Characterization of nuclear localization and export signals of the major tegument protein VP8 of bovine herpesvirus-1.
Bovine herpesvirus-1 (BHV-1) VP8 is found in the nucleus immediately after infection. Transient expression of VP8 fused to yellow fluorescent protein (YFP) in COS-7 cells confirmed the nuclear localization of VP8 in the absence of other viral proteins. VP8 has four putative nuclear localization signals (NLS). Deletion of pat4 ((51)RRPR(54)) or pat7 ((48)PRVRRPR(54)) NLS2 abrogated nuclear accumulation, whereas deletion of (48)PRV(50) did not, so pat4 NLS2 is critical for nuclear localization of VP8. Furthermore, NLS1 ((11)RRPRR(15)), pat4 NLS2, and pat7 NLS2 were all capable of transporting the majority of YFP to the nucleus. Finally, a 12-amino-acid peptide with the sequence RRPRRPRVRRPR directed all of YFP into the nucleus, suggesting that reiteration of the RRPR motif makes the nuclear localization more efficient. Heterokaryon assays demonstrated that VP8 is also capable of shuttling between the nucleus and cytoplasm of the cell. Deletion mutant analysis revealed that this property is attributed to a leucine-rich nuclear export sequence (NES) consisting of amino acids (485)LSAYLTLFVAL(495). This leucine-rich NES caused transport of YFP to the cytoplasm. These results demonstrate that VP8 shuttles between the nucleus and cytoplasm.
Zheng
Chunfu
C
Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3.
Brownlie
Robert
R
Babiuk
Lorne A
LA
van Drunen Littel-van den Hurk
Sylvia
S
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D007242:Herpesvirus 1, Bovine / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport; D016415:Sequence Alignment; D014760:Viral Fusion Proteins / Q000378:metabolism
D036022:Capsid Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; C442561:VP8 protein, Bovine herpesvirus 1; D014760:Viral Fusion Proteins