title: Identification and characterization of a novel bipartite nuclear localization signal in the hepatitis B virus polymerase.
abstract: OBJECTIVE
  To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle.
  
  
  METHODS
  Sequence analysis was performed to predict functional motives within P. Phosphorylation of P was analyzed by in vitro phosphorylation. Phosphorylation site and nuclear localization signal (NLS) were destroyed by site directed mutagenesis. Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding. Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.
  
  
  RESULTS
  We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinase II (CKII) phosphorylation site located within the terminal protein domain (TP) of the HBV polymerase. Inhibition of CKII impairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase. Binding of the import factor karyopherin-α2 to the polymerase depends on its CKII-mediated phosphorylation of the bipartite NLS. In HBV-infected primary Tupaia hepatocytes CKII inhibition in the early phase (post entry phase) of the infection process prevents the establishment of the infection.
  
  
  CONCLUSIONS
  Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.
authors.affiliation: Joachim Lupberger, Stephanie Schaedler, Eberhard Hildt, Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Lupberger
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schaedler
authors.forename: Alexander
authors.initials: A
authors.lastname: Peiran
authors.forename: Eberhard
authors.initials: E
authors.lastname: Hildt
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II / Q000378:metabolism; D002467:Cell Nucleus / Q000821:virology*; D017124:Conserved Sequence; D015684:Gene Products, pol / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D009154:Mutation; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014417:Tupaia; D014779:Virus Replication; D028901:alpha Karyopherins / Q000378:metabolism
chemical_list: D015684:Gene Products, pol; D019913:Nuclear Localization Signals; C066027:P protein, Hepatitis B virus; D028901:alpha Karyopherins; D047390:Casein Kinase II
keywords: Casein kinase II; Hepatitis B virus; Nuclear localization signal; Replication; Trafficking