed Scopus (79) Google Scholar, 8.Clarkson W.D. Corbett A.H. Paschal B.M. Kent H.M. McCoy A.J. Gerace L. Silver P.A. Stewart M. J. Mol. Biol. 1997; 272: 716-730Crossref PubMed Scopus (65) Google Scholar), but the precise role of these components is unclear. An additional protein, hsp/hsc70, has been shown to be required for import of some substrates (e.g. the large T-antigen protein (9.Shi Y. Thomas J.O. Mol. Cell. Biol. 1992; 12: 2186-2192Crossref PubMed Scopus (300) Google Scholar)), but not of others (e.g. the glucocorticoid receptor (10.Yang J. DeFranco D.B. Mol. Cell. Biol. 1994; 14: 5088-5098Crossref PubMed Scopus (103) Google Scholar)).Most viruses that replicate in the nucleus must use the NPC to introduce their genome into the nucleoplasm. Although it is known that the gated channel of the NPC can expand up to ∼25 nm in diameter to allow signal-mediated transport (11.Gerace L. Curr. Opin. Cell Biol. 1992; 4: 637-645Crossref PubMed Scopus (94) Google Scholar), this is still considerably smaller than the diameters of many viruses known to replicate in the nucleus. For example, the diameters of SV40, adenovirus, and herpesvirus are 45–50, 60–90, and 120–200 nm, respectively. Enveloped viruses such as human immunodeficiency virus and influenza manage to circumvent the physical barrier of the NPC by releasing the genome from their large capsids during entry into the cytoplasm. The genome is subsequently transported through the NPC as a flexible protein-nucleic acid complex that appears to utilize a classical NLS-mediated pathway (12.O'Neill R.E. Jaskunas R. Blobel G. Palese P. Moroianu J. J. Biol. Chem. 1995; 270: 22701-22704Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 13.Gallay P. Stitt V. Mundy C. Oettinger M. Trono D. J. Virol. 1996; 70: 1027-1032Crossref PubMed Google Scholar). Most non-enveloped viruses must undergo restructuring for the import of their genomes into the nucleus. Both the DNA and associated capsid proteins of SV40 are transported into the nucleus after infection of cells, but the structural state of the nucleocapsid during import has not yet been characterized (14.Yamada M. Kasamatsu H. J. Virol. 1993; 67: 119-130Crossref PubMed Google Scholar). However, the DNA viruses such as adenovirus, baculovirus, and hepadnavirus all appear to only partially uncoat at various stages of entry into the host cell (15.Waris M. Halonen P. J. Chromatogr. 1987; 397: 321-325Crossref PubMed Scopus (25) Google Scholar, 16.Shenk T. Fundamental Virology. Lippincott-Raven, New York1996: 979-1016Google Scholar). In the case of adenovirus, the early events of infection such as cell attachment and internalization have been partially defined (17.Bergelson J.M. Cunningham J.A. Droguett G. Kurt-Jones E.A. Krithivas A. Hong J.S. Horwitz M.S. Crowell R.L. Finberg R.W. Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2605) Google Scholar, 18.Wickham T.J. Mathius P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1944) Google Scholar, 19.Wickham T.J. Filardo E.J. Cheresh D.A. Nemerow G.R. J. Cell Biol. 1994; 127: 257-264Crossref PubMed Scopus (364) Google Scholar, 20.Greber U.F. Willetts M. Webster P. Helenius A. Cell. 1993; 75: 477-486Abstract Full Text PDF PubMed Scopus (670) Google Scholar), but little is known about the transport of adenovirus DNA into the nucleus. As adenovirus is a strong candidate for gene therapy applications (21.Robbins P.D. Tahara H. Ghivizzani S.C. Trends Biotechnol. 1998; 16: 35-40Abstract Full Text PDF PubMed Scopus (200) Google Scholar), it is important to obtain a detailed understanding of the interactions between this virus and the host cell.Adenovirus is a non-enveloped double-stranded DNA virus containing 11–15 different virus-encoded polypeptides and a linear genome of ∼36 kilobase pairs (22.Stewart P.L. Fuller S.D. Burnett R.M. EMBO J. 1993; 12: 2589-2599Crossref PubMed Scopus (297) Google Scholar). The total mass of the nucleocapsid is estimated to be 150 MDa (22.Stewart P.L. Fuller S.D. Burnett R.M. EMBO J. 1993; 12: 2589-2599Crossref PubMed Scopus (297) Google Scholar). The major capsid protein is the hexon coat protein. The capsid contains 240 hexon trimers, which form an icosahedron with 12 vertices and 20 facets. At the vertices are the fiber proteins, which contain a primary cell-surface attachment site (17.Bergelson J.M. Cunningham J.A. Droguett G. Kurt-Jones E.A. Krithivas A. Hong J.S. Horwitz M.S. Crowell R.L. Finberg R.W. Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2605) Google Scholar). Anchoring each fiber to the capsid is the penton protein, which contains a secondary binding site whose recognition is a precursor to internalization (18.Wickham T.J. Mathius P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1944) Google Scholar, 19.Wickham T.J. Filardo E.J. Cheresh D.A. Nemerow G.R. J. Cell Biol. 1994; 127: 257-264Crossref PubMed Scopus (364) Google Scholar). Stabilizing the capsid are several other minor proteins that either associate with hexons alone (proteins VI, VIII, and IX) or strengthen the association between hexon and penton proteins (proteins IIIa and IX). The DNA is condensed with proteins V, VII, and μ and the covalently attached terminal protein, which together form the viral core.The adenovirus capsid is a very stable structure that protects the viral genome against harsh environmental conditions. The virus enters the cell by receptor-mediated internalization in coated pits, where the combination of the energy of binding and the acidic environment of the early endosome is thought to cause a series of cooperative structural changes in the capsid (20.Greber U.F. Willetts M. Webster P. Helenius A. Cell. 1993; 75: 477-486Abstract Full Text PDF PubMed Scopus (670) Google Scholar). These structural changes appear to facilitate fusion with the endosomal membrane and penetration into the cytoplasm (23.Patterson S. Oxford J.S. Vaccine. 1986; 4: 79-90Crossref PubMed Scopus (13) Google Scholar) as well as potentiate the loss of certain capsid proteins. By the time it is delivered to the cytoplasm, the virus loses the fiber penton, peripentonal hexon proteins, and the capsid-stabilizing proteins (proteins IIIa, VIII, and IX) (20.Greber U.F. Willetts M. Webster P. Helenius A. Cell. 1993; 75: 477-486Abstract Full Text PDF PubMed Scopus (670) Google Scholar). The nucleocapsid resulting from these rearrangements docks with the NPC, where it appears to undergo further disassembly, allowing its DNA to enter the nuclear interior (20.Greber U.F. Willetts M. Webster P. Helenius A. Cell. 1993; 75: 477-486Abstract Full Text PDF PubMed Scopus (670) Google Scholar). Because cellular nucleoprotein particles (e.g. messenger ribonucleoproteins) can be transported through the NPC by the machinery that mediates protein transport (4.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (911) Google Scholar), adenovirus similarly might exploit the cellular nuclear protein import machinery for the import of its DNA into the nuclei of infected cells.Here we use a digitonin-permeabilized cell assay to analyze the interaction of nucleocapsid derived from adenovirus type 2 with the NPC and the transport of viral DNA into the nucleus. Our results indicate that adenovirus DNA import utilizes components of the classical protein import machinery and hsp/hsc70. Furthermore, our findings, in conjunction with previous work, indicate that the major capsid protein (hexon) has an important role in viral DNA import. Since recombinant factors for classical NLS-mediated import are insufficient to support adenovirus DNA import, additional components appear to be involved. We discuss the possibility that these are involved in nucleocapsid uncoating at the NPC.DISCUSSIONHere we describe an assay for the import of adenovirus DNA into the nuclei of permeabilized HeLa cells. In this assay, a large amount of nucleocapsid becomes associated with the NPC within 30 min, as documented by both immunofluorescence staining and thin-section electron microscopy. A significant amount of viral DNA is subsequently imported into the nucleus by 45–60 min, as monitored by in situ hybridization. We have used this assay to define the requirements for adenovirus DNA import and to begin a mechanistic analysis of this process. As discussed in detail below, our experiments indicate that the docking of adenovirus to the NPC is a prerequisite for DNA import and that docking (and possibly subsequent steps) involves the hexon protein and components of the machinery for nuclear import of proteins containing basic amino acid-type NLSs. Although we could reconstitute adenovirus DNA import in vitro with cytosol and demonstrated a requirement for hsp/hsc70, the four classical nuclear protein import factors together with hsc70 are insufficient to reconstitute viral DNA import in permeabilized cells.Our results showing that WGA blocks the docking of adenovirus at the NPC and the internalization of viral DNA are consistent with the effects of WGA in vivo (29.Greber U.F. Suomalainen M. Stidwill R.P. Boucke K. Ebersold M.W. Helenius A. EMBO J. 1997; 16: 5998-6007Crossref PubMed Scopus (241) Google Scholar). Furthermore, our electron microscopic data showing that nucleocapsids dock directly at the NPC prior to the appearance of adenovirus DNA in the nucleus closely resemble electron microscopic observations made for in vivoadenovirus infection (34.Dales S. Chardonnet Y. Virology. 1973; 56: 465-483Crossref PubMed Scopus (105) Google Scholar, 35.Puvion-Dutilleul F. Puvion E. Microsc. Res. Tech. 1995; 31: 22-43Crossref PubMed Scopus (11) Google Scholar, 36.Cepko C.L. Sharp P.A. Cell. 1982; 31: 407-415Abstract Full Text PDF PubMed Scopus (74) Google Scholar). Thus, our assay reflects some of the basic features of adenovirus DNA import that have been described previously with in vivo studies and is likely to be physiologically relevant.Nucleocapsid docking at the NPC appears to be a necessary precondition for DNA import since blocking the association of nucleocapsid with the nuclear envelope in vitro either with WGA or with an excess of a protein conjugate containing a classical NLS also inhibits the import of viral DNA into the nucleus. Further support for nucleocapsid docking at the NPC as a requirement for DNA import is the finding that the kinetics of DNA import in vitro (this study) andin vivo (29.Greber U.F. Suomalainen M. Stidwill R.P. Boucke K. Ebersold M.W. Helenius A. EMBO J. 1997; 16: 5998-6007Crossref PubMed Scopus (241) Google Scholar) are significantly slower than the kinetics of nucleocapsid binding at the NPC. This conclusion is also supported by immunofluorescence localization experiments involving cells infected with adenovirus that suggest that the DNA-associated protein VII (and presumably the associated DNA) is released from the capsid only after the nucleocapsid docks at the nuclear envelope (29.Greber U.F. Suomalainen M. Stidwill R.P. Boucke K. Ebersold M.W. Helenius A. EMBO J. 1997; 16: 5998-6007Crossref PubMed Scopus (241) Google Scholar).A variety of data suggest that the hexon coat protein is the principal targeting determinant for adenovirus binding to the nuclear envelope.A priori, the fact that the hexon has karyophilic properties, as shown in our in vitro studies, and is the major protein on the surface of the viral capsid both in our in vitro assay and in vivo (22.Stewart P.L. Fuller S.D. Burnett R.M. EMBO J. 1993; 12: 2589-2599Crossref PubMed Scopus (297) Google Scholar) implicates it as the principal agent responsible for docking to the NPC. Further evidence for this is the fact that excess basic amino acid-type NLSs competitively inhibit the import of purified hexon protein as well as the docking of the nucleocapsid to the NPC. It is unclear whether the role of the hexon protein is simply to dock the nucleocapsid to the NPC or to engage more distal components of the NLS-mediated import machinery for the import of the adenovirus DNA-protein complex into the nucleus. Other candidate proteins for this second function are proteins VII and V, which are associated with viral DNA in the nucleus and may be co-imported with the latter (29.Greber U.F. Suomalainen M. Stidwill R.P. Boucke K. Ebersold M.W. Helenius A. EMBO J. 1997; 16: 5998-6007Crossref PubMed Scopus (241) Google Scholar).Although it is known that hexon trimers efficiently enter the nucleus during viral assembly (36.Cepko C.L. Sharp P.A. Cell. 1982; 31: 407-415Abstract Full Text PDF PubMed Scopus (74) Google Scholar), an NLS on the hexon has not yet been defined. However, scrutiny of the primary structure of the hexon protein reveals several sequences that have basic amino acid-type NLS character. One is at position 279 in the amino acid sequence, and others occur at positions 567, 780, and 807. Although there are fewer basic amino acids in each of these sequences than in the prototypical NLS of the SV40 T-antigen, the amino acid sequence starting at position 279 shares at least a superficial similarity with a number of sequences known to have NLS function (for a comprehensive list, see Ref. 37.Boulikas T. Crit. Rev. Eukaryotic Gene Expression. 1993; 3: 193-227PubMed Google Scholar). However, it must be pointed out that the hexon may not possess a linear, continuous stretch of basic amino acids that serves as an NLS. Instead, some of the sites mentioned above may combine in the folded three-dimensional structure to form an active NLS. Finally, although less likely, the hexon may altogether lack an intrinsic NLS, but could rely on specifically binding to a host cytosolic protein that does possess an NLS.Previous observations have suggested a role for hsp/hsc70 in the infective pathway of adenovirus. Immunolocalization studies in infected cells have shown that hexon protein and hsp/hsc70 associate after release from the early endosome (31.Niewiarowska J. D'Halluin J.C. Belin M.T. Exp. Cell Res. 1992; 201: 408-416Crossref PubMed Scopus (38) Google Scholar), raising the possibility that hsp/hsc70 may act on the nucleocapsid before docking with the NPC. One possible function of this binding could be to induce conformational changes in the nucleocapsid that expose NLSs. Consistent with this model, previous studies have shown that the hexon protein undergoes conformational changes during uncoating (16.Shenk T. Fundamental Virology. Lippincott-Raven, New York1996: 979-1016Google Scholar). Although purified hexon is imported into the nucleus and must therefore have an easily accessible NLS, this signal may be concealed in the nucleocapsid until a conformational change involving hsp/hsc70 or some other component exposes it. It is tantalizing to speculate that the hexon in the intact adenovirus nucleocapsid has a concealed NLS to protect the latter from the host's immune system since most basic amino acid-type NLSs are charged, hydrophilic, exposed, and thus highly antigenic.A second possible function for hsc70 could be to disassemble the nucleocapsid at the NPC. Because capsid disassembly must occur before viral DNA can be imported (20.Greber U.F. Willetts M. Webster P. Helenius A. Cell. 1993; 75: 477-486Abstract Full Text PDF PubMed Scopus (670) Google Scholar), it may be that this step is blocked by the antibodies to hsp70. Docking with components of the NPC may catalyze the conversion of nucleocapsid to a disassembled state, and hsc70 could stabilize the disassembled state, consistent with its role as a chaperone protein (38.Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3090) Google Scholar). Alternatively, hsp/hsc70 could actively unfold the capsid. Finally, hsc70 may act directly on components of the NPC itself, and it is their activity that is essential for the translocation of viral DNA into the nucleus. In yeast, hsp/hsc70 has been suggested to play a role in the binding of the NLS both to its receptor and on the NPC itself during protein import (39.Shulga N. Roberts P. Gu Z. Spitz L. Tabb M.M. Nomura M. Goldfarb D.S. J. Cell Biol. 1996; 135: 329-339Crossref PubMed Scopus (186) Google Scholar). Our data do not allow us to precisely identify the site of hsp/hsc70 activity in the import pathway of adenovirus. It may be that hsp/hsc70 plays a role in several steps in the infective pathway of adenovirus discussed above.Permeabilized cell nuclear import assays similar to the one we describe for adenovirus have been employed in the study of two lipid enveloped viruses that replicate in the nuclei of infected cells: influenza virus (12.O'Neill R.E. Jaskunas R. Blobel G. Palese P. Moroianu J. J. Biol. Chem. 1995; 270: 22701-22704Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and woodchuck hepadnavirus (40.Kann M. Bischof A. Gerlich W.H. J. Virol. 1997; 71: 1310-1316Crossref PubMed Google Scholar). A simple ribonucleoprotein complex of influenza virus, consisting of nucleoprotein and the RNA genome, is sufficient for the import of the influenza genome in vitro and for the production of infectious virus in vivo (12.O'Neill R.E. Jaskunas R. Blobel G. Palese P. Moroianu J. J. Biol. Chem. 1995; 270: 22701-22704Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Similarly, a complex of hepadnavirus DNA and viral polymerase is sufficient to transport the hepadnavirus genome into the nucleus in vitro (40.Kann M. Bischof A. Gerlich W.H. J. Virol. 1997; 71: 1310-1316Crossref PubMed Google Scholar). A combination of the cytosolic factors that can reconstitute import of proteins with basic amino acid-type NLSs in permeabilized cells is also sufficient to reconstitute import of the influenza RNA in vitro (12.O'Neill R.E. Jaskunas R. Blobel G. Palese P. Moroianu J. J. Biol. Chem. 1995; 270: 22701-22704Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar).Our work indicates that adenovirus requires a more complex set of interactions with the host cell than these other viruses. In particular, when the adenovirus nucleocapsid is introduced into a permeabilized cell nuclear transport assay, a combination of the cytosolic factors that support nuclear import of substrates with basic amino acid-type NLSs together with hsc70 is insufficient to reconstitute adenovirus DNA import, although DNA import is supported by complete cytosol. The unknown cytosolic factors required for adenovirus DNA import could be involved in nucleocapsid uncoating at the NPC and/or in translocation through the pore. It is expected that thein vitro assay reported here will permit detailed biochemical analysis of these components, which are expected to be key to adenovirus infectivity. Proteins are imported into the nucleus through aqueous channels that span the nuclear envelope called nuclear pore complexes (NPCs).1 Whereas ions and molecules less than ∼20–40 kDa can diffuse passively through the NPC, larger proteins are transported by saturable pathways that are energy- and signal-dependent. The classical signals that specify nuclear protein import (nuclear localization sequences (NLSs)) are short stretches of amino acids rich in basic residues, although other classes of NLSs have been described recently (1.Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 2.Standiford D.M. Richter J.D. J. Cell Biol. 1992; 118: 991-1002Crossref PubMed Scopus (33) Google Scholar). The transport of NLS-bearing proteins into the nucleus requires the participation of soluble cytosolic factors (3.Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (766) Google Scholar). The advent of a cytosol-dependent in vitro assay for nuclear import (3.Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (766) Google Scholar) has led to the identification of a number of these factors (4.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (911) Google Scholar), namely importin-α (also called the NLS receptor and karyopherin-α), importin-β (also called p97 and karyopherin-β), Ran (TC4), and NTF2 (also called p10 and pp15). An initial step in the import of proteins containing classical NLSs occurs in the cytoplasm, where the substrate binds to the receptor importin-α. Subsequently, this complex docks at the cytoplasmic face of the NPC together with importin-β. The complex is then translocated through a central gated channel of the NPC into the nuclear interior. Movement of the import complex through the NPC requires Ran and NTF2 (5.Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar, 6.Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (638) Google Scholar, 7.Paschal B.M. Delphin C. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7679-7683Crossref PubMed Scopus (79) Google Scholar, 8.Clarkson W.D. Corbett A.H. Paschal B.M. Kent H.M. McCoy A.J. Gerace L. Silver P.A. Stewart M. J. Mol. Biol. 1997; 272: 716-730Crossref PubMed Scopus (65) Google Scholar), but the precise role of these components is unclear. An additional protein, hsp/hsc70, has been shown to be required for import of some substrates (e.g. the large T-antigen protein (9.Shi Y. Thomas J.O. Mol. Cell. Biol. 1992; 12: 2186-2192Crossref PubMed Scopus (300) Google Scholar)), but not of others (e.g. the glucocorticoid receptor (10.Yang J. DeFranco D.B. Mol. Cell. Biol. 1994; 14: 5088-5098Crossref PubMed Scopus (103) Google Scholar)). Most viruses that replicate in the nucleus must use the NPC to introduce their genome into the nucleoplasm. Although it is known that the gated channel of the NPC can expand up to ∼25 nm in diameter to allow signal-mediated transport (11.Gerace L. Curr. Opin. Cell Biol. 1992; 4: 637-645Crossref PubMed Scopus (94) Google Scholar), this is still considerably smaller than the diameters of many viruses known to replicate in the nucleus. For example, the diameters of SV40, adenovirus, and herpesvirus are 45–50, 60–90, and 120–200 nm, respectively. Enveloped viruses such as human immunodeficiency virus and influenza manage to circumvent the physical barrier of the NPC by releasing the genome from their large capsids during entry into the cytoplasm. The genome is subsequently transported through the NPC as a flexible protein-nucleic acid complex that appears to utilize a classical NLS-mediated pathway (12.O'Neill R.E. Jaskunas R. Blobel G. Palese P. Moroianu J. J. Biol. Chem. 1995; 270: 22701-22704Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 13.Gallay P. Stitt V. Mundy C. Oettinger M. Trono D. J. Virol. 1996; 70: 1027-1032Crossref PubMed Google Scholar). Most non-enveloped viruses must undergo restructuring for the import of their genomes into the nucleus. Both the DNA and associated capsid proteins of SV40 are transported into the nucleus after infection of cells, but the structural state of the nucleocapsid during import has not yet been characterized (14.Yamada M. Kasamatsu H. J. Virol. 1993; 67: 119-130Crossref PubMed Google Scholar). However, the DNA viruses such as adenovirus, baculovirus, and hepadnavirus all appear to only partially uncoat at various stages of entry into the host cell (15.Waris M. Halonen P. J. Chromatogr. 1987; 397: 321-325Crossref PubMed Scopus (25) Google Scholar, 16.Shenk T. Fundamental Virology. Lippincott-Raven, New York1996: 979-1016Google Scholar). In the case of adenovirus, the early events of infection such as cell attachment and internalization have been partially defined (17.Bergelson J.M. Cunningham J.A. Droguett G. Kurt-Jones E.A. Krithivas A. Hong J.S. Horwitz M.S. Crowell R.L. Finberg R.W. Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2605) Google Scholar, 18.Wickham T.J. Mathius P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1944) Google Scholar, 19.Wickham T.J. Filardo E.J. Cheresh D.A. Nemerow G.R. J. Cell Biol. 1994; 127: 257-264Crossref PubMed Scopus (364) Google Scholar, 20.Greber U.F. Willetts M. Webster P. Helenius A. Cell. 1993; 75: 477-486Abstract Full Text PDF PubMed Scopus (670) Google Scholar), but little is known about the transport of adenovirus DNA into the nucleus. As adenovirus is a strong candidate for gene therapy applications (21.Robbins P.D. Tahara H. Ghivizzani S.C. Trends Biotechnol. 1998; 16: 35-40Abstract Full Text PDF PubMed Scopus (200) Google Scholar), it is important to obtain a detailed understanding of the interactions between this virus and the host cell. Adenovirus is a non-enveloped double-stranded DNA virus containing 11–15 different virus-encoded polypeptides and a linear genome of ∼36 kilobase pairs (22.Stewart P.L. Fuller S.D. Burnett R.M. EMBO J. 1993; 12: 2589-2599Crossref PubMed Scopus (297) Google Scholar). The total mass of the nucleocapsid is estimated to be 150 MDa (22.Stewart P.L. Fuller S.D. Burnett R.M. EMBO J. 1993; 12: 2589-2599Crossref PubMed Scopus (297) Google Scholar). The major capsid protein is the hexon coat protein. The capsid contains 240 hexon trimers, which form an i" @default.
• W2029784758 created "2016-06-24" @default.
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• W2029784758 date "2000-02-01" @default.
• W2029784758 modified "2023-09-30" @default.
• W2029784758 title "Nuclear Import of Adenovirus DNA in Vitro Involves the Nuclear Protein Import Pathway and hsc70" @default.
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