Identification of the nuclear localization signals within the Epstein-Barr virus EBNA-6 protein

• Published in: Journal of general virology 2004-01, Vol.85 (1), p.165-172
• Main Authors: Krauer, Kenia, Buck, Marion, Flanagan, James, Belzer, Deanna, Sculley, Tom
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Cell Nucleus - metabolism; EBNA-6 protein; Epstein-Barr virus; Epstein-Barr Virus Nuclear Antigens - chemistry; Epstein-Barr Virus Nuclear Antigens - genetics; Epstein-Barr Virus Nuclear Antigens - metabolism; Fundamental and applied biological sciences. Psychology; Gene Deletion; Green Fluorescent Proteins; HeLa Cells; Herpesvirus 4, Human - metabolism; Humans; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Microbiology; Microscopy, Confocal; Miscellaneous; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Localization Signals; Recombinant Fusion Proteins; Virology
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/vir.0.19549-0

Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston Road, Brisbane 4029, Queensland, Australia Correspondence Tom Sculley tomS{at}qimr.edu.au 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.