Characterization of the nuclear localization and nuclear export signals of bovine herpesvirus 1 VP22.
The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130PRPR133, was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130PRPR133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204LDRMLKSAAIRIL216, was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.
Zheng
Chunfu
C
Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, Saskatchewan S7N 5E3, Canada.
Brownlie
Robert
R
Babiuk
Lorne A
LA
van Drunen Littel-van den Hurk
Sylvia
S
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics; D007242:Herpesvirus 1, Bovine / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000821:virology; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004279:DNA, Viral; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C434023:tegument protein VP22, bovine herpesvirus 1; C054562:yellow fluorescent protein, Bacteria