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
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ABSTRACT |
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INTRODUCTION |
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Analysis of the EBNA-6 amino acid sequence has revealed features common to many viral and cellular transcription factors. These include a region which resembles a basic DNA-binding domain adjacent to a potential leucine-zipper motif (b-zip) and a transactivation domain rich in glutamine/proline residues, which has similarities to the mammalian transcription factor Sp1 (Bain et al., 1996; Radkov et al., 1997
; Marshall & Sample, 1995
). The EBNA-6 protein has also been identified as an immortalizing oncoprotein which can cooperate with activated (Ha-)ras in cotransformation assays and can override Rb-mediated pathways (Allday et al., 1993
). EBNA-6 is a hydrophilic, proline-rich, charged protein that is targeted exclusively to the cell nucleus, and cellular fractionation experiments have shown that EBNA-6 is associated with the nuclear matrix, and to a lesser extent is present in the nucleoplasm (Petti et al., 1990
). Sample & Kieff (1990)
showed that EBNA-6 localized to subnuclear granules within the cell nucleus.
Subsequent to translation, the fate of a protein depends largely on whether its amino acid sequence contains sorting signals directing the protein to specific cellular locations. The active transport of proteins in both directions across the nuclear envelope requires the presence of specific targeting sequences within the protein (Gorlich et al., 1996). There are at least three types of nuclear localization signals (NLS) on proteins and these signals are characteristically rich in the basic amino acids lysine and arginine and usually contain proline (Dingwall & Laskey, 1991
). The first type of NLS is a continuous stretch of amino acids, which can be located almost anywhere in the protein sequence. The archetypal NLS is that of the SV40 large tumour antigen (PPKKRKV) (Huber et al., 1996
). The second type of NLS is a bipartite sequence, or a signal patch, that consists of a three-dimensional arrangement on the protein surface. An example of this is Xenopus laevis nucleoplasmin, where there are two clusters of basic amino acid residues separated by an intervening 1012 aa spacer (Dingwall & Laskey, 1992
). The third type are less well conserved sequences with few basic residues such as that of the adenovirus E1A (KRPRP) (Lyons et al., 1987
). However, nuclear localization signals can be masked by phosphorylation close to or within an NLS. This phosphorylation inactivates the NLS through charge or conformational effects (Hennekes et al., 1993
; Ohta et al., 1989
). NLS are not cleaved off after transport into the nucleus, which is presumably because nuclear proteins need to be imported repeatedly, once after every cell division (Chaudhary & Courvalin, 1993
). Proteins containing NLS are recognized by the transport machinery and are imported through the nuclear pore complex, whereas proteins lacking NLS remain in the cytoplasm (Moll et al., 1991
).
EBNA-6 has several localized concentrations of arginine or lysine residues that are potential signatures of nuclear localization and although it is recognized that EBNA-6 is targeted to the nucleus, the NLS responsible have not been identified.
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METHODS |
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pBS-GFP-E6.
The EBNA-6 cDNA fragment (B95-8 virus) was excised from vector pGBT9-E6 (Young et al., 1997) using BsrGI and BstEII. The excised fragment was then Klenow treated and ligated into pBS-GFP that had previously been restricted with BsrGI and Klenow treated. (pBS-GFP was a gift from M. Vogel, Medicine, Microbiology and Hygiene, Regensburg University; pBS-GFP-E6 was originally prepared by D. Young, QIMR.)
pBS-GFPL.
The pBS-GFP plasmid was adapted to include a linker containing the restriction enzyme sites BsrGI, StuI, BamHI, EcoRV, HpaI, XhoI and NotI. This was prepared by annealing 1 µg of two self-complementary 41 base oligomers (Custom-made; Life Technologies, Australia) and then ligating this fragment into pBSGFP that had been restricted with BsrGI and NotI.
pBS-GFPL-E61206.
Plasmid pBS-GFP-E6 was cut with HpaI and NotI and the E6 fragment was inserted into pBS-GFPL cut with HpaI and NotI.
pBS-GFP-E61395/601992.
The pBS-GFPL-E61206 construct was digested with ClaI and NarI, resulting in a 618 bp fragment, which was Klenow treated and then ligated to pBSGFPL that had been HpaI restricted. This resulted in a fusion of GFP with aa 396600 of EBNA-6 containing NLS24.
pBSGFP-E6184992.
The vector pBS-GFP-E6 was digested with XbaI, Klenow treated, and then digested with EcoRV and religated resulting in a fusion of GFP with aa 22183 of EBNA-6 which contained NLS1.
pEBO-GFP-E6396625.
NLS24 were removed from EBNA-6 by excising a 696 bp fragment from the vector pBSGFP-E6 by restriction with ClaI and NarI. The restricted ends were then Klenow treated and religated. The plasmid was then digested with HindIII and NotI and the EBNA-6 region inserted into EBO-pLPP, restricted with the same enzymes. This resulted in removal of aa 396625 of EBNA-6.
pBS-GFPL-E61206/396625.
The pBSGFPL-E6396625 construct was digested with HpaI and NotI, and the resulting fragment was inserted into pBS-GFPL that had been restricted with HpaI and NotI. This resulted in the expression of a fusion of GFP with EBNA-6 aa 207395 joined with aa 626992 and contained NLS5.
pBS-GFP-E61206/396625/941992.
The 1·5 kb StuI fragment, excised from pBS-GFPL-E61206/396625, was then ligated into pBS-GFPL previously restricted with HpaI. This resulted in the expression of a GFP fusion linked to aa 207395 joined with aa 626940 of EBNA-6, which lacked all computer-predicted NLS.
Site directed mutagenesis.
In vitro mutagenesis of double-stranded DNA templates was performed as described in Sambrook & Russell (2001) with some modifications. Briefly, a PCR reaction was performed with 550 ng plasmid DNA and 125 ng of each primer using Pfu Turbo polymerase under the following buffer conditions: 20 mM Tris/HCl pH 8·8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0·1 % Triton X-100, 0·1 mg nuclease-free BSA ml-1 in a 50 µl reaction. Cycling conditions were as follows: 95 °C 30 s, 55 °C 1 min, 68 °C 2 min per kb of plasmid DNA for 1618 cycles, followed by 72 °C for 10 min. PCR primers are listed in Table 1
. Following the PCR reaction plasmid DNA was digested with 20 U DpnI for 3 h at 37 °C. The PCR mixture (4 µl) was then transformed into E. coli DH5
competent cells by electroporation and the bacteria were then plated onto LB plates containing ampicillin. Resultant bacterial colonies were selected, plasmid DNA was prepared and confirmation of mutagenized bases determined by restriction enzyme digestion and DNA sequencing. A list of the resultant NLS mutations is shown in Table 2
.
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Direct fluorescence microscopy.
HeLa cells expressing GFP-E6 fusion proteins were grown on coverslips and fixed with ice-cold acetone (2 min, -20 °C), and then mounted with Vectashield mounting medium onto glass slides. HeLa cells were scanned using either a Bio-Rad MRC 600 confocal microscope with images acquired using COMOS (Confocal Microscope Operating Software Version 6.03) or a Leica confocal microscope, model TCS SP2 and images recorded using Leica Confocal Software. The acquired images were then analysed and processed for presentation using CAS (Confocal Assistant Software Version 3.10), in which single optical sections are shown.
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RESULTS |
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Constructs of EBNA-6, linked to GFP, were prepared such that they contained at least one of these NLS, as well as one construct which lacked all of the computer-predicted NLS, to determine whether other previously unrecognized NLS were present in the protein. The integrity of the constructs was determined by DNA sequencing and immunoblotting. To ensure easy visualization of both the nucleus and cytoplasm the constructs were transfected into HeLa cells. All of the constructs were transiently expressed in HeLa cells and the cellular location of the fusion proteins was determined by confocal microscopy. In contrast to GFP alone, which was distributed diffusely in both the cytoplasm and the nucleus, each of the constructs containing a predicted NLS localized to the nucleus of cells. However, the fusion protein in which all computer-predicted NLS were removed (GFP-E61206/396625/941992), was present only in the cytoplasm of cells (Fig. 1
). These results indicated that the predicted NLS present in EBNA-6 were likely to be functional and that there were unlikely to be any additional NLS present within the EBNA-6 coding region (at least within the sequence included in GFP-E6
1206/396625/941992).
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DISCUSSION |
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The EBNA-6 protein is targeted exclusively to the cell nucleus, and cellular fractionation experiments have shown that it is present in the nucleoplasm and associates with the nuclear matrix while localization studies demonstrate that it localizes to discrete subnuclear granules within the cell nucleus (Petti et al., 1990; Sample & Kieff, 1990
). There are five potential NLS containing arginine and lysine residues within EBNA-6. The data presented here show that NLS1, NLS2 and NLS5 are all independently capable of targeting protein to the nucleus. The pattern 4 C-terminal NLS5 sequence (KRPR) is the shortest known nuclear targeting signal, and is also found in EBNA-1, EBNA-2, and in adenovirus E1A (Lyons et al., 1987
; Ambinder et al., 1991
; Le Roux et al., 1993
). The removal of all NLS (pGFP-E6
1206/396625/941992) resulted in the truncated EBNA-6 protein being present solely throughout the cytoplasm, indicating that there are no additional functional NLS within residues 207395 or 626940 (Fig. 1
). In addition, mutation of NLS1, NLS2 and NLS5 resulted in GFP-EBNA-6 fusion proteins being cytoplasmic, indicating that no additional functional NLS were present in sequences 22183, 396600 or 941992 (Fig. 2
). Taken together the results revealed that, other than NLS1, NLS2 and NLS5, it is unlikely that EBNA-6 contains any other functional NLS.
Two types of EBV exist (type-I or type-II) which show sequence divergence within the genes encoding the EBNA-LP, -2, -3, -4 and -6 gene products (Adldinger et al., 1985; Dambaugh et al., 1984
; Sample et al., 1986
; Sculley et al., 1989
). Since EBNA-6 is a nuclear protein it might be expected that there would be conservation of the functional NLS between the two virus types. Computer analysis of the Ag-876 type-II EBNA-6 protein sequence showed conservation of NLS14 but not NLS5 (Fig. 3
), suggesting that the type-II EBNA-6 protein probably only has two functional NLS (NLS1 and NLS2). Intriguingly, the sequences for the two nonfunctional NLS (NLS3 and NLS4) were almost perfectly conserved in the type-II EBNA-6 protein, raising the possibility that they may serve another function. NLS3 is contained within the repression domain of EBNA-6, while NLS4 is within the DP-103 interaction domain and these sequences may play crucial roles within each of these domains rather than functioning as NLS.
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ACKNOWLEDGEMENTS |
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Received 5 August 2003;
accepted 1 October 2003.