Download Identification of the nuclear localization signals within the Epstein

Journal of General Virology (2004), 85, 165–172
DOI 10.1099/vir.0.19549-0
Identification of the nuclear localization signals
within the Epstein–Barr virus EBNA-6 protein
Kenia Krauer, Marion Buck, James Flanagan, Deanna Belzer
and Tom Sculley
Correspondence
Tom Sculley
Queensland Institute of Medical Research and ACITHN University of Queensland, 300 Herston
Road, Brisbane 4029, Queensland, Australia
[email protected]
Received 5 August 2003
Accepted 1 October 2003
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.
INTRODUCTION
Epstein–Barr virus (EBV) is a DNA tumour virus that has
the capacity to infect and transform B-lymphocytes. Following infection, the co-ordinate expression of a number
of viral nuclear antigens (EBNAs) and membrane proteins
(LMP-1 and -2) acts to maintain B-cell growth and transformation (for review see Kieff, 1996). EBNA-6 is essential
in the transformation and immortalization process.
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 leucinezipper 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 EBNA6 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
0001-9549 G 2004 SGM
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 10–12 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
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K. Krauer and others
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.
METHODS
Construction of GFP-EBNA-6 deletion mutants. The correct
orientation and insertion of fragments in each of the deletion
constructs of EBNA-6 were confirmed by DNA sequencing and
immunoblotting.
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 mg 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-E6D1–206. 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-E6D1–395/601–992. The pBS-GFPL-E6D1–206 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
396–600 of EBNA-6 containing NLS2–4.
pBSGFP-E6D184–992. 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 22–183 of EBNA-6 which
contained NLS1.
pEBO-GFP-E6D396–625. NLS2–4 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 396–625 of
EBNA-6.
pBS-GFPL-E6D1–206/396–625. The pBSGFPL-E6D396–625 con-
struct 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 EBNA6 aa 207–395 joined with aa 626–992 and contained NLS5.
pBS-GFP-E6D1–206/396–625/941–992. The 1?5 kb StuI fragment,
excised from pBS-GFPL-E6D1–206/396–625, was then ligated into
pBS-GFPL previously restricted with HpaI. This resulted in the
expression of a GFP fusion linked to aa 207–395 joined with aa
626–940 of EBNA-6, which lacked all computer-predicted NLS.
Site directed mutagenesis. In vitro mutagenesis of doublestranded DNA templates was performed as described in Sambrook
& Russell (2001) with some modifications. Briefly, a PCR reaction
was performed with 5–50 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 ml21
in a 50 ml reaction. Cycling conditions were as follows: 95 uC 30 s,
55 uC 1 min, 68 uC 2 min per kb of plasmid DNA for 16–18 cycles,
followed by 72 uC 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 uC. The PCR mixture (4 ml) was then transformed
into E. coli DH5a 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.
Cell lines, maintenance and DNA transfection. HeLa cells were
maintained in RPMI 1640 supplemented with 10 % foetal calf serum,
benzylpenicillin (0?7 mg ml21) and streptomycin (1 mg ml21) at
37 uC in a 5 % CO2 atmosphere. HeLa cells were transfected using
ExGen 500 (Progen) according to the manufacturer’s protocol.
Briefly, cells were plated 24 h prior to transfection at a cell density
of 2?46105 cells per well in 3 ml RPMI 1640 containing 10 % FCS.
Plasmid DNA (5 mg) was diluted with 300 ml 150 mM NaCl and
then 15?5 ml ExGen 500 was added. After vortexing and incubation
Table 1. Primers used for site-directed mutagenesis
NLS mutated
NLS1a
NLS1b
NLS2
NLS3
NLS5
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Primers
59-GCAGCGCATCAGGGCAAGGGCGGCAAGACGGGCTGCCTTG-39
59-CAAGGCAGCCCGTCTTGCCGCCCTTGCCCTGATGCGCTGC-39
59-GCGCATCAGGGCAGCGGCGGCAGCACGGGCTGCC-39
59-GGCAGCCCGTGCTGCCGCCGCTGCCCTGATGCGC-39
59-CGTGTGCCTGCAAAGGCACCGGGGAAACTGCCTTGGCC-39
59-GGCCAAGGCAGTTTCCCCGGTGCCTTTGCAGGCACACG-39
59-CCACCGCCTTCCCGTGCGGCAAGGGGAGCGTGTGTTG-39
59-CAACACACGCTCCCCTTGCCGCACGGGAAGGCGGTGG-39
59-GCCACCACGCCAAAAGGGGCTCGAGTAGAAGAAAGTTCTC-39
59-GAGAACTTTCTTCTACTCGAGCCCCTTTTGGCGTGGTGGC-39
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Journal of General Virology 85
NLS in EBNA-6
Table 2. Summary of EBNA-6 NLS mutants
Amino acid residues are indicated with the single-letter code. Bold
letters indicate the mutated amino acid residues within each NLS.
NLS
NLS1
NLS1a mut
NLS1b mut
NLS2
NLS2 mut
NLS3
NLS3 mut
NLS5
NLS5 mut
Sequence
RIRRRRRRR
RIRARAARR
RIRAAAAAR
PAKKPRK
PAKAPGK
PPPSRRRRG
PPPSRAARG
PKRPRVE
PKGARVE
for 10 min at room temperature, the mixture was added directly to
the HeLa cells. The plates were centrifuged at 1200 r.p.m. for 5 min
and then placed into an incubator. Cells were analysed for transient
gene expression at 24 h after incubation at 37 uC in a 5 % CO2
atmosphere.
Direct fluorescence microscopy. HeLa cells expressing GFP-E6
fusion proteins were grown on coverslips and fixed with ice-cold
acetone (2 min, 220 uC), 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.
RESULTS
Nuclear localization signals in EBNA-6
Five potential NLS, as defined by either the bipartite
consensus (Robbins & McMichael, 1991) or the Nakai
consensus (Nakai & Kanehisa, 1992), were identified within
the EBNA-6 protein sequence using PSORT (Pedro’s
BioMolecular Research Tools – http://www.public.iastate.
edu/~pedro/rt_1.html). NLS1, which comprises multiple
overlapping pattern 4 sequences, is located within the
N-terminal region of EBNA-6, aa 72–80 (RIRRRRRRR).
The second NLS, aa 412–418 (KKPRK), has overlapping
pattern 4 and pattern 7 sequences as has the third NLS
(NLS3) at aa 494–500 (PPSRRRR). A bipartite NLS (NLS4)
is located at aa 533–549 (RKHQDGFQRSGRRQKRAA).
The fifth NLS, which has overlapping pattern 4 and pattern
7 sequences, is located at the C-terminal end of the protein
(NLS5) and encompasses aa 939–945 (PKKRPRVE).
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
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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-E6D1–206/396–625/941–992), 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 GFPE6D1–206/396–625/941–992).
Mutagenesis of the EBNA-6 NLS
To determine if the predicted NLS within each of the
GFP-EBNA-6 constructs was responsible for their nuclear
localization, site-directed mutants in the basic residues
within each of the predicted NLS were generated. The
mutations generated within each of the NLS are shown in
Table 2. Because of the number of consecutive arginine
residues present in NLS1 two mutants were generated, one
with three of the arginine residues replaced with alanine
residues and the second mutant with five of the arginine
residues replaced with alanine residues. Each of the mutagenized constructs was transiently transfected into HeLa cells
and the cellular localization of the mutant proteins was
determined by confocal microscopy (Fig. 2). Substitution of
the arginine residues at position 75, 77 and 78 by alanine
residues (NLS1a mut) had no effect upon nuclear localization and additional substitutions of arginine residues 76
and 79 (NLS1b mut) was required to abrogate the nuclear
localization of the GFP-E6aa184–992 protein.
Mutation of NLS3 had no effect upon the nuclear localization of the GFP-E6aa396–600 protein whereas substitution of lysine residue 414 and arginine residue 416 within
NLS2 by alanine and glycine residues, respectively, was
sufficient to abrogate the nuclear localization of the
GFP-E6aa396–600 protein. This result confirmed that the
sequence KKPRK was a functional NLS and that NLS3
and NLS4 were nonfunctional. Substitution of arginine
residue 941 by glycine and proline residue 942 by alanine
was sufficient to destroy NLS5 and prevent the nuclear
localization of the GFP-E6aa643–992 protein.
DISCUSSION
EBNA-6 is required for the transformation of B-lymphocytes and is likely to function as a transcriptional regulator
as sequence analysis revealed a region homologous to the
basic leucine-zipper motif that is found in many mammalian transcription factors. Studies have shown changes in
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K. Krauer and others
Fig. 1. Schematic representation of the full-length EBNA-6 and a series of EBNA-6 deletions fused to GFP and their
subcellular localization. The locations of computer-predicted NLS are indicated. HeLa cells were transiently transfected with
constructs expressing the fusion proteins and were analysed 24 h post-transfection by confocal fluorescence microscopy.
Fig. 2. Cellular localization of GFP-EBNA-6 deletions following mutation of NLS sequences. HeLa cells were transiently
transfected with plasmids expressing each of the EBNA-6 constructs and 24 h post-transfection the subcellular localization of
each of the fusion proteins was determined by confocal fluorescence microscopy.
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Journal of General Virology 85
NLS in EBNA-6
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K. Krauer and others
viral and cellular genes, such as IL-1b (Krauer et al., 1998),
pleckstrin (Kienzle et al., 1996), LMP1 and CD21, a B-cell
activation antigen (Sample et al., 1994), following expression of the EBNA-3 family proteins. EBNA-6 has also
been shown to interact with a variety of cellular proteins,
including the human metastatic suppressor protein Nm23H1, a 152 aa 17 kDa nuclear protein highly conserved in
eukaryotes (Subramanian et al., 2001), histone deacetylase
through the N terminus of EBNA-6 (Radkov et al., 1999),
RBP-Jk/2N (Robertson et al., 1995) and prothymosin alpha
(ProTa), which is a small non-histone highly acidic nuclear
protein that localizes to regions of the nucleus that are
involved in transcription (Subramanian & Robertson,
2002). EBNA-6 can activate the human B-myb promoter
through the E2F response element, can co-operate with
(Ha-)ras in co-transformation assays and can override a
pRb-mediated pathway that inhibits proliferation. EBNA-6
also plays a role in the disruption of cell-cycle checkpoints
(Parker et al., 1996).
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 (pGFPE6D1–206/396–625/941–992) resulted in the truncated
EBNA-6 protein being present solely throughout the
cytoplasm, indicating that there are no additional functional NLS within residues 207–395 or 626–940 (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 22–183, 396–600 or 941–992 (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 EBNALP, -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 NLS1–4 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)
170
Fig. 3. Comparison of EBNA-6 NLS in Type I and II isolates
of EBV. Amino acid residues are indicated with the singleletter code. Bold letters indicate differences in sequence
between each NLS. Underline indicates that this NLS was not
computer-predicted.
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.
It is not understood why a single protein contains multiple
NLS. However, it has been suggested that multiple NLS may
function more efficiently (Roberts et al., 1987; Knauf et al.,
1996), or that different NLS may have different specificities
in different cell types (Liu et al., 1998). Alternatively, some
NLS can also function under different conditions, such as
one of the multiple NLS in XPG nuclease (Xeroderma
pigmentosum type G), which can regulate the localization
of the protein to the nucleus following UV irradiation
(Knauf et al., 1996). Proteins with multiple NLS include
the tumour suppressor menin (Guru et al., 1998), the
high-mobility group transcription factors SRY and SOX9
(Sudbeck & Scherer, 1997), the proto-oncogene c-Abl
(transforming gene of Abelson murine leukaemia virus)
(Wen et al., 1996), tumour suppressor p53 (Shaulsky et al.,
1990) and human c-Myc, which contains one strong and
one weak NLS (Dang & Lee, 1988). The presence of multiple NLS in EBNA-6 could be needed if, prior to transport
into the nucleus, EBNA-6 interacts with proteins in the
cytoplasm or if EBNA-6 is modulated by phosphorylation,
either of which could mask one or more of the NLS. Other
possible reasons for multiple NLS could involve differential
cellular regulation, or simply enhancement of nuclear accumulation (Roberts et al., 1987).
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Journal of General Virology 85
NLS in EBNA-6
ACKNOWLEDGEMENTS
efficient nuclear targeting of the Epstein–Barr virus nuclear antigen
3A. J Virol 67, 1716–1720.
The authors would like to acknowledge Paula Hall and Grace
Chowjnowski, Queensland Institute of Medical Research, for assistance
with confocal microscopy. This work was supported by grants from the
NHMRC, Australia.
Liu, K. D., Gaffen, S. L. & Goldsmith, M. A. (1998). JAK/STAT
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