Characterization of the nuclear localization signal of the borna disease virus polymerase.
Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. BDV proteins involved in replication and transcription must pass through the nuclear envelope to associate with the genomic viral RNA. The RNA-dependent RNA polymerase (L) of BDV is postulated to be the catalytic enzyme of replication and transcription. We demonstrated previously that BDV L localizes to the nucleus of BDV-infected cells and L-transfected cells. Nuclear localization of the protein presupposes the presence of a nuclear localization signal (NLS) within its primary amino acid sequence or cotransport to the nucleus with another karyophilic protein. Because L localized to the nucleus in the absence of other viral proteins, we investigated the possibility that L contains an NLS. The minimal sequence required for nuclear localization of L was identified by analyzing the subcellular distribution of deletion mutants of L fused to a flag epitope tag or beta-galactosidase. Although the majority of the L fusion proteins localized to the cytoplasm of transfected BSR-T7 cells, a strong NLS (844RVVKLRIAP852) with basic and proline residues was identified. Mutation of this sequence resulted in cytoplasmic distribution of L, confirming that this sequence was necessary and sufficient to drive the nuclear localization of L.
Emerging Diseases Laboratory, Department of Neurology, University of California-Irvine, 92697-4292, USA.
Michelle Portlance
MP
Walker
W Ian
WI
Lipkin
D000595:Amino Acid Sequence; D001483:Base Sequence; D001891:Borna disease virus / Q000201:enzymology* / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D006801:Humans; D019913:Nuclear Localization Signals / Q000235:genetics*; D010449:Peptide Mapping; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion; D001616:beta-Galactosidase / Q000235:genetics
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D012324:RNA-Dependent RNA Polymerase; D001616:beta-Galactosidase