(Received for publication, August 19, 1996, and in revised form, October 30, 1996)
From the The Epstein-Barr virus (EBV) is implicated in the
induction of several malignancies. The nuclear antigen 1 (EBNA1) is the only viral protein that is expressed consistently in all EBV-associated tumors. EBNA1 is involved in the replication and maintenance of the
viral episome in the infected cell and exhibits oncogenic activity in
transgenic mice. Here we report the identification of the nuclear
transporter karyopherin The Epstein-Barr virus (EBV)1 is the
etiological agent of infectious mononucleosis and is implicated in the
induction of several malignancies including Burkitt's lymphoma,
nasopharyngeal carcinoma, Hodgkin's disease, T-cell lymphoma, and
polyclonal B-cell lymphoma that arise under immunosuppression (reviewed
in Ref. 1). The expression of EBV-encoded proteins is most stringently
restricted in primary Burkitt's lymphoma in that only EBNA1 and the
nontranslated Epstein-Barr virus-encoded small nuclear RNAs are
transcribed. EBNA1 binds to the viral origin of replication (oriP)(2,
3) and is the only virus protein necessary and sufficient to replicate and maintain the EBV genome in the infected cell (4). In addition, experiments using transgenic animals show that EBNA1 by itself is able
to induce tumors in vivo (5, 6). EBNA1 is a multifunctional phosphoprotein (7) with a variety of properties, including sequence-specific binding to DNA (8, 9), formation of homodimers (10),
nuclear localization (11), and stimulation of cellular gene expression
(12).
Import of proteins into the nucleus is an active process that can be
divided into at least two steps: 1) binding of proteins with a nuclear
localization signal (NLS) to a cytosolic NLS-receptor and translocation
to the nuclear pore complex; and 2) transport of NLS-bearing proteins
into the nucleus. The NLS-receptor consists of two subunits, a 60-kDa
protein that is called karypherin Burkitt's lymphoma invariably carry a chromosomal translocation
involving the c-myc proto-oncogene (15). EBNA1 stimulates the expression of the recombination activating genes 1 and 2 (RAG1 and RAG2, respectively), which are
necessary for V(D)J recombination in B-cells (16). The presence of
EBNA1 in B-cells and the concomitant stimulation of
RAG1/RAG2 expression might ultimately lead to the chromosomal changes observed in Burkitt's lymphoma. Karyopherin Human 293 GP cells derived from
embryonal kidney cells by transformation with the adenovirus type 5 E1a
protein (19) were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. B95-8 is an EBV-positive
marmoset cell line (20), iB4 is a human lymphoblastoid cell line
transformed by the B95-8 virus (21), and BJAB is an EBV-negative
B-cell tumor cell line from a tumor initially diagnosed as BL which,
however, does not carry a chromosomal translocation involving the
c-myc locus (22). BL41 is an EBV-negative Burkitt's
lymphoma cell line (23), and BL41-P3HR1 was established by infection of
BL41 with the transformation-defective P3HR1 virus (24). The BL41 cell
line was a generous gift from U. Zimber-Strobl, GSF, Munich, Germany.
The B-cells were maintained in RPMI 1640 medium (Life Technologies,
Inc.) supplemented with 10% fetal calf serum (Seromed), 40 IU/ml
penicillin, and 50 µg/ml streptomycin and were subcultured routinely
once per week. The insect cell line SF158 (25) was kept at 27 °C in
TC100 medium supplemented as above. Recombinant baculoviruses
expressing EBNA1 and EBNA2 were described earlier (26).
The rat monoclonal antibodies (mAbs) 1H4 directed against EBNA1 and R3
directed against EBNA2A have been described (27, 28). The mAb pab122
directed against p53 was a generous gift from K. Roemer,
Universitätskliniken, Homburg. For the production of rat
monoclonal antibodies against Rch1, an EcoRI-resistant DNA
fragment encoding amino acids 116-515 of Rch1 from clone pGADGH Rch1
122 (see below) was ligated to the EcoRI digested vector pATH10 (29). (We note that the EcoRI site at codon 515 in
clone Rch1-122 was not predicted by the original cDNA sequence; the mutation leading to the new restriction site did not change the amino
acid composition of Rch1 in our clone Rch1-122). The resulting plasmid
pATH-Rch1 was used to express the amino acids 116-515 of Rch1 fused
in-frame to the Trp E protein in Escherichia coli strain
BL21/DE 3 (lon For a review of the yeast
"two-hybrid" system, see Phizicky and Fields (31). The GAL4
DNA-binding domain (GAL4DBD) fusions were constructed in the vector
pGBT9 (32). GAL4 DBD EBNA1 CT (330-641) was constructed by polymerase
chain reaction (PCR)-mediated amplification of the DNA fragment
encoding amino acids 330-641 of EBNA1, using oligonucleotide primers
EBNA1 Eco (5 The amplified fragments were digested with EcoRI and
SalI (underlined in the primers EBNA1 Eco and
EBNA1 SalI, respectively) and ligated to
EcoRI/SalI-digested pGBT9. Additional fragments of EBNA1 were inserted into the vector pGBT9 as described above. The
amino acid encoded by the various PCR products are given in parentheses.
The primers used for the PCR amplification of EBNA1 cDNA fragments
are: GAL4 DBD EBNA1 Eco (330-494), EBNA1 Eco, and EBNA1 1480R
(5 Plasmids pGADGH Rch1 122 and pGADGH Rch1 142 encoding amino acids
116-529 and 38-529 of Rch1, respectively, were isolated during the
yeast two-hybrid screening. For the expression in E. coli of
the full-length Rch1 protein, which consists of 529 amino acids, the
complete cDNA of Rch1 was PCR-amplified from the HeLa cDNA
library originally used for the two-hybrid screen using the oligonucleotide primers: N-term Rch1,
5 The resulting DNA fragment was digested with NdeI and
BamHI (underlined in the above primers) and
ligated to the vector pET3a (33) to yield vector pET3a-Rch1, which
directs the expression of Rch1 as a non-fusion protein in E. coli. The anticodon CTA, which corresponds to the TAG stop-codon
of Rch1, is emphasized in boldface.
Plasmid pCEP4 R for the eukaryotic expression of an N-terminally
truncated Rch1 was constructed using the PCR primers: Rch1-C-5 Yeast transformation was
performed according to the method of Schiestl and Geitz (34). The yeast
strain HF7c (35) was transformed with plasmid construct GAL4 DBD EBNA1
CT (330-641), and transformants were plated on Trp Extracts of 1.5 × 107 EBV-positive and EBV-negative cells were separated by
centrifugation through a 15-30% sucrose gradient as described (39).
Fifteen fractions collected by bottom puncture were precipitated in
ethanol and subjected to 10% SDS-PAGE (polyacrylamide gel
electrophoresis) followed by immunoblotting with mAbs 2G7 and 1H4
directed against Rch1 and EBNA1, respectively.
Transfections
were performed by the calcium phosphate method, as described previously
(40). After 48 h, transfected cells were fractionated. Cytoplasmic
and nuclear fractions were analyzed by Western blot with anti-EBNA1 and
anti-Rch1 mAb. To determine the subcellular localization of EBNA1 and
Rch1, transfected 293 GP cells were lysed by Dounce homogenization
exactly as described by Niedermann et al. (41). The nuclear
and cytosolic fractions were analyzed with the EBNA1-specific mAb 1H4
and the Rch1-specific mAb 2G7.
The association of EBNA1 with Rch1 was
analyzed by a modified far-Western blotting procedure (42, 43).
Extracts from baculovirus-infected SF158 cells were prepared by
suspending 1 × 107 SF158 cells infected with
wild-type or recombinant baculovirus expressing either EBNA1-bac or
EBNA2-bac in 0.5 ml lysis buffer (26). Cells were suspended, sonicated,
and boiled in SDS-gel loading buffer. After centrifugation (13,000 × g, 4 °C, 10 min), extracts derived from 1 × 106 cells/gel track were separated by SDS-PAGE and
transferred to nitrocellulose (Schleicher and Schuell) in a buffer that
consisted of 25 mM Tris/HCl, 0.195 M glycine,
pH 8.8. The membranes were preincubated for 30 min in HBB (HEPES
binding buffer: 20 mM HEPES, pH 7.5, 5 mM
MgCl2, and 1 mM KCl) that contained 5% nonfat
dried milk. The membrane was then incubated for 18 h at 4 °C
with 0.05-0.1 ml/cm2 HBB supplemented with 5% nonfat
dried milk and 5 mM dithiothreitol that contained 0.1 ml of
pET-Rch1 extract/ml HBB. All subsequent incubation steps were carried
out in PBS, 5% nonfat dried milk, and intermitting washing steps were
performed with PBS/0.1% Triton X-100. Bound Rch1 was visualized by
incubating the membrane with mAb 2G7 at a dilution of 1:20, followed by
incubation with peroxidase-coupled goat-anti-rat antibody
(Sigma) at a dilution of 1:500.
EBNA1 varies considerably in length between different EBV
isolates due to heterogeneity in the so-called "glycine-alanine" repeats present in the N-terminal half of the molecule (see Fig. 1). The glycine-alanine repeats were shown to
down-modulate the T-cell response against EBV-infected cells (44). The
remaining functions of EBNA1 are mostly attributable to sequences in
the C-terminal part of the protein (11), as outlined in Fig. 1. For
screening in the yeast two-hybrid system, a DNA fragment of the strain
B95-8 encoding amino acids 330-641 EBNA1 obtained by PCR
amplification was fused in-frame to the DNA-binding domain of GAL4 in
the "bait"-vector pGBT9. The extent of this fragment and of
additional, shorter C-terminal fragments of EBNA1 that were generated
are shown Fig. 1. We then screened 7 × 106 individual
clones of a HeLa cDNA library for the presence of interacting
partner proteins expressed as fusion proteins with the
GAL4-transactivation domain. Colonies that grew under selection were
segregated and rescreened, and a total of 11 positive clones were
obtained. Further analysis of these clones revealed that they either
contained an insert of 2.0 or 2.1 kilobases; two representative isolates designated clones 142 and 122, respectively, were selected for
further analysis. By DNA sequencing, we determined that the cDNA
inserts encoded either amino acids 38-529 or 116-529 of the previously described Rch1 gene, which is predicted to encode
a protein of 529 amino acids (17, 18). Because the NLS was shown to
encompass amino acids 379-386 of EBNA1 (11), we constructed additional
bait-vectors that either did or did not contain the NLS of EBNA1, as
outlined in Fig. 1. When the bait vector expressed amino acids 330-423
or 330-494 as a fusion protein with the GAL4 DNA-binding domain, the
resulting proteins interacted with the corresponding Rch1 proteins from
the cDNA clones obtained during the initial screen, whereas bait
vectors encompassing either amino acids 410-641 or 483-641 did not
yield positive signals (data not shown). These results are in line with
the observation that the NLS of EBNA1 resides between amino acids
379 and 386.
We also carried out the converse experiment to test which region of
Rch1 is responsible for binding to EBNA1. A DNA fragment encoding amino
acids 116-515 of Rch1, which was used for the production of monoclonal
antibodies against Rch1, was inserted into the vector pGAD424. We then
tested this construct with the original, EBNA1-expressing bait vector
in the two-hybrid system. We obtained positive signals (data not shown)
and, therefore, conclude that the minimal region of interaction with
EBNA1 comprises amino acids 251-515 of Rch1.
For further
biochemical analysis, we generated monoclonal antibodies against Rch1.
A DNA fragment encoding amino acids 116-515 of Rch1 was expressed in
E. coli as a Trp E-fusion protein and used to immunize Lou/c
rats. The subsequent production of monoclonal antibodies against Rch1
was carried out essentially as described earlier (27, 28). A clone,
designated 2G7 (rat IgG2a), that reacted only with the fusion protein
(data not shown) was subcloned and used in all of the experiments.
Nuclear and cytoplasmic extracts from the Burkitt's lymphoma cell line
Jijoye, which contains the type 2 strain of EBV and extracts from the
EBV-negative lymphoma cell line BJAB, were analyzed in a Western blot
shown in Fig. 2. The EBNA1-specific antibody 1H4-1
reacted only with a protein of the predicted size in the nuclear
extract of the cell line Jijoye, as shown in Fig. 2A, lane
2. In contrast, the Rch1-specific antibody stained a band with a
molecular mass of approximately 58 kDa in the nuclear and cytoplasmic
extracts of both cell lines. The presence of Rch1 in the cytoplasm and
the nucleus of the cell lines was as expected because the present model
suggests that Rch1 shuttles between the two cell compartments (13,
14).
A cDNA encoding the full-length Rch1 protein was
PCR-amplified from the HeLa cDNA library (Clontech) originally used
for the two-hybrid screen and expressed in E. coli using the
vector pET3a-Rch1. As can be seen in Fig. 3, the
antibodies against Rch1 reacted with a protein of the expected size in
the extract derived from E. coli transformed with the
pET3a-Rch1 construct, whereas the bacterial control extract did not
yield a signal. The E. coli-expressed Rch1 was subsequently
used as a probe in the far-Western analysis (43, 44). We showed that
the monoclonal antibodies used for the detection of the proteins do not
cross-react. For this purpose, EBNA1 and Rch1 from bacteria and insect
cells, respectively, were analyzed in a standard Western blot
experiment shown in Fig. 3, lanes 1-4. The antibody 1H4-1
reacted only with EBNA1 (Fig. 3, lanes 1 and 2),
whereas the antibody 2G7 reacted only with Rch1 (Fig. 3, lanes
3 and 4). For the far-Western experiment, EBNA1 expressed in insect cells using a baculovirus construct (26) was
separated by SDS-PAGE, transferred to nitrocellulose membrane, and
incubated with E. coli extract containing the Rch1 probe. Bound Rch1 was visualized with mAb 2G7. Extracts from insect cells infected with wild-type baculovirus (Fig. 3, lane 5) or with
a baculovirus expressing the EBV-encoded nuclear antigen 2A (26) (Fig.
3, lane 6) served as negative control. The presence of EBNA2 in the insect cell extract was confirmed separately by Western blot
analysis (data not shown). Although we observed some binding of Rch1 to
cellular proteins in the extract from wild-type or EBNA2-containing
extract, we could clearly detect binding of Rch1 to EBNA1 (Fig. 3,
lane 7). The position of EBNA1 in Fig. 3 is indicated by an
arrow. In the control extracts, no binding to a protein of
the same size as EBNA1 was detectable. Rch1 did not bind to EBNA2,
which migrates with an apparent molecular weight of 85 kDa. As an
additional control, E. coli extract from cells transformed
with the parental pET3a plasmid (Fig. 3, lane 8) or from
cells expressing Rch1 (Fig. 3, lane 9) was used to
preincubate the Rch1-specific antibody 2G7. As can be seen,
preincubation with control extract did not change the signal, whereas
preincubation with Rch1 abolished binding to the membrane.
The nuclear localization signal of EBNA1 comprises amino acids
379-386, and we assume that the electrophoresis and transfer to the
membrane does not significantly alter this sequence, which is still
recognizable by native Rch1 in the far-Western analysis. However, when
we carried out the reverse experiment by transfering Rch1 to the
membrane and probing the blot with EBNA1 from insect cells, we were
unable to detect binding of EBNA1. The region of Rch1 responsible for
binding to EBNA1 might be larger than the NLS of EBNA1 and may also be
conformation-dependent. Thus, the denaturing conditions
during SDS-gel electrophoresis may permanently destroy the tertiary
structure of the protein, precluding binding of the soluble EBNA1
probe.
We also asked whether Rch1 would yield a signal with EBNA1 from B
cells. When we carried out a far-Western experiment analogous to the
one described above, we did not observe binding to the EBNA1 from B
cells. We assume that the failure to observe binding was due to the low
amount of EBNA1 present in B cells as compared to the
baculovirus-infected insect cells. Alternatively, the EBNA1 protein
from the B cells might be modified after the transport to the nucleus.
This modification might inhibit the rebinding of Rch1 to EBNA1.
However, because we were able to show an association of EBNA1 with Rch1
by: (i) inhibition of nuclear transport of EBNA1 by expression of an
N-terminally truncated Rch1; and (ii) colocalization of the two
proteins in a sucrose gradient (see below), this matter was not further
pursued.
It was shown recently that the matrix protein of the human
immunodeficiency virus (HIV) is transported to the nucleus by Rch1, and
that translocation of the matrix protein to the nucleus is impaired by
expression of the C-terminal amino acids 244-529 of Rch1 (45). From
other lines of experimentation, it is clear that Rch1 binds to the
protein(s) to be transported to the nucleus through its C-terminal
portion, whereas the N-terminal amino acids 1-55 tether the resulting
protein complex to karyopherin
We wanted to test whether the presence of EBNA1 in B-cells
interfered with the formation of complexes between Rch1 and cell proteins. For this purpose, cell extracts were analyzed by sucrose gradient centrifugation as described previously (39). We compared the
EBV-negative cell lines BL41 and BJAB (both cell lines were isolated
from patients diagnosed with Burkitt's lymphoma) with the
EBNA1-expressing cell lines BL41-P3HR1 (BL41 cells infected with the
P3HR1 strain of EBV) and the human lymphoblastoid cell line iB4
(generated from uninfected primary human lymphoid cells by infection
with the B95-8 virus). The results obtained with the cell lines BJAB
and iB4 are shown in Fig. 5. The position of molecular
mass standard proteins separated on a gradient, which was analyzed in
parallel, is indicated above the top panel; the lanes designated
"U" contain unfractionated whole-cell extract as a control. As can
be seen in the top panel, the major part of Rch1 from EBV-negative
B-cell line BJAB was found in the top of the gradient (fractions
10-15), corresponding to uncomplexed, monomeric Rch1 (fractions
13-15) or low molecular mass complexes (fractions 10-12). A smaller
but sizable fraction of Rch1, however, was detectable in fractions 1-5
corresponding to very high molecular mass complexes. In contrast, when
cell extract derived from the EBV-positive iB4-cell line was analyzed,
we found that Rch1 had a different migration behavior (Fig. 5,
middle panel). The major portion of Rch1 was now detectable
in fractions 10-12 instead of fractions 13-15, corresponding to a
molecular mass of approximately 150-250 kDa. When the fractions from
the same gradient were tested for the presence of EBNA1 (Fig. 5,
bottom panel), we found that the peak of EBNA1 reactivity
was also detectable in fractions 10-12, indicating that Rch1 is in a
complex with EBNA1. EBNA1 and Rch1 have calculated molecular weights of
Mr 72,000 and Mr 58,000, respectively. Assuming that the majority of the EBNA1 forms homodimers
(49), this result would indicate that dimeric EBNA1 binds either one or
two molecules of Rch1, resulting in a molecular mass of about 200 kDa.
In our experiments, we observed a molecular weight of approximately
Mr 150,000-200,000. It is also possible,
however, that monomeric EBNA1 binds to Rch1.
We obtained the same results with the cell lines BL41 and BL41-P3HR1
(data not shown). In the BL41-P3HR1 cells, we found a lower amount of
EBNA1 as compared to iB4. In BL41-P3HR1, we also noted a shift of the
low molecular weight form of Rch1 to the higher molecular weight
fractions that coincided with the EBNA1-containing fractions. We also
noted a slight reduction of the very high molecular weight form of
Rch1.
The result that Rch1 and EBNA1 colocalize in conjunction with the
results obtained from the far-Western analysis and the fact that the
truncated Rch1 impedes the import of EBNA1 into the nucleus further
strengthens the notion that the proteins form a complex. Furthermore,
we reproducibly observed a reduction in the amount of Rch1 detectable
in fractions 1-5 corresponding to high molecular mass complexes. There
are two possible explanations for this result. Either the presence of
EBNA1 prevents the binding of Rch1 to high molecular mass complexes, or
EBNA1 inhibits the synthesis of proteins contained in those
complexes.
Transformation of B lymphocytes in vitro by the
Epstein-Barr virus is mediated through a set of viral proteins that are
found in the nucleus or the membrane of the cell. By genetic means, it
was shown that the nuclear antigens EBNA1, -2, -3A, -3C, and -5 as well
as the latent membrane protein 1 are absolutely essential for this
transformation process (reviewed in Ref. 50). It is generally assumed
that viral oncogenes bind to cellular factors that regulate cell
growth, thereby changing their biochemical and biological activities;
for instance, binding of the multifunctional SV40 large T-antigen to
the the p53 and p105rb proteins is thought to induce the molecular
changes resulting in cell transformation (reviewed in Refs. 51). For
EBNA-2, a variety of cellular partners, mostly proteins involved in
gene regulation, have been identified; EBNA-5 appears to target the p53
and p105rb proteins, and latent membrane protein 1 forms a
complex with the tumor necrosis factor receptor family of growth factor
receptors (52). Thus far, no cellular proteins that might be targeted by EBNA1 to carry out its various functions have been described. We
have used the yeast two-hybrid system to identify such cellular proteins to gain a deeper understanding of the EBV-mediated
transformation. Here we show that EBNA1 binds to karyopherin Karyopherin One point of concern was whether the observed binding of EBNA1 to Rch1
was specific or just simply the result of a fortuitous binding due to
the presence of a possibly "promiscuous" nuclear localization site
in EBNA1. We assume the latter not to be the case because the EBNA1
bait used for the screening in the yeast two-hybrid system did not
react with the control vector pTD1. This construct encodes the
GAL4-transactivation domain fused to amino acids 84-708 of the SV40
large T antigen, which encompasses the NLS of large T antigen. Also,
when using the complete EBNA-3C gene instead of the EBNA1 as
a bait, we observed no binding to Rch1 in the yeast two-hybrid
assay.2 By definition, EBNA3C is a nuclear
antigen encoded by EBV that must contain an NLS. In addition, we also
did not observe binding of Rch1 to the nuclear antigen EBNA-2 in a
far-Western experiment, whereas we observed strong binding to EBNA1.
Lastly, the expression of a truncated Rch1 in 293 GP cells specifically
impeded the nuclear import of EBNA1 but not of p53. We, therefore, deem
it unlikely that the observed binding of the nuclear protein EBNA1 to
the nuclear transporter Rch1 is purely artifactual.
Finally, we found that Rch1 migrated with different mobilities during
sucrose gradient centrifugation, depending on the presence or absence
of EBNA1. In the EBNA1-negative cell lines BJAB and BL41, Rch1 was
localized mainly in low molecular weight fractions corresponding to
uncomplexed, monomeric Rch1 but also in fractions corresponding to very
high molecular weight. In the EBNA1-positive cell lines iB4 and
BL41-P3HR1, the low molecular weight Rch1 was shifted to the
EBNA1-containing fractions with intermediate molecular weight, whereas
the amount of Rch1 in the complexes with very high molecular weight was
reduced. This latter effect was more pronounced in the iB4 cells that
expressed more EBNA1 than the BL41-P3HR1 cells. These results indicate
that EBNA1 might inhibit binding to the high molecular weight complexes
by a squelching mechanism. Although the reduction of high molecular
weight complexes was less pronounced, we clearly observed a shift of
the Rch1 protein in the low molecular weight fractions to a fraction of
intermediate molecular weight, which also contained EBNA1. The presumed
complex formation would then prevent binding to other proteins present in the cells. From the Western blot experiment, we estimated that the
amount of Rch1 in the cell is at least 50-fold higher than the amount
of EBNA1, but the signals obtained with the different antibodies might
not reflect the true amount of each protein in the cell. However, the
shift in Rch1 mobility might be due to the fact that exactly the
portion of the Rch1 molecules that are otherwise uncomplexed are now
bound by EBNA1. Ultimately, the presence of EBNA1 appears to reduce the
binding of Rch1 to cellular factors, either by directly binding to Rch1
or by a qualitative change in the expression of these proteins. It is
tempting to speculate that the reduced binding of Rch1 to such proteins
might impede their import to the nucleus, thus preventing their
activity. These proteins could be involved in controlling the proper
execution of the recombinatorial events during B-cell maturation. In
this respect, it is noteworthy that the oho-31 tumor
suppressor gene in Drosophila melanogaster is a homologue of
Rch1. The inactivation of oho-31 leads to tumors of the
hematopoietic organs and the genital disk in the fly (55). The
oho-31 gene product is predominantly cytosolic in interphase
but strongly accumulates in the nucleus during mitosis. Of the latent
gene products of EBV, EBNA1 is the only protein that is found to be
associated with the chromatin during mitosis (56) and is thought to
ensure the equal distribution of the viral DNA to the progeny cells. At
this point, we assume that the binding of EBNA1 to Rch1 results in a
quantitative change of the Rch1 protein available for nuclear transport
that is not available for transport of its normal partner proteins.
Additional experiments will be carried out to clarify whether the
specific transport of EBNA1 by Rch1 is necessary for EBV-mediated
transformation or whether the nuclear transport of EBNA1 is the sole
prerequisite for its function in cell transformation.
We thank Dr. J. S. Lipsick, University of
Stanford, CA, for helpful suggestions and critical reading of the
manuscript. We also thank J. Hearing, SUNY, Stony Brook, NY, for
communicating results prior to publication.
Institut für Mikrobiologie und
Hygiene,
2 as a cellular partner of EBNA1 using the
yeast "two-hybrid system." Karyopherin
2 is also called importin
or Rch1. The binding to karyopherin
2 was mediated through a
C-terminal region of EBNA1 encompassing the nuclear localization
signal, whereas clones of EBNA1 devoid of the nuclear localization
signal failed to bind to karyopherin
2. The interaction was
biochemically confirmed by far-Western analysis using bacterially
expressed karyopherin
2 and karyopherin
2-specific monoclonal
antibodies. The nuclear transport of EBNA1 was impaired by expression
of N-terminally truncated karyopherin
2. Zone velocity sedimentation
in a sucrose gradient indicated that: (i) EBNA1 and Rch1 colocalize;
and (ii) the association of karyopherin
2 with high molecular weight
protein complexes might be impeded by the presence of EBNA1.
2 (alternatively, it is called
importin
in vertebrates and Srp1 in yeast) and a 95-kDa protein,
called karyopherin
(also known as importin
in vertebrates and
Kap95p in yeast). Karyopherin
2 serves as the NLS-receptor, whereas
karyopherin
functions as an adapter that mediates binding to
nucleoporins. Two additional proteins, GTPase Ran/TC4 and p15 (NTF2)
are required for transport into the nucleus. For recent reviews about
nuclear transport, see Görlich and Mattaj (13) and Hurt (14).
2
was originally identified and molecularly cloned by virtue of its
ability to bind to the recombination activating protein 1 (RAG1) using
the yeast two-hybrid system (17, 18); therefore, it is alternatively
called Rch1 (
ag
o
ort
). In the present communication, we show that EBNA1 also binds to the nuclear transport protein karyopherin
2. For brevity, we refer to karyopherin
2/importin
in the following report as Rch1.
Cells and Antibodies
)(30). This particular strain of E. coli was
chosen for the expression using the pATH vectors because the absence of
protease resulted in a higher yield of fusion proteins. The
gel-purified protein was used to immunize Lou/c rats. The fusion and
screening for Rch1-specific monoclonal antibodies was done exactly as
described (28) using an irrelevant Trp E fusion protein as a control.
The Rch1-specific clone 2G7 (IgG2a) was subcloned and used for
additional experiments.
-CCG
GGTGGAGGAGGCAGTGGAGGC) and EBNA1 Sal
(5
-CACGC
-CACGC
TCACCTAGCCAGGAGAGCTCTTAA); GAL4 DBD EBNA1 Sal (483-529), EBNA1 1450F
(5
-CCG
TTAAGAGCTCTCCCTGGCT), and EBNA1 Sal;
GAL4 DBD EBNA1 CTØNLS (410-529), EBNA1 NLS5
(5
-TGTAGGG
GATTATT), and EBNA1 Sal; and GAL4 DBD
EBNA1 NLS (330-423), EBNA1 Eco, and EBNA1 NLS3
(5
-AGGCTCAC
GGCCAC).
-GCTCTAGAGGATCCCTCATA
TCCACCAACGAGAATGCTAAT; and
C-term Rch1,
5
-CGC
AAGCTTCTAAAAGTTAAAGGTCCCAGG).
, 5
-CGC
CCCGGGAATTCATAACCATGGATGCTGTTGAGCAGATT;
and Rch1-C-3
,
5
-CGC
AAGCTTCTAAAAGTTAAAGGTCCCAGG. The resulting DNA fragment encoding amino acids 251-529 of Rch1 was digested with
BamHI (underlined) and ligated to vector pCEP4 (Invitrogen). The primer Rch1-C-5
encodes an ATG initiator methionine codon (boldface) to allow the expression of the truncated Rch1 in eukaryotic cells.
selective-defined media. The GAL4 DBD EBNA1 CT (330-641) transformant was subsequently transformed with a HeLa cDNA library (Clontech), and selection was done on
Trp
Leu
His
selective-defined
media as described previously (36). Colonies that showed moderate to
intense growth were streaked on
Trp
Leu
His
selective-defined
media and tested for
-gal expression by a colony lift filter assay
(37). Library-derived plasmids were recovered by transformation of
competent bacteria with total yeast DNA, followed by selection for
ampicillin resistance as described previously (38).
Identification of Karyopherin 2/Rch1 as a Cellular Target of
EBNA1
Fig. 1.
A, functional domains of EBNA1. The
standard EBNA1 protein, which consists of 641 amino acids (Baer
et al. (57)) is shown at the top. The various
domains as described by Ambinder et al. (11) are depicted in
black. Down-modulation of the cytotoxic T-cell
(CTL) response against EBV-infected cells by the
glycine-alanine repeat (GLY/ALA) was described by Masucci
et al. (44). The NLS of EBNA1 comprises amino acids
379-386. The fragments of EBNA1 used in the yeast "two-hybrid"
system are shown in gray. The numbers indicate the amino
acids residues of EBNA1 encoded by the various constructs.
B, functional domains of karyopherin 2/Rch1. The Rch1
protein, which consists of 529 amino acid residues, is shown at the
top. The functional domains of Rch1 as described by Gallay et al. (45) and Görlich et al. (48) are
shown in black. The various parts of Rch1 expressed in
different constructs are shown in gray. The numbers indicate
the Rch1-specific amino acid residues encoded by the different
constructs.
[View Larger Version of this Image (20K GIF file)]
Fig. 2.
Subcellular localization of EBNA1 and Rch1.
A, the nuclear and cytosolic fractions of the EBV-positive
cell line Jijoye (lanes 2 and 3) or the
EBV-negative cell line BJAB (lanes 4 and 5) were
separated by SDS-PAGE and transferred to the membrane and stained with
the EBNA1-specific mAb 1H4. Lane 1 contains whole-cell extract of insect cells infected with a recombinant baculovirus expressing EBNA1. B, the extracts described in A
were tested with the Rch1-specific mAb 2G7. The position of the
molecular mass marker proteins (in kDa) is indicated by
arrows on the left of A. The marker
proteins were, in descending order: phosphorylase b, bovine
serum albumin, ovalbumin, and carbonic anhydrase (Pharmacia Biotech
Inc.). The arrows on the left of B
indicate the position of phosphorylase b and bovine serum
albumin.
[View Larger Version of this Image (33K GIF file)]
Fig. 3.
Binding of Rch1 to EBNA1 as detected by
far-Western analysis. EBNA1 expressed in insect cells using a
recombinant baculovirus (26)(lanes designated bac-EBNA1) and
Rch1 expressed in E. coli (lanes designated
pET-Rch1) were analyzed by Western blotting using the
EBNA1-specific mAb 1H4 or Rch1-specific mAb 2G7. For the far-Western
analysis, extracts of insect cells infected either with wild-type
baculovirus (lane designated wt-bac), an EBNA2A-expressing baculovirus (lane designated bac-EBNA2), or the
EBNA1-expressing baculovirus (lanes designated bac-EBNA1)
were separated by SDS-PAGE, transferred to nitrocellulose, and
incubated with bacterially expressed Rch1 as a probe. Bound Rch1 was
visualized using the mAb 2G7. In the competition experiment, the mAb
2G7 was preincubated either with bacterial extract containing either no
Rch1 (lane 8) or recombinant Rch1 (lane 10). The
marker proteins (in kDa) were, in descending order: phosphorylase b,
bovine serum albumin, ovalbumin, and carbonic anhydrase (Pharmacia
Biotech Inc.). The arrow on the right side of the
figure shows the position of the EBNA1 protein.
[View Larger Version of this Image (56K GIF file)]
, which is responsible for the
transport of the resulting complex to the nuclear pore complex (46,
47). Deletion of the N-terminal part of Rch1 thus precludes binding to
karyopherin
(48). In addition, the truncated Rch1 competes with the
full-length Rch1 for binding to the target proteins and inhibits their
nuclear translocation. Therefore, we decided to carry out a similar
experiment to inhibit nuclear transport of EBNA1 with a truncated Rch1.
A DNA fragment encoding amino acids 251-529 fused to an initiator methionine start codon was generated by PCR amplification and inserted
into the vector pCEP 4. This vector allows the episomal replication of
the plasmid by supplying the latent origin of replication of EBV
together with EBNA1, which in turn induces the replication by binding
to oriP of EBV; the vector directs the expression of an additional gene
driven by the CMV immediate-early promoter. The resulting vector, pCEP
4 R, expressing both EBNA1 and the truncated Rch1, was transiently
introduced into the cell line 293 GP, which allows high level
expression of the vector-encoded proteins by calcium phosphate
precipitation. Fourty-eight h after the transfection, the 293 GP cells
were fractionated and analyzed by Western blotting. As shown in Fig.
4, we could detect the truncated Rch1 protein in
addition to the full-length, cellular Rch1 in the cytoplasmic fraction
(fig. 4, lane 10) but not in the nuclear fraction of 293 GP
cells transfected with vector pCEP 4 R (Fig. 4, lane 9). In
contrast, the full-length Rch1 was detectable in both the cytoplasmic
and the nuclear fraction as predicted. When we used the parental vector
pCEP 4, we only observed the full-length Rch1 in both cell compartments
(Fig. 4, lanes 7 and 8). The same extracts were
then tested for the distribution of EBNA1. Using the parental vector,
we could detect approximately equal amounts of EBNA1 in both the
cytoplasm (Fig. 4, lane 2) and the nucleus (Fig. 4,
lane 3), probably because a very high level of protein expression was induced. In contrast, we found that the cytoplasmic extracts derived from cells expressing the truncated Rch1 contained at
least 80% of EBNA1, with the remaining portion detectable in the
nucleus (Fig. 4, lanes 5 and 4, respectively). To
ensure that the unexpectedly large amount of EBNA1 detected in the
cytoplasmic fraction was not due to an experimental error, we also
analyzed the same fractions for the presence of p53, which is a nuclear protein. The major portion of p53 was detectable in the nuclear fraction with about 10-15% of total p53 detectable in the cytoplasmic fraction, which might constitute the freshly synthesized p53. We
conclude from this observation that the nuclear transport of EBNA1, but
not of p53, is specifically mediated by Rch1. In addition to the
retention of EBNA1 in the cytoplasmic fraction, we also observed a
reduction in the overall expression of EBNA1 (Fig. 4, compare
lanes 2 and 3 with lanes 4 and
5). We take this as an additional indication that the
specific nuclear transport of EBNA1 by the wild-type Rch1 is inhibited
by the truncated Rch1. The reduced amount of EBNA1 transported to the
cell nucleus ultimately results in a strongly reduced rate of the
(EBNA1-dependent) replication rate of the vector pCEP 4 R,
which in turn yields lower levels of EBNA1 protein.
Fig. 4.
N-terminally truncated Rch1 inhibits nuclear
import of EBNA1. Human 293 GP cells were transfected with control
plasmid pCEP 4 expressing EBNA1 and plasmid pCEP 4 R expressing both
EBNA1 and truncated Rch1. The truncated Rch1 consists of amino acids 251-529 of Rch1. The cytosolic and nuclear fractions (lanes designated C or N, respectively) of cells transfected with
the parental vector (lanes designated 293 GP, pCEP 4) or the
vector expressing the truncated Rch1 (lanes 293 GP, pCEP 4 R) were analyzed by Western blot analysis using the EBNA1-specific
mAb 1H4 (lanes 1-5) or the Rch1-specific mAb 2G7
(lanes 6-10). Extract from insect cells expressing
recombinant EBNA1 (lane 1) or bacterially expressed Rch1
(lane 6) served as a control. The marker proteins were as in
Fig. 1; the positions of EBNA1, full-length Rch1, and truncated Rch1
are indicated by arrows on the right side of the
figure.
[View Larger Version of this Image (56K GIF file)]
Fig. 5.
Zone velocity sedimentation of Rch1 protein
and EBNA1 protein. Extracts of EBV-negative cells, BJAB
(A), and EBV-positive cells, iB4 (B and
C), were separated by centrifugation through a 5-30%
sucrose gradient (39). Fifteen fractions (1, bottom: 30%;
15, top: 5%) were collected and were subjected to 10%
SDS-PAGE followed by immunoblotting with anti-Rch1 mAb (A
and B) and anti-EBNA1 mAb (C). Sedimentation
standards (29 kDa, fraction 15; 67 kDa, fraction 13; 150 kDa, fraction
11) are indicated by arrowheads above panel (A).
U, unfractionated cell extract. The positions of molecular
mass marker proteins (in kDa, see Fig. 1) and of Rch1 and EBNA1 are
indicated.
[View Larger Version of this Image (55K GIF file)]
2,
which is also called importin
or Rch1. To our knowledge, this is
the first description of such a cellular partner protein of EBNA1.
2 is involved in the nuclear import of proteins (13,
14). By another line of experimentation, it was shown that a protein
termed Rch1, which is identical to karyopherin
2, binds to the
recombination activation protein RAG1 (17, 18). Furthermore, it was
shown that the presence of EBNA1 in B lymphocytes induces the
expression of the recombination activating genes RAG1 and
RAG2 (16). The proteins, in turn, are necessary and
sufficient to induce the V(D)J-recombination, which is a crucial step
in B-cell maturation. However, the same mechanism leading to the
rearrangement of the immunoglobulin loci in B cells might be active
during the chromosomal translocation involving the immunoglobulin loci
and the c-myc gene, which ultimately leads to the genesis of
Burkitt's lymphoma (53). At this point, it remains unclear whether the
transport of both RAG1 and EBNA1 by the nuclear transporter Rch1 and
the stimulation of RAG1 (and RAG2) expression by EBNA1 is coincidental.
EBNA1 has an intrinsic property to bind to DNA, and it is known that
EBNA1 can juxtapose segments of DNA that are otherwise apart from each
other (49, 54). It is conceivable that EBNA1 might be involved in the
formation of DNA complexes that undergo illegitimate recombination as
observed in Burkitt's lymphoma. This recombination event in turn might
be induced by the EBNA1-stimulated expression of RAG1 and RAG2.
*
This work was supported by the Deutsche
Forschungsgemeinschaft through Sonderforschungsbereich 399, Project B1.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-6841-163983; Fax: 49-684-63980; E-mail:
graesser{at}med-rz.uni-sb.de.
1
The abbreviations used are: EBV, Epstein-Barr
virus; NLS, nuclear localization signal; mAb, monoclonal antibody; PCR,
polymerase chain reaction; PAGE, polyacrylamide gel
electrophoresis.
2
N. Fischer, unpublished observations.
6
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.