title: Functional characterization of nuclear trafficking signals in pseudorabies virus pUL31.
abstract: The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes.
  
  
  OBJECTIVE
  The molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Paßvogel
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Barbara G
authors.initials: BG
authors.lastname: Klupp
authors.affiliation: Institute of Infectology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Granzow
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Fuchs
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany thomas.mettenleiter@fli.bund.de.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Mettenleiter
mesh_terms: D000818:Animals; D002460:Cell Line; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D011485:Protein Binding; D021381:Protein Transport; D011817:Rabbits; D017384:Sequence Deletion; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; C000592811:UL31 protein, pseudorabies virus; C403420:UL34 protein, Human herpesvirus 1; D014764:Viral Proteins