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Phosphorylation-dependent Binding of Hepatitis B Virus Core Particles to the Nuclear Pore Complex

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      Abstract

      Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) α and β. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.

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      Most cited references 59

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      Detection of specific sequences among DNA fragments separated by gel electrophoresis

       E.M. Southern (1975)
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        Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors

         A Adam,  L Gerace,  R. Marr (1990)
        We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N- ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.
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          Nucleocytoplasmic transport.

          Active transport of proteins and RNAs between the nucleus and cytoplasm is a major process in eukaryotic cells. Recently, factors that recognize transport substrates and mediate nuclear import or export have been characterized, revealing interactions that target substrates to the nuclear pore complexes, through which translocation occurs. Translocation requires energy, and for the import process this energy is at least partly consumed by the action of the small guanosine triphosphatase Ran. In the first half of the review, some of the well-established general background information on nucleocytoplasmic transport is discussed. The second half describes recent information on the mechanistic details of nuclear import and export as well as major unresolved issues such as how directionality is conferred on either import or export. The whole review is slanted toward discussion of metazoan cells.
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            Author and article information

            Affiliations
            [* ]Institute of Medical Virology, Justus Liebig University, D-35392 Giessen, Germany; and []Yale University School of Medicine, Department of Cell Biology, New Haven, Connecticut 06520-8002
            Journal
            J Cell Biol
            The Journal of Cell Biology
            The Rockefeller University Press
            0021-9525
            1540-8140
            5 April 1999
            : 145
            : 1
            : 45-55
            2148218
            10189367
            Categories
            Regular Articles

            Cell biology

            nucleocapsid, phosphorylation, nuclear pore, core, hepatitis b virus

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