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Journal of Virology, June 1999, p. 5214-5219, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Epstein-Barr Virus (EBV) Nuclear Protein 2-Induced Disruption
of EBV Latency in the Burkitt's Lymphoma Cell Line Akata: Analysis
by Tetracycline-Regulated Expression
Shigeyoshi
Fujiwara,*
Yoshikazu
Nitadori,
Hiroyuki
Nakamura,
Takashi
Nagaishi, and
Yasushi
Ono
Department of Microbiology, Nihon University
School of Medicine, Oyaguchikami-machi, Itabashi-ku, Tokyo
173-8610, Japan
Received 4 December 1998/Accepted 16 March 1999
 |
ABSTRACT |
The Burkitt's lymphoma (BL) cell line Akata retains the latency I
program of Epstein-Barr virus (EBV) gene expression and cross-linking
of its surface immunoglobulin G (IgG) by antibodies results in
activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was
artificially expressed by a constitutive expression vector, the Cp EBNA
promoter remained inactive and accordingly the latency III program was
not induced. In contrast, expression of LMP2A and activity of the Fp
lytic promoter were activated. Consistent with this Fp activity, the
rate of spontaneous activation of the EBV replicative cycle was
increased significantly, suggesting the possibility that EBNA2 can
induce EBV replication. The efficiency of anti-IgG-induced activation
of the viral replication was reduced in Akata cells expressing EBNA2.
To obtain more direct evidence for EBNA2-induced activation of the EBV
replicative cycle, this protein was next expressed by a
tetracycline-regulated expression system. EBNA2 was undetectable with
low doses (<0.5 µg/ml) of tetracycline, while its expression was
rapidly induced after removal of the antibiotic. This induced
expression of EBNA2 was immediately followed by expression of EBV
replicative cycle proteins in up to 50% of the cells, as shown by
indirect immunofluorescence and immunoblot analysis. These results
suggest an unexpected potential of EBNA2 to disrupt EBV latency and to
activate viral replication.
| |
TEXT |
Epstein-Barr virus (EBV) (for
reviews, see references 18 and
25) is a ubiquitous herpesvirus, endemic in human
populations throughout the world. EBV has been associated with the
pathogenesis of a number of malignancies, including Burkitt's lymphoma
(BL), nasopharyngeal carcinoma (NPC), Hodgkin's disease, peripheral T-cell lymphoma, gastric carcinoma, and immunoblastic lymphoma in
immunosuppressed patients. EBV is also the cause of infectious mononucleosis, a self-limiting lymphoproliferative disorder. In vitro,
EBV infection of human mature B lymphocytes results in morphological
transformation resembling lymphocyte activation and establishment of
lymphoblastoid cell lines (LCLs) with capability of unlimited growth in culture.
Two different programs of latent EBV gene expression have been
described in B cells that are latently infected with the virus. The
latency I program is exemplified by BL cells in vivo and is characterized by selective expression of the EBV nuclear antigen 1 (EBNA1), BARF0, and occasionally the latent membrane protein 2A (LMP2A)
(10, 27). The other program, latency III, seen in
immunoblastic lymphomas in immunosuppressed patients and
EBV-immortalized LCLs in vitro, is characterized by expression of six
different EBNAs (EBNAs 1, 2, 3A, 3B, 3C, and LP), three LMPs (LMPs 1, 2A, and 2B), and BARF0 (reviewed in reference 18).
EBNA2 is essential for the transformation of B lymphocytes (3, 12,
17) and plays a central role in latency III by up-regulating
promoters for all these latent EBV genes (1, 6, 15, 16, 33, 36,
39, 44). EBNA2 exerts its transcriptional activation function by
masking the transcriptional repression domain of the recombination
signal-binding protein J
(RBP-J) (11, 13, 14, 43).
Although typical BL cells exhibit the latency I program in vivo, this
program is not usually retained in vitro and is replaced by the latency
III program after long-term culture (10, 27). In this
context, the Akata BL line (34) is exceptional in that
latency I has been maintained through long-term in vitro culture.
Another unique property of Akata cells is that they have a tendency to
lose EBV genomes spontaneously and to give rise to virus-negative
sublines (30). Akata cells express surface immunoglobulin G
(IgG) molecules, and their cross-linking by antibodies results in
activation of EBV replication, through signal transduction pathways
involving Ca2+ mobilization and activation of protein
kinase C (5, 35). In contrast, EBV genomes in LCLs with the
latency III phenotype are not significantly activated by ligation of
surface immunoglobulin molecules. To examine the effects of EBNA2 on
EBV gene expression and anti-IgG-induced viral replication in Akata
cells, the EBNA2 gene was introduced by gene transfer experiments.
Establishment of Akata clones stably expressing EBNA2.
For
stable and constitutive expression of EBNA2, the expression plasmid
pOH-SGE2 was constructed. An AccII-DraI fragment
(B95-8 coordinates 48472 to 50303) of EBV DNA including the entire
EBNA2 coding region was cloned into the EcoRI site of the
eukaryotic expression vector pSG5 (Stratagene) after ligation with
an EcoRI linker, and the resulting construct was termed
pSGE2. EBV Ori-P DNA fragment (SphI-SacII
fragment corresponding to B95-8 coordinates 7333 to 9516) was cloned
into the SmaI site of the plasmid vector pBluescript SK(
)
(Stratagene) by blunt-end ligation. This construct was then opened by
digestion with ClaI and SalI and ligated with a
ClaI-SalI fragment containing the simian virus 40 (SV40) early promoter-driven hygromycin B phosphotransferase gene
(8), and the resulting plasmid was termed pOH. A fragment
containing the SV40 promoter, 
-globin intron, EBNA2 gene, and
poly(A) signal was excised from pSGE2 by digestion with SalI
and ligated with pOH that had been digested by the same enzyme, to
generate pOH-SGE2. pOH-SG2E is a control plasmid with its EBNA2 gene
put in a reverse direction with respect to the SV40 promoter of pSG5.
When EBNA1 is provided in trans, Ori-P is the only
cis element required for episomal persistence of a plasmid
(40, 41). Since Akata cells produce EBNA1, expressed from
their endogenous EBV genomes, pOH-SGE2 was expected to be maintained in
Akata cells as multiple copies of episomes, thereby facilitating
efficient expression of EBNA2.
pOH-SGE2 or pOH-SG2E was transfected into Akata cells by
electroporation, and Akata clones capable of growing with 300 µg of
hygromycin B per ml were selected. These clones were further examined
for expression of EBNA2 by the labeled streptavidin biotin (LSAB)
method using the PE2 anti-EBNA2 monoclonal antibody (42). In
total, 220 hygromycin-resistant clones were screened, and two clones
were identified in which the majority of the cells were positive for
EBNA2 (Fig. 1). Concurrently, two
hygromycin-resistant Akata clones were isolated after transfection with
pOH-SG2E and used as negative controls.
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FIG. 1.
Constitutive expression of EBNA2 in Akata clones
harboring pOH-SGE2. (A) Immunoblot analysis. Cellular lysates from
Akata cells (lane 1), Akata clones harboring pOH-SG2E (lanes 2 and 3),
Akata clones harboring pOH-SGE2 (lanes 4 and 5), B95-8 cells (lane 6),
and an LCL (lane 7) were examined by immunoblot analysis with the PE2
anti-EBNA2 monoclonal antibody. Cell lysates representing 5 × 105 cells were analyzed in each lane. Horseradish
peroxidase-conjugated antibody to mouse immunoglobulins was used as
secondary antibody, and the membrane was developed by the enhanced
chemiluminescence method (Pharmacia). (B) Immunoenzymatic staining.
Smears of Akata cells (a), an Akata clone harboring pOH-SG2E (b), and
an Akata clone harboring pOH-SGE2 (c) were examined with the PE2
antibody by the LSAB method (DAKO), following the protocol supplied by
the manufacturer.
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|
Influence of EBNA2 expression on other latent EBV genes.
Since
EBNA2 is known to up-regulate transcription from latent EBV promoters,
such as Cp and LMP1 and LMP2 promoters (1, 6, 15, 16, 33, 36, 39,
44), activities of some of these promoters as well as expression
of latent EBV genes characteristic to latency III were examined by the
reverse transcription-PCR method as described previously
(24). The results are shown in Fig.
2A and indicated that Cp was not
detectably activated by expression of EBNA2, and this is consistent
with the absence of detectable levels of EBNA3A and EBNA3B mRNAs. The
EBNA2 mRNAs transcribed from the endogenous Akata EBV genome were not
detected either, confirming that EBNA2 detected in these cells is
indeed expressed from the transfected plasmid. Consistent with the
latency I program, the Qp-derived EBNA1 mRNA was detected in both Akata transfectants and controls. LMP1 was detected by the S12 antibody (21) in an Akata transfectant clone expressing EBNA2, but
its level was much lower than those in B95-8 and LCLs (Fig. 2B). In contrast, mRNAs transcribed from the Fp promoter and those coding for
LMP2A were evident in EBNA2-positive cells and not in control cells
(Fig. 2A). EBNA2-induced LMP2A expression is consistent with previous
work (44), yet activation of Fp was unexpected and suggested
a possibility that expression of EBNA2 is associated with activation of
EBV replication, since this promoter was recently shown to be activated
in lytic EBV infection (20, 28).
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FIG. 2.
Analysis of latent EBV gene expression in Akata cells
expressing EBNA2. (A) Reverse transcription-PCR analyses. Messenger
RNAs coding for EBNA2, EBNA3A, EBNA3B, and LMP2A, Qp-derived EBNA1
mRNA, and activities of Fp and Cp were assayed with the primers listed
below. Two Akata transfectant clones harboring pOH-SGE2 (lanes 5 and
6), and two control Akata clones harboring pOH-SG2E (lanes 3 and 4)
were examined. As references, B95-8 cells (lane 1) and EBV-negative
Ramos cells (lane 2) were also examined. The sequences and the B95-8
EBV nucleotide coordinates of PCR primers used are as follows: EBNA2
primers, 5'-AGAGGAGGTGGTAAGCGGTTC-3' (nucleotide [nt]
14802 to 14822) and 5'-TGACGGGTTTCCAAGACTATCC-3' (nt 48584 to 48563); EBNA3A primers, 5'-TTAGGAAGCGTTTCTTGAGC-3' (nt
67483 to 67502) and 5'-TCTTCCATGTTGTCATCCAGGG-3' (nt 92292 to 92271); EBNA3B primers, 5'-TTAGGAAGCGTTTCTTGAGC-3' (nt
67483 to 67502) and 5'-CATAATCTGGTGGGTCCTCGG-3' (nt 95431 to
95411); LMP2A primers, 5'-ATGACTCATCTCAACACATA-3' (nt 166874 to 166893) and 5'-gacgaattcTTTCCAGTGTAAGGCAGTAG-3' (nt 1639 to 1620); primers for Qp-initiated EBNA1 mRNA,
5'-GTGCGCTACCGGATGGCG-3' (nt 62440 to 62457) and
5'-CATTTCCAGGTCCTGTACCT-3' (nt 107986 to 107967); primers
for Fp-initiated mRNAs, 5'-ACCCTCCTGTCACCACCTCC-3' (nt 62284 to 62303) and 5'-ATGCCCTGAGACTACTCTCT-3' (nt 67563 to
67544); and primers for Cp-initiated mRNAs,
5'-CATCTAAACCGACTGAAGAA-3' (nt 11470 to 11479 and nt 11626 to 11635) and 5'-CCCTGAAGGTGAACCGCTTA-3' (nt 14832 to
14813). The sequences and the B95-8 nucleotide coordinates of the
probes used are as follows: EBNA2 probe,
5'-GAGAGTGGCTGCTACGCATT-3' (nt 47885 to 47904); probe for
EBNA3A and EBNA3C, 5'-AGAGAGTAGTCTCAGGGCAT-3' (nt 67544 to
67563); LMP2A probe, 5'-gacggatccATGCTTGTGCTCCTGATACT-3' (nt
548 to 567); probe for Qp-initiated EBNA1 mRNA,
5'-AGAGAGTAGTCTCAGGGCAT-3' (nt 67544 to 67563); probe for
Fp-initiated mRNAs, 5'-TTAGGAAGCGTTTCTTGAGC-3' (nt 67483 to
67502); and probe for Cp-initiated mRNAs,
5'-TGGGCGACCGGTGCCTTCTT-3' (nt 14740 to 14721). Lowercase
letters represent non-EBV sequences attached for cloning purposes. (B)
Immunoblot analysis. Two Akata transfectant clones harboring pOH-SGE2
(lanes 5 and 6) and control Akata clones harboring pOH-SG2E (lanes 3 and 4) were examined for expression of LMP1 by the S12 monoclonal
antibody. As references, EBV-negative BJAB cells (lane 1), Akata cells
(lane 2), B95-8 cells (lane 7), and an LCL (lane 8) were also examined.
The arrowhead indicates the truncated form of LMP1 characteristic of
lytic infection.
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|
Spontaneous disruption of EBV latency in Akata cells expressing
EBNA2.
To test if expression of EBNA2 has any influence on
spontaneous and anti-IgG-induced disruption of EBV latency, the two
Akata transfectant clones expressing EBNA2 and two control clones were cultured with or without goat affinity-purified antibodies to human IgG
(whole molecule) (Cappel) and activation of viral cycle was assessed by
indirect immunofluorescence (Table 1).
Without anti-IgG, less than 0.1% of the control transfectant cells
expressed the early antigens (EA). Upon stimulation with anti-IgG, EA
was induced in 15 to 29% of these control cells. In contrast, an
unexpectedly large fraction (1.1 to 4.8%) of the EBNA2-expressing
cells were shown already positive with EA even without treatment
with anti-IgG. After addition of anti-IgG, however, the increase in the
number of EA-positive cells was significantly smaller than that of
control cells (1.4 to 8.1%).
Tetracycline-regulated expression of EBNA2 in Akata cells.
The
results described above suggested that EBNA2 has a potential to induce
EBV cycle, and EBV replication is generally presumed to exert
deleterious effects on cells. It was therefore suspected that
significant numbers of EBNA2-positive cells were lost after transfection with pOH-SGE2. The unexpectedly low rate (2 of 220) of
EBNA2-expressing clones among hygromycin-resistant clones (see above),
despite the presence of both the EBNA2 gene and the hygromycin resistance gene on the same plasmid, supported this notion. To obtain
more direct evidence of EBNA2-induced activation of EBV replication in
Akata cells, a tetracycline-regulated expression system was used
(9, 31). Two plasmids, pTet-SGE2 and pTAk-Hyg, were
constructed according to the scheme described by Floettmann and others
(7). pSGE2 was digested with ClaI and
SalI and the fragment containing the -globin intron,
EBNA2 gene, and poly(A) signal was isolated. This fragment was then
inserted by blunt-end ligation in the EcoRV site of
pTet-Splice (GIBCO-BRL), which contains the tetracycline-responsive
promoter, and the resulting plasmid was termed pTet-SGE2. A hygromycin
resistance gene (ClaI-SalI fragment)
(8) was inserted by blunt-end ligation in the
NotI site of pTet-tTAk (GIBCO-BRL), which encodes the
tetracycline-regulated transactivator, and the resulting construct was
designated pTAk-Hyg. Akata cells cotransfected with pTet-SGE2 and
pTAk-Hyg were kept in the presence of 0.5 µg of tetracycline per ml
and were selected for resistance to hygromycin. Hygromycin-resistant
clones were further examined for EBNA2 expression after tetracycline
was removed from the culture medium. A number of Akata clones that
expressed EBNA2 in a tetracycline-regulated manner were isolated, and
three such clones are shown in Fig. 3A.
In the presence of 0.5 µg of tetracycline per ml, EBNA2 was not
detected either by immunoblot analysis or immunoenzymatic staining.
Upon removal of tetracycline, EBNA2 expression was efficiently induced
and the level of its expression was higher than the average among
EBV-immortalized LCLs (Fig. 3A). Dose response analysis indicated that
a threshold level of tetracycline concentration exists around 10 to 20 ng/ml and that two- to fourfold dilution spanning this range gave
almost full induction (data not shown). When the cells were examined at
various times after removal of tetracycline, EBNA2 was first detected
at 12 h and reached the plateau by 48 h (Fig. 3C).
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FIG. 3.
Tetracycline-regulated expression of EBNA2 in Akata
cells. (A) Immunoblot analysis. Cells of three Akata transfectant
clones (lanes 2 through 7) were maintained for 48 h with (lanes 2, 4, and 6) or without (lanes 3, 5, and 7) tetracycline, and cell lysates
were analyzed by immunoblotting with the PE2 monoclonal antibody. As
references, untransfected Akata cells (lane 1), B95-8 cells (lane 8),
and two LCLs (lanes 9 and 10) were also analyzed. (B) Immunoenzymatic
staining. Cell smears of an Akata transfectant clone harboring
pTet-SGE2 and pTAk-Hyg were cultured for 48 h with (a) or without
(b) tetracycline and examined with the PE2 antibody by the LSAB method.
(C) Time course of EBNA2 expression after removal of tetracycline.
Cells of an Akata transfectant clone harboring pTet-SGE2 and pTAk-Hyg
were washed twice with fresh tetracycline-free culture medium and then
resuspended in the same medium (0 h). Thereafter, cell lysates were
prepared at the indicated times after the start of culture and analyzed
by immunoblot analysis.
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Disruption of EBV latency by inducible EBNA2 expression.
Smears of an Akata transfectant clone were prepared at 72 h after
removal of tetracycline and examined for expression of EA and viral
capsid antigens (VCA) by indirect immunofluorescence. The results are
shown in Fig. 4A and indicate that EA was
induced in more than 50% of the cells and that VCA was induced in
around 25% of the cells. Similar levels of EA and VCA were detected in three other Akata transfectant clones after the removal of tetracycline (data not shown). Proliferation of these cells was significantly retarded, and their viability declined in several days after induction of EBNA2 (data not shown). Expression of EBV lytic-cycle proteins similar to those induced by anti-IgG antibodies was also detected by
immunoblot analysis of three Akata transfectant clones (Fig. 4B). As
expected, no such replication cycle proteins were induced in control
Akata transfectants harboring pTAk-Hyg alone (Fig. 4A and B). These
lytic-cycle proteins were first detected at a low level at 18 h
after the removal of tetracycline, increased until 36 h, and
remained at a plateau until 96 h (Fig. 4C). The BZLF1 protein, an
immediate-early protein critical to activation of EBV replication from
the latent state, was also shown to be induced (Fig. 4D). A separate
immunoblot analysis using a monoclonal antibody to the BFRF3
protein, an immunodominant component of the EBV capsid, indicated that
the 18-kDa band seen in Fig. 4B and C corresponds to this protein (data
not shown).
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FIG. 4.
Expression of EBV replicative cycle proteins following
induced expression of EBNA2. (A) Immunofluorescence. An Akata
transfectant clone harboring both pTet-SGE2 and pTAk-Hyg were
maintained in culture medium with (a) or without (b and c) tetracycline
and examined by indirect immunofluorescence. Serum from an NPC patient
was used to detect EA (a and b) and that from a healthy EBV carrier was
used to detect VCA (c). As a reference, a control Akata transfectant
clone harboring pTAk-Hyg alone was maintained in tetracycline-free
medium and examined with serum from a patient with NPC (d). (B)
Immunoblot analysis. Three Akata transfectant clones harboring
pTet-SGE2 and PTAk-Hyg (lanes 3 through 8) and a control Akata clone
harboring pTAk-Hyg alone (lanes 9 and 10) were washed twice with
tetracycline-free fresh medium and cultured for 48 h in the same
medium (lanes 3, 5, 7, and 9) or medium containing tetracycline (lanes
4, 6, 8, and 10) and examined by immunoblot analysis with pooled sera
from patients with NPC. As a reference, Akata cells before (lane 1) and
after (lane 2) treatment with anti-IgG antibodies were also examined.
(C) Time course of induction of EBV replicative cycle proteins. Protein
samples prepared at the indicated times after removal of tetracycline
from culture medium were probed with pooled sera from patients with
NPC. The protein samples are identical to those shown in Fig. 3C. (D)
Time course of synthesis of the BZLF1 protein. Protein samples prepared
at the indicated times after removal of tetracycline from culture
medium were probed with the BZ.1 monoclonal antibody. The protein
samples are identical to those shown in Fig. 3C.
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|
In this study, EBNA2 was artificially expressed in Akata cells first by
the vector pOH-SGE2 that achieved its constitutive
expression. The
result of this experiment indicated that in Akata
cells expressing
EBNA2 (i) the rate of spontaneous activation
of the EBV replicative
cycle was increased significantly, (ii)
the efficiency of the
anti-IgG-induced viral cycle was decreased,
and (iii) the Cp promoter
was not activated and therefore the
latency III program was not
induced. The decreased rate of anti-IgG-induced
viral cycle is not due
to down-regulation of surface IgG expression,
since flow cytometrical
analysis with fluorescein isothiocyanate-conjugated
anti-human
IgG indicated that EBNA2 did not alter its level on
the cell
surface (data not shown). Instead, it is better explained
by the
EBNA2-induced up-regulation of LMP2A. LMP2A has been shown
to block
Ca
2+ mobilization and thereby to impede activation of the
EBV cycle
triggered by cross-linking of surface immunoglobulins
(
22).
The Cp promoter, a hallmark of latency III, was not
induced by
EBNA2, and this is consistent with the recent finding that
DNA
methylation around Cp is a decisive factor in the maintenance
and
possibly establishment of latency I (
23,
26,
29).
Inducible EBNA2 expression by the tetracycline-regulated system
confirmed and gave more decisive evidence for the disruption
of EBV
latency induced by the protein. The EBV replicative cycle
was more
efficiently induced (>50%) as compared with the experiments
with the
noninducible vector pOH-SGE2 (1.1 to 4.8%). Considering
the
deleterious effects of the EBV replicative cycle on cells,
clones with
lower rates of EBV activation should have an advantage
after
transfection with pOH-SGE2. The lower rates of EBV activation
in the
pOH-SGE2 transfectant clones are thus likely to be a result
of
selection. The relationship between the EBNA2 dose and activation
of
EBV cycle remains to be
elucidated.
Akata is a rare exception among BL cell lines in that its latency I
phenotype was not replaced by latency III after long-term
in vitro
culture. The EBNA2-induced disruption of the EBV cycle
may provide an
explanation for this unique property of the cell
line. Similar to other
BL-derived cell lines, occasional Akata
cells may express EBNA2
spontaneously, yet instead of inducing
the latency III phenotype, EBNA2
expression will result in activation
of the EBV replicative cycle and
cell death. This scenario may
be also relevant to the mechanism of
spontaneous loss of EBV genomes
from Akata cells. If the rate of
spontaneous EBNA2 expression
and consequent viral replication is beyond
a certain level, cells
that have lost EBV genomes should have an
advantage for survival.
It will be interesting to test if EBNA2
activates viral cycles
in the other few BL cell lines that retain the
latency I
phenotype.
The effect of EBNA2 on the expression of other EBV and cellular genes
has been analyzed mainly with gene transfer experiments
with
constitutive expression vectors. These experiments provided
evidence that EBNA2 plays a central role in the latency III program
by
transactivating the Cp EBNA promoter and promoters for LMP1
and LMP2
(
1,
6,
15,
16,
33,
36,
39,
44). It
was also demonstrated
that, in cooperation with LMP1, EBNA2 is
responsible for the induction
of the activated B-cell phenotype
(
2-4,
12,
17,
19,
32,
37,
38). In these previous
experiments, the EBNA2 gene was
transferred mainly to EBV-negative
cell lines, and, to our knowledge,
its effects on replicative-cycle
genes have not been investigated. The
present study, in contrast,
employed an inducible expression vector and
chose EBV-positive
BL cells with the latency I phenotype as recipients.
This unconventional
approach has been essential for the unexpected
finding of the
EBNA2-induced EBV cycle. This EBV-activating potential
of EBNA2
may be dependent on cellular phenotype, because the protein is
expressed in LCLs of the latency III phenotype without apparently
inducing EBV replication. It is also plausible that EBNA2 acts
differently on gene regulation depending on its level of expression
or
the presence of other latent EBV proteins or
both.
The molecular mechanism involved in the EBNA2-induced EBV cycle remains
an open question. Since the RBP-J
-binding motif (GTGGGAA)
is not found close to immediate-early EBV genes such as BZLF1
and
BRLF1, it does not appear likely that EBNA2 directly transactivates
them. A more likely mechanism is that EBNA2 primarily
transactivates
certain cellular genes and that their products
change the cellular
environment to enhance EBV activation. Although the
significance
of EBNA2-induced activation of viral cycle in the
physiology of
EBV is not known now, it may shed light on an unknown
area of
the EBNA2
function.
| |
ACKNOWLEDGMENTS |
We thank Kenzo Takada for EBV DNA clones.
This work was supported by the High-Tech Research Center grant from the
Japanese Ministry of Education, Science, and Culture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Nihon University School of Medicine, Oyaguchikami-machi, Itabashi-ku, Tokyo 173-8610, Japan. Phone: 81-3-3972-8111, ext. 2263. Fax: 81-3-3972-9560. E-mail: shige{at}med.nihon-u.ac.jp.
Present address: Department of Pediatrics, Surugadai Hospital,
Nihon University School of Medicine, Kandasurugadai, Chiyoda-ku, Tokyo
101-8309, Japan.
| |
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Journal of Virology, June 1999, p. 5214-5219, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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