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Short Communication |
1 Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Queensland 4029, Australia
2 Griffith Medical Research Centre, 300 Herston Road, Brisbane, Queensland 4029, Australia
Correspondence
Anita Burgess
anitaB{at}qimr.edu.au
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ABSTRACT |
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) and is also associated with several human malignancies, including Burkitt's lymphoma (BL), nasopharyngeal carcinoma and immunoblastic B-cell lymphomas in immunocompromised individuals (Macsween & Crawford, 2003). EBV infections result in a lifelong carrier state whereby the virus exists in a latent state in B cells. EBV is able to efficiently transform and immortalize human B cells in vitro, resulting in the generation of lymphoblastoid cell lines. Despite the presence of the complete viral genome in EBV-immortalized B lymphocytes, only a limited number of viral genes are expressed. These include the latent proteins EBV nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C and LP, two latent membrane proteins (LMP-1 and -2) and the EBER RNAs and the BamHI A rightward transcripts (BARTs).
EBNA3A, 3B and 3C share a similar genomic organization, they have molecular masses between 135 and 165 kDa and are hydrophilic, proline-rich, charged proteins. Each of the EBNA3 proteins interacts with the DNA-binding protein RBP-J
/RBP-2N (also known as CBF1) (Johannsen et al., 1996; Krauer et al., 1996; Robertson et al., 1995; Young et al., 1997), indicating that they play a role in transcriptional regulation (Krauer et al., 1998; Marshall & Sample, 1995). The EBNA3B protein is non-essential for EBV-mediated B-cell growth transformation in vitro (Hsu et al., 2005), although the persistent expression of EBNA3B against negative selective pressure by cytotoxic T cells in vivo is consistent with an important role for this gene product. EBNA3B has been shown to be capable of disrupting a drug-induced G2/M checkpoint (Krauer et al., 2004b), to upregulate expression of the cytoskeletal protein vimentin and the activation antigen CD40 and to cause downregulation of the BL-associated antigen BLA (CD77) (Silins & Sculley, 1994, 1995).
The EBNA3B protein is targeted exclusively to the cell nucleus and localization studies have shown that it localizes to discrete subnuclear granules within the cell nucleus (Petti et al., 1990; Sample & Kieff, 1990). Following translation, nuclear proteins are imported from the cytoplasm through the nuclear-pore complex into the nucleus. Proteins must contain, or be bound to proteins that contain, at least one nuclear-localization signal (NLS) to permit transport into the nucleus. Proteins lacking these recognition signals remain cytoplasmic or may be directed to other cellular compartments. Three main types of NLS have been described. These are generally rich in the basic amino acids lysine and arginine and often contain proline. The first category of NLS consists of one of two types (pattern 4 or pattern 7). Pattern 4 is composed of either four basic residues or three basic residues and either a proline or a histidine. Pattern 7 NLSs are 7 aa in length, they begin with a proline and contain four basic residues. The second category of NLS is a bipartite NLS (Suzuki et al., 1995), which begins with two basic residues followed by a 10-residue spacer and then a basic region containing at least three basic residues in the last 5 aa. The third type of NLS is an N-terminal signal found in the yeast protein Mat alpha2. This NLS has not been well studied to date and is not included as yet in current NLS-prediction programs. In this study, we have defined the NLSs present within the EBNA3B protein and demonstrated that EBNA3B co-localizes in the nucleus with both EBNA3A and EBNA3C.
Four potential NLSs, as defined by the Nakai consensus (Nakai & Kanehisa, 1992), were identified within the EBNA3B protein sequence by using PSORT II (Nakai & Kanehisa, 1992; Nakai & Horton, 1999). NLS-A, a pattern 4 sequence NLS, is located within the N-terminal region at aa 155–158 (RKKP). A second NLS located within close proximity to the first, NLS-B, is present at aa 160–166 (PIVKQRR) and is a pattern 7 NLS. NLS-C is located at aa 430–434 (HRKKK) and comprises two overlapping pattern 4 sequences, whereas NLS-D, aa 867–874 (PPSKRAKI), consists of two overlapping pattern 7 sequences.
Deletion constructs of EBNA3B, linked to enhanced green fluorescent protein (EGFP), were prepared such that they covered the entire EBNA3B sequence. Each of the constructs contained at least one of these predicted NLSs except for one construct (pEGFPC3–EBNA3B
1–532/775–938) that lacked all computer-predicted NLSs. The integrity of each of the constructs was determined by DNA sequencing and immunoblotting (data not shown). To ensure easy visualization of both the nucleus and cytoplasm, all of the constructs were expressed transiently in HeLa cells by using ExGen 500 (Promega) according to the manufacturer's instructions and the cellular location of the fusion proteins was determined by confocal microscopy (Leica, model TCS SP2) 24 h after transfection. In contrast to EGFP alone, which was distributed diffusely in both the cytoplasm and the nucleus, each of the proteins containing a predicted NLS localized to the nuclei of cells, whereas the protein encoded by pEGFPC3–EBNA3B1–532/775–938 did not contain a predicted NLS and was mostly excluded from the nucleus (Fig. 1).
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with some modifications. Briefly, a PCR was performed with 10–100 ng plasmid DNA and 125 ng each primer using Pfu Turbo polymerase. Following the PCR, plasmid DNA was digested with 20 U DpnI for 3 h at 37 °C. The PCR mixture (4 µl) was then transformed into Escherichia coli XL-1 Blue competent cells by using electroporation and the bacteria were then plated onto LB plates containing kanamycin. Resultant bacterial colonies were selected, plasmid DNA was prepared and confirmation of mutated bases was determined by restriction-enzyme digestion and DNA sequencing. Details of primers used for site-directed mutagenesis are available from the authors on request.
Initially, combinations of the first two predicted NLSs (NLS-A and -B) within the protein encoded by pEGFP–EBNA3B335–938 were mutated. Each of the mutated constructs was transiently transfected into HeLa cells and the cellular localization of the mutant proteins was determined by confocal microscopy (Fig. 2a). Mutagenesis of NLS-A or NLS-B alone or in combination was insufficient to prevent the protein from localizing to the nucleus, indicating that an additional NLS must be present. Analysis of the amino acid sequence by eye identified a motif similar to a pattern 4 NLS at aa 243–246 (RRAR). This was the only region likely to be able to act as an NLS within this sequence. Mutagenesis of the RRAR region to GGAG in the pEGFP–EBNA3B335–938 construct, which had NLS-A and NLS-B already mutated, resulted in complete cytoplasmic distribution of the protein, demonstrating that the RRAR motif was a functional NLS. To determine whether either NLS-A or NLS-B was also functional, mutagenesis of NLS-A and NLS-B was performed in the pEGFP–EBNA3B335–938 construct, which already had the RRAR motif mutated to GGAG. Analysis of the cellular distribution of the expressed proteins demonstrated that NLS-A was non-functional, whereas NLS-B was a functional NLS.
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1–327/533–938 plasmid to change two lysine residues to alanine residues (Fig. 2b
). Prior to mutation, this construct localized almost exclusively to the nucleus, whereas following mutagenesis, there was an increase in the amount of protein localizing to the cytoplasm. Additional residues were then mutated such that all four basic residues were changed to alanines, which resulted in the mutated protein residing primarily in the cytoplasm, showing that NLS-C was also functional.
NLS-D was mutated within the pEGFPC1–EBNA3B1–775 plasmid by changing the three basic residues to alanines (Fig. 2c). This resulted in exclusion of the protein from the nucleus and demonstrated that this NLS was functional. The location of each of the identified NLSs within the EBNA-3B protein sequence is shown in Fig. 3(c).
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The EBNA3 proteins are targeted exclusively to the cell nucleus, are present in the nucleoplasm and localize to discrete subnuclear bodies within the cell nucleus. Plasmid constructs of each of the EBNA3s, either FLAG-tagged or linked to EGFP, were prepared and were co-transfected into HeLa cells (Fig. 3). The FLAG–EBNA3A1–279 construct was used for co-localization studies, as this construct has less-diffuse staining than the full-length protein and we have shown that both the full-length protein and FLAG–EBNA3A1–279 protein localize to the same structures in the nucleus (data not shown). The expressed, tagged EBNA3 proteins were visualized by immunofluorescence using anti-FLAG antibody (Sigma) diluted 1 : 1000 in 10 % fetal calf serum/PBS followed by anti-mouse–Cy3 secondary antibody (Jackson ImmunoResearch) diluted 1 : 100. Cells were examined by confocal microscopy and showed co-localization of EBNA3B and 3C in all cells examined, and extensive co-localization between EBNA3B and 3A in all cells examined. Whilst there was co-localization, there were always regions of EBNA3A and 3C that did not coincide with EBNA3B (arrowed in Fig. 3). These results demonstrate that a significant proportion of the EBNA3B protein localizes to the same nuclear structures as EBNA3A and 3C when overexpressed following transient transfection.
Whilst EBNA3B is not required for transformation of B lymphocytes by EBV, it is one of several EBV-encoded proteins thought to be involved in transcriptional regulation of both viral and cellular genes and is able to interact with the DNA-binding protein RBP-J (Krauer et al., 1996). It has been shown to upregulate expression of vimentin, CD40 and bcl-2 (Silins & Sculley, 1994, 1995) and to be capable of disrupting a drug-induced G2/M checkpoint (Krauer et al., 2004b). EBNA3B is related to EBNA3A and EBNA3C, both of which are essential for EBV transformation of B lymphocytes. EBNA3B is found in both type I and type II EBV strains and has homologues in members of the genus Lymphocryptovirus, found to infect Old World primates (Jiang et al., 2000). It has therefore been conserved throughout evolution of the virus and probably plays an important role in the viral life cycle.
By utilizing computer programs, four NLSs were identified within the EBNA3B sequence. The data presented here show that only three of the four predicted NLSs were able to direct truncated forms of EBNA3B to the nucleus. In addition to the computer-predicted NLSs, an additional NLS was identified at aa 243–246 in the N-terminal region of the EBNA3B protein. Mutagenesis of all four identified NLSs within the full-length EBNA3B protein resulted in the protein being restricted to the cytoplasm of cells, demonstrating that there were no additional, unidentified NLSs present in the EBNA3B protein. However, two of the NLSs were unable to direct EBNA3B to the nucleus of cells, suggesting that they may be buried within the protein. However, as these sequences are able to direct deletion constructs of EBNA3B to the nucleus of cells, they have been listed as NLSs in the EBNA3B sequence (Fig. 3c). The NLS-1 sequence in EBNA3B was found to be partially functional, resulting in nuclear accumulation in approximately half of the cells expressing EBNA3B. This NLS falls within the region of EBNA3B that is thought to be involved in its interaction with RBP-J and it is possible that this interaction may mask the NLS.
Like EBNA3B, both EBNA3C (Krauer et al., 2004a) and EBNA3A have multiple NLSs. Multiple NLSs may enhance their function or have different specificities in different cell types or function under different conditions (Knauf et al., 1996; Liu et al., 1998; Roberts et al., 1987). The EBNA3B protein has not been studied extensively and, therefore, there are few data concerning binding partners of EBNA3B. However, EBNA3B has homology with EBNA3A and 3C and, as these proteins bind multiple partners, it is likely that EBNA3B will have a similar capacity. Interaction of EBNA3B with protein partners may prevent an NLS from functioning, which may be the case for NLS-1, and therefore multiple NLSs may be required within EBNA3B to ensure that it localizes to the nucleus, even when bound to other proteins.
All three members of the EBNA-3 family are found exclusively in the cell nucleus, but are excluded from the nucleolus (Petti et al., 1990). It is known that the EBNA-3 proteins bind to specific nuclear structures, but not whether they each bind to the same structures. EBNA3A and EBNA3C have been shown to co-localize to nuclear structures (Krauer et al., 2004c) and this study shows that EBNA3B also associates with EBNA3A and 3C within a subset of nuclear structures when overexpressed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 October 2005;
accepted 29 November 2005.
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