title: Dissection of a novel nuclear localization signal in open reading frame 29 of varicella-zoster virus.
abstract: Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin alpha- and beta-dependent mechanism. These data are further supported by the demonstration that a glutathione S-transferase-karyopherin alpha fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin alpha. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.
authors.affiliation: Integrated Program in Cellular, Molecular and Biophysical Studies and the Department of Microbiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Stallings
authors.forename: Saul
authors.initials: S
authors.lastname: Silverstein
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005982:Glutathione Transferase / Q000378:metabolism; D006562:Herpes Zoster / Q000821:virology*; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000502:physiology; D008970:Molecular Weight; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology*; D028861:Nuclear Pore Complex Proteins / Q000502:physiology; D016366:Open Reading Frames; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000502:physiology; D028901:alpha Karyopherins / Q000378:metabolism / Q000502:physiology; D028961:beta Karyopherins / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D014764:Viral Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C094493:ran-binding protein 2; D005982:Glutathione Transferase