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Short Communication |
1 Queensland Institute of Medical Research and Griffith Medical Research Centre, Griffith University, 300 Herston Road, Brisbane, QLD 4029, Australia
2 Queensland University of Technology, School of Life Sciences, GPO Box 2434, Brisbane, QLD 4001, Australia
Correspondence
Anita Burgess
Anita.Burgess{at}qimr.edu.au
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Present address: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia. 
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). Transformation of B cells by EBV in vitro generates continuously proliferating lymphoblastoid cell lines, where manipulation of the host cells' biochemical and signalling pathways may lead to oncogenic changes in these cells (Young & Murray, 2003). The entire EBV genome encodes more than 100 gene products; however, only nine viral proteins are expressed in EBV-transformed cells and, therefore, these proteins are thought to be responsible for the transformation process (Okano & Gross, 1996; Rowe et al., 1989). Significantly, the use of recombinant viruses has shown that only six of these proteins are essential for transforming B cells, including Epstein–Barr nuclear antigen 3A (also known as EBNA3) (Tomkinson et al., 1993). EBNA3A, a nuclear phosphoprotein of 944 aa, has been shown to interact with RBP-J
, a cellular transcription factor in the Notch pathway that regulates lymphoid development and function (Tamura et al., 1995; Zhao et al., 1996). More recently, EBNA3A has been shown to bind a transcriptional regulator called the co-repressor carboxyl terminal-binding protein (CtBP), as well as the cell-cycle protein Chk2 (Krauer et al., 2004a; Touitou et al., 2001). EBNA3A is targeted to the nucleus and associates with nuclear substructures, processes that are likely to be important for the function of EBNA3A and fundamental to the transformation process (Cludts & Farrell, 1998; Tomkinson et al., 1993).
Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC) and macromolecules larger than 40–60 kDa can only enter the nucleus by active transport, a process commonly facilitated by soluble nuclear receptors. There are three main classes of nuclear receptors; however, they all operate via a similar mechanism involving recognition of a protein's nuclear-localization signal (NLS) sequence. The receptor–protein complex then interacts with components of the NPC to allow the passage of receptor–protein complexes (Weis, 2003). Classical NLS sequences are often short (fewer than 12 aa) and usually consist of one (monopartite) or two (bipartite) clusters of basic amino acids (Hodel et al., 2001), although glycine-rich regions (Cokol et al., 2000) and RGR motifs (Claus et al., 2003) have also been shown to facilitate nuclear localization. Two of the most common types of NLS are pattern 4 and pattern 7. Pattern 4 is composed of either four basic residues or three basic residues and either a proline or histidine. Pattern 7 NLSs begin with a proline and are followed within three residues by three out of four basic residues. Le Roux et al. (1993) used deletion constructs of EBNA3A to identify an NLS essential for the nuclear localization of EBNA3A. However, Krauer et al. (2004b) have shown that an EBNA3A mutant that did not contain the NLS identified by Le Roux et al. (1993) was still targeted to the nucleus, suggesting that the EBNA3A protein contained more than one functional NLS. In this study, we have identified five additional functional NLSs within the EBNA3A protein.
Le Roux et al. (1993) showed that a motif of 10 aa (RDRRRNPASR) between residues 146 and 155 of EBNA3A was involved in the nuclear localization of the protein. These authors suggested that the EBNA3A protein only contained this one functional NLS. However, expression of a truncated EBNA3A280–944 protein [that does not contain the NLS defined by Le Roux et al. (1993)] was shown previously to be nuclear in HeLa cells (Krauer et al., 2004b), indicating that there must be an additional NLS(s) present within the EBNA3A protein. To identify other potential NLSs within EBNA3A, an analysis was performed by using the PSORT II program (Nakai & Horton, 1999). The PSORT II program identified two potential NLSs in EBNA3A; a pattern 4 NLS (aa 63–66, NLS1) and a pattern 7 NLS (aa 375–381, NLS3). It is noteworthy that the program did not recognize the functional NLS identified by Le Roux et al. (1993) as a consensus NLS. To identify additional NLSs within EBNA3A, a series of deletion constructs linked to enhanced green fluorescent protein (EGFP) was prepared. These constructs were transfected into HeLa cells and their subcellular localization was determined by confocal microscopy (Fig. 1). All expressed proteins were found in the nucleus of cells (EGFP–EBNA3A820–944 was both cytoplasmic and nuclear, with a pattern similar to that of EGFP alone). These results showed that there must be at least two additional NLSs present in EBNA3A, as both the EGFP–EBNA3A280–567 and EGFP–EBNA3A568–819 proteins were nuclear. The integrity of each of the constructs was determined by DNA sequencing and immunoblotting (Fig. 2).
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). Examination of the amino acid sequence of EBNA3A280–567 revealed the presence of another motif (RAGKR) that could possibly function as an NLS. The RAGKR motif was mutated, alone and in combination with NLS3, and the resulting mutated proteins were expressed in HeLa cells and their localization was determined by confocal microscopy (Fig. 3a). The results show that mutation of either NLS3 or the RAGKR motif (hence referred to as NLS4) alone was not sufficient to exclude the EBNA3A280–567 protein from the nucleus, whereas mutation of both motifs resulted in the EBNA3A280–567 protein being restricted to the cytoplasm of cells, demonstrating that both motifs were functional NLSs.
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); however, there were ten arginine residues present within the first 24 aa of the EBNA3A568–819 sequence, suggesting that this was probably the region responsible for the nuclear localization of the protein. PCR was used to generate an EGFP–EBNA3A592–819 protein (missing the first 24 aa of EGFP–EBNA3A568–819) and this protein was expressed in HeLa cells and was found to be present in both the cytoplasm and nucleus, in a pattern similar to that observed for EGFP alone (data not shown). This indicated that there was an NLS(s) present within aa 568–592 of EBNA3A. Examination of aa 568–592 revealed two sequences (RRAR and RDRLAR) that could potentially act as NLSs. Each of these sequences was mutated alone or together and the mutated proteins were expressed in HeLa cells and their localization was determined by confocal microscopy. Mutation of either NLS sequence alone resulted in only a small amount of the protein being present in the cytoplasm of cells [indicated by an arrow in Fig. 3(b)
], whereas mutation of both sequences resulted in the protein being excluded from the nucleus, demonstrating that both the RRAR and RDRLAR sequences (hence referred to as NLS5 and NLS6, respectively) were capable of functioning as NLSs (Fig. 3b).
Next, we determined whether NLS1 was also functional. Because of a lack of appropriate restriction-enzyme sites within the 5' region of the EBNA3A gene sequence, site-directed mutagenesis was utilized to introduce stop codons into this region. Three consecutive stop codons were introduced into the pEGFP–EBNA3A plasmid following nt 417 of EBNA3A, resulting in expression of the first 139 aa of EBNA3A (EGFP–EBNA3A1–139), which contained NLS1, but not the NLS defined by Le Roux et al. (1993). Next, the NLS1 sequence was mutated in the EBNA3A1–139 protein from KRKR to KGGG by using site-directed mutagenesis. All mutations were verified by DNA sequencing. Each construct was transfected into HeLa cells and the cellular localization of the expressed EGFP-tagged proteins was determined by confocal microscopy. The EGFP–EBNA3A1–139 protein was found to be nuclear, whilst mutation of NLS1 within EGFP–EBNA3A1–139 resulted in the protein being excluded from the nucleus, demonstrating that NLS1 was indeed functional (Fig. 3c).
EBNA3A is a hydrophilic, proline-rich, charged protein that is able to interact with the DNA-binding protein RBP-J/RBP-2N (also known as CBF1) (Johannsen et al., 1996; Krauer et al., 1996; Robertson et al., 1996; Young et al., 1997), which has led to the suggestion that it plays a role in transcriptional regulation (Krauer et al., 1998; Marshall & Sample, 1995). EBNA3A is essential for in vitro transformation of B lymphocytes and, by using a yeast two-hybrid system, EBNA3A has been shown to bind to proteins such as Xap-2, also known as the p38 subunit of the aryl hydrocarbon receptor complex, and the epsilon subunit of the chaperonin-containing T-complex protein 1 (Kashuba et al., 2000). Xap-2 is preferentially cytoplasmic, but was found to translocate to the nucleus upon expression of EBNA3A. Also, EBNA3A has been found to interact with a novel human uridine kinase/uracil phosphoribosyltransferase, which also translocates to the nucleus upon co-expression of EBNA3A (Kashuba et al., 2002). Hickabottom et al. (2002) demonstrated that EBNA3A could also bind to CtBP, a cellular protein that is important in the regulation of the cell cycle and in the development and transformation of cells. These authors showed that EBNA3A was able to cooperate with activated Ras to transform rodent fibroblasts and that this transformation effect was dependent on the EBNA3A–CtBP physical interaction.
EBNA3A is targeted exclusively to the cell nucleus and localizes to discrete subnuclear granules within the cell nucleus (Petti et al., 1990). Le Roux et al. (1993) showed that a motif of 10 aa (RDRRRNPASR) between residues 146 and 155 was involved in the nuclear localization of EBNA3A and they suggested that this was the only NLS within EBNA3A. However, an EGFP-tagged EBNA3A280–944 protein lacking the NLS identified by Le Roux and colleagues was targeted to the nucleus, indicating that EBNA3A must contain an additional NLS(s) (Fig. 1). Generation of a series of EGFP–EBNA3A deletion mutants showed that there were at least two additional NLSs present in EBNA3A, as both the EGFP–EBNA3A280–567 and EGFP–EBNA3A568–819 proteins were targeted independently to the nucleus. The PSORT II program, an algorithm that uses a database of experimentally identified amino acid sequence motifs that direct proteins to their proper subcellular compartment (Nakai & Horton, 1999), identified two NLSs in EBNA3A: a pattern 4 NLS (residues 63–66) and a pattern 7 NLS (residues 375–381), both of which were shown to be functional (Fig. 3). Even though experimental data for the identification of NLSs are expanding rapidly, the diverse range of sequences that can act as an NLS makes producing a definitive list of consensus NLS sequences difficult (Hodel et al., 2001) and three of the NLSs defined within EBNA3A in this study, as well as the NLS defined by Le Roux et al. (1993), were not detected by the PSORT II program.
Two types of EBV exist (type I and type II) that show sequence divergence within the genes encoding the EBNA-LP, -2, -3A, -3B and -3C gene products (Adldinger et al., 1985; Dambaugh et al., 1984; Sample et al., 1986; Sculley et al., 1989). As EBNA3A is a nuclear protein, it might be expected that there would be conservation of the functional NLSs between the two virus types. Computer analysis of the Ag-876 type II EBNA3A protein sequence showed conservation of all the identified NLSs except NLS4, suggesting that the type II EBNA3A protein probably only has five functional NLSs. It is not understood why a single protein contains multiple NLSs; however, EBNA3A has multiple binding partners, with many of these partner proteins being present in the cytoplasm and recruited to the nucleus in the presence of EBNA3A. Under these circumstances, it is possible that interaction with cytoplasmic proteins may mask one or more of the NLSs in the EBNA3A protein and multiple NLSs would then be required to ensure that EBNA3A is targeted efficiently to the nucleus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Claus, P., Döring, F., Gringel, S., Müller-Ostermeyer, F., Fuhlrott, J., Kraft, T. & Grothe, C. (2003). Differential intranuclear localization of fibroblast growth factor-2 isoforms and specific interaction with the survival of motoneuron protein. J Biol Chem 278, 479–485.
Cludts, I. & Farrell, P. J. (1998). Multiple functions within the Epstein-Barr virus EBNA-3A protein. J Virol 72, 1862–1869.
Cokol, M., Nair, R. & Rost, B. (2000). Finding nuclear localization signals. EMBO Rep 1, 411–415.[CrossRef][Medline]
Dambaugh, T., Hennessy, K., Chamnankit, L. & Kieff, E. (1984). U2 region of Epstein–Barr virus DNA may encode Epstein–Barr nuclear antigen 2. Proc Natl Acad Sci U S A 81, 7632–7636.
Hickabottom, M., Parker, G. A., Freemont, P., Crook, T. & Allday, M. J. (2002). Two nonconsensus sites in the Epstein-Barr virus oncoprotein EBNA3A cooperate to bind the co-repressor carboxyl-terminal-binding protein (CtBP). J Biol Chem 277, 47197–47204.
Hodel, M. R., Corbett, A. H. & Hodel, A. E. (2001). Dissection of a nuclear localization signal. J Biol Chem 276, 1317–1325.
Johannsen, E., Miller, C. L., Grossman, S. R. & Kieff, E. (1996). EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJ in Epstein-Barr virus-transformed B lymphocytes. J Virol 70, 4179–4183.[Abstract]
Kashuba, E., Kashuba, V., Pokrovskaja, K., Klein, G. & Szekely, L. (2000). Epstein–Barr virus encoded nuclear protein EBNA-3 binds XAP-2, a protein associated with hepatitis B virus X antigen. Oncogene 19, 1801–1806.[CrossRef][Medline]
Kashuba, E., Kashuba, V., Sandalova, T., Klein, G. & Szekely, L. (2002). Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase. BMC Cell Biol 3, 23–23.[CrossRef][Medline]
Krauer, K. G., Kienzle, N., Young, D. B. & Sculley, T. B. (1996). EpsteinBarr nuclear antigen-3 and -4 interact with RBP-2N, a major isoform of RBP-J in B lymphocytes. Virology 226, 346–353.[CrossRef][Medline]
Krauer, K. G., Belzer, D. K., Liaskou, D., Buck, M., Cross, S., Honjo, T. & Sculley, T. (1998). Regulation of interleukin-1
transcription by Epstein–Barr virus involves a number of latent proteins via their interaction with RBP. Virology 252, 418–430.[CrossRef][Medline]
Krauer, K. G., Burgess, A., Buck, M., Flanagan, J., Sculley, T. & Gabrielli, B. (2004a). The EBNA-3 gene family proteins disrupt the G2/M checkpoint. Oncogene 23, 1342–1353.[CrossRef][Medline]
Krauer, K. G., Buck, M., Belzer, D. K., Flanagan, J., Chojnowski, G. M. & Sculley, T. B. (2004b). The Epstein–Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein. Virology 318, 280–294.[CrossRef][Medline]
Le Roux, A., Berebbi, M., Moukaddem, M., Perricaudet, M. & Joab, I. (1993). Identification of a short amino acid sequence essential for efficient nuclear targeting of the Epstein-Barr virus nuclear antigen 3A. J Virol 67, 1716–1720.
Macsween, K. F. & Crawford, D. H. (2003). Epstein-Barr virus – recent advances. Lancet Infect Dis 3, 131–140.[CrossRef][Medline]
Marshall, D. & Sample, C. (1995). Epstein-Barr virus nuclear antigen 3C is a transcriptional regulator. J Virol 69, 3624–3630.[Abstract]
Nakai, K. & Horton, P. (1999). PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24, 34–35.[CrossRef][Medline]
Okano, M. & Gross, T. (1996). Epstein-Barr virus-associated hemophagocytic syndrome and fatal infectious mononucleosis. Am J Hematol 53, 111–115.[CrossRef][Medline]
Petti, L., Sample, C. & Kieff, E. (1990). Subnuclear localization and phosphorylation of Epstein-Barr virus latent infection nuclear proteins. Virology 176, 563–574.[CrossRef][Medline]
Robertson, E., Lin, J. & Kieff, E. (1996). The amino-terminal domains of Epstein-Barr virus nuclear proteins 3A, 3B, and 3C interact with RBPJ. J Virol 70, 3068–3074.[Abstract]
Rowe, M., Young, L. S., Cadwallader, K., Petti, L., Kieff, E. & Rickinson, A. B. (1989). Distinction between Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 2B) isolates extends to the EBNA 3 family of nuclear proteins. J Virol 63, 1031–1039.
Sample, J., Hummel, M., Braun, D., Birkenbach, M. & Kieff, E. (1986). Nucleotide sequences of mRNAs encoding Epstein–Barr virus nuclear proteins: a probable transcriptional initiation site. Proc Natl Acad Sci U S A 83, 5096–5100.
Sculley, T. B., Apolloni, A., Stumm, R., Moss, D. J., Mueller-Lantzsch, N., Misko, I. S. & Cooper, D. A. (1989). Expression of Epstein-Barr virus nuclear antigens 3, 4, and 6 are altered in cell lines containing B-type virus. Virology 171, 401–408.[CrossRef][Medline]
Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T. & Honjo, T. (1995). Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J/Su(H). Curr Biol 5, 1416–1423.[CrossRef][Medline]
Tomkinson, B., Robertson, E. & Kieff, E. (1993). Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. J Virol 67, 2014–2025.
Touitou, R., Hickabottom, M., Parker, G., Crook, T. & Allday, M. J. (2001). Physical and functional interactions between the corepressor CtBP and the Epstein-Barr virus nuclear antigen EBNA3C. J Virol 75, 7749–7755.
Weis, K. (2003). Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112, 441–451.[CrossRef][Medline]
Young, L. S. & Murray, P. G. (2003). Epstein–Barr virus and oncogenesis: from latent genes to tumours. Oncogene 22, 5108–5121.[CrossRef][Medline]
Young, D. B., Krauer, K. G., Kienzle, N. & Sculley, T. B. (1997). Both A type and B type Epstein–Barr virus nuclear antigen 6 interact with RBP-2N. J Gen Virol 78, 1671–1674.[Abstract]
Zhao, B., Marshall, D. R. & Sample, C. E. (1996). A conserved domain of the Epstein-Barr virus nuclear antigens 3A and 3C binds to a discrete domain of J. J Virol 70, 4228–4236.[Abstract]
Received 9 February 2006;
accepted 30 May 2006.
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1–279/568–944 plasmid expressing the EGFP–EBNA3A280–567 protein, as well as the pEGFP–EBNA3A