Identification of the nuclear localization signals within the Epstein-Barr virus EBNA-6 protein.
Epstein-Barr virus nuclear antigen (EBNA)-6 is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. Previous studies have shown that EBNA-6 acts as a transcriptional regulator of viral and cellular genes; however at present, few functional domains of the 140 kDa EBNA-6 protein have been completely characterized. There are five computer-predicted nuclear localization signals (NLS), four monopartite and one bipartite, present in the EBNA-6 amino acid sequence. To identify which of these NLS are functional, fusion proteins between green fluorescent protein and deletion constructs of EBNA-6 were expressed in HeLa cells. Each of the constructs containing at least one of the NLS was targeted to the nucleus of cells whereas a construct lacking all of the NLS was cytoplasmic. Site-directed mutation of these NLS demonstrated that only three of the NLS were functional, one at the N-terminal end (aa 72-80), one in the middle (aa 412-418) and one at the C-terminal end (aa 939-945) of the EBNA-6 protein.
Krauer
Kenia
K
Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston Road, Brisbane 4029, Queensland, Australia.
Buck
Marion
M
Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston Road, Brisbane 4029, Queensland, Australia.
Flanagan
James
J
Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston Road, Brisbane 4029, Queensland, Australia.
Belzer
Deanna
D
Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston Road, Brisbane 4029, Queensland, Australia.
Sculley
Tom
T
Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston Road, Brisbane 4029, Queensland, Australia.
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins
D019309:Epstein-Barr Virus Nuclear Antigens; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins