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Journal of Virology, December 1999, p. 9858-9866, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role of the Epstein-Barr Virus Rta Protein in
Activation of Distinct Classes of Viral Lytic Cycle Genes
Tobias
Ragoczy1 and
George
Miller1,2,*
Departments Molecular Biophysics and
Biochemistry,1
Pediatrics,2 and Epidemiology
and Public Health,3 Yale University School
of Medicine, New Haven, Connecticut 06520
Received 28 May 1999/Accepted 23 August 1999
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ABSTRACT |
Initiation of the Epstein-Barr virus (EBV) lytic cycle is
controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta,
stimulate their own expression, reciprocally stimulate each other's
expression, and activate downstream viral targets. It has been
difficult to examine the individual roles of these two transactivators
in EBV-infected lymphocytes, as they are expressed simultaneously upon
induction of the lytic cycle. Here we show that the Burkitt lymphoma
cell line Raji represents an experimental system that allows the study
of Rta's role in the lytic cycle of EBV in the absence and presence of
ZEBRA. When expressed in Raji cells, exogenous Rta does not activate
endogenous BZLF1 expression, yet Rta remains competent to transactivate
certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and
a late gene, BLRF2, are maximally activated by Rta itself in the
absence of detectable ZEBRA. The use of the Z(S186A) mutant form of
ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes,
such as BMRF1 and BHRF1, that are activated in synergy by Rta and
ZEBRA. It has already been documented that of the two activators, only
ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third
class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA
exhibits an inhibitory effect on Rta's capacity to stimulate the late
gene, BLRF2. Consequently ZEBRA may function to repress Rta's
potential to activate some late genes. Raji cells thus allow
delineation of the combinatorial roles of Rta and ZEBRA in control of
several distinct classes of lytic cycle genes.
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INTRODUCTION |
The reactivation of Epstein-Barr
virus (EBV) from latency is associated with the expression of two
immediate-early genes, BZLF1 and BRLF1, whose products are
transcriptional activators that drive the lytic cascade of the virus
(12, 36, 44, 51, 54). These two genes lie in overlapping
transcriptional units and are expressed simultaneously during induction
of the lytic cycle (35, 50). Until recently ZEBRA, the
product of BZLF1, had been thought to be the only viral protein capable
of initiating the lytic cycle (12, 22, 45, 51). Recently
Rta, the product of BRLF1, was also shown to be able to disrupt latency
in epithelial cells and in certain B-cell lines (44, 54). In
those cells Rta leads to activation of Zp, the promoter of BZLF1,
expression of ZEBRA, and thereby stimulation of early lytic genes, DNA
replication, and late gene expression.
In the past there had been suggestions that cell line and cell type
differences played a role in determining the extent to which Rta is
able to induce the lytic cycle. Both early and recent studies failed to
find any evidence that Rta mediated disruption of latency in Raji
cells, a Burkitt lymphoma (BL) cell line (1, 13). Bogedain
et al. suggested that Rta might be more active than ZEBRA in activating
early lytic cycle genes in lymphoblastoid cell lines, while Rta had
little effect in BL cell lines (6). Zalani et al. reported
that Rta was able to induce the lytic cycle in an epithelial
cell-specific manner but not in B cells (54). We have shown
that Rta does have the capability to activate lytic cycle genes in
B-cell lines such as the HH514-16 derivative of the P3JHR-1 (HR1) BL
cell line (44).
These varied and sometimes contradictory results have caused us to
examine more closely the effects of viral and cellular background on
the activity of Rta. Here we describe the activity of Rta in the BL
cell line Raji and compare its behavior to that in the HH514-16 cell
line. In HH514-16 cells Rta activates the promoter of BZLF1 (Zp) and
that of its own gene, BRLF1 (Rp) (44). However, we show here
that in Raji Rta activates neither of these two promoters and fails to
stimulate detectable ZEBRA expression. Nonetheless, Rta is a competent
transactivator of other downstream lytic cycle genes in Raji cells.
These genes include one class which responds maximally to Rta and
another group which requires the concomitant activities of Rta and
ZEBRA. Moreover, in the absence of ZEBRA, Rta can bypass a requirement
for DNA synthesis in stimulating expression of the late gene BLRF2 in
Raji cells. ZEBRA exerts an inhibitory effect on Rta's action on late genes.
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MATERIALS AND METHODS |
Cell lines.
Cells were maintained in 5% CO2 at
37°C in RPMI 1640 supplemented with 8% fetal calf serum. HH514-16 is
a clonal derivative of the P3J-HR-1 B-cell line derived from an
EBV-positive BL that is permissive for viral replication
(43); Raji is a human B-cell line derived from a BL
containing an EBV strain that is defective for DNA replication and late
gene expression (42); B95-8 is a marmoset B-cell line
transformed with EBV (37).
Antisera.
Anti-Rta is a polyclonal rabbit antiserum raised
to an N-terminal fragment of Rta (44). Anti-ZEBRA and
anti-BLRF2 are polyclonal rabbit antisera raised to TrpE-BZLF1 and
TrpE-BLRF2 fusion proteins (26, 48). R3 is a mouse
monoclonal antibody to BMRF1 (39), and SJ is a human serum
with reactivity to EBNA1 and BFRF3 (48).
Chemical induction and PAA treatment.
Cells were subcultured
3 days prior to drug treatment or transfection. Drug treatment
consisted of the addition of 10 ng of tetradecanoylphorbol-13-acetate
(TPA) per ml and 3 mM sodium butyrate to the culture medium. In assays
for late gene expression, the viral polymerase inhibitor
phosphonoacetic acid (PAA) was added to the culture medium to 0.4 mM at
the same time as the drug treatment or immediately following
transfection (52).
Expression vectors and reporter plasmids.
The Rta and ZEBRA
expression vectors pRTS/Rta, pBXG1-genomic Z (pBXG1/genZ), and CMV
(cytomegalovirus)-genomic Z(S186A) [pCMV/genZ(S186A)] and their
parent vectors pRTS, pBXG1, and pHD1013 have been described previously
(14, 18, 44). The RpCAT constructs were generated by cloning
the PCR-amplified sequence spanning nucleotides 106123 to 107143 of the
EBV genome (relative to the B95-8 prototype [2]) into
the XbaI and SphI sites of the chloramphenicol
acetyltransferase (CAT) expression plasmid pCAT basic (Promega). PCRs
were performed with total cellular DNA from Raji, HH514-16, and B95-8
cells. The ZpCAT constructs were made in a similar manner by cloning the PCR-amplified BZLF1 promoter fragment (nucleotides 103182 to 103742 of the EBV genome) into pCAT basic. The cloning sites were incorporated
into the PCR primers. E4CAT, containing a minimal adenovirus E4
promoter, Z3E4CAT, containing three consecutive ZIIIB sites upstream of
this minimal promoter, and the luciferase control vector pGL2 basic+HMP
have been described elsewhere (9, 49).
Transfections.
Transfections were carried out by
electroporation (49). For Western and Northern analyses, 10 µg of plasmid DNA (5 µg of each expression vector or 5 µg of
expression vector plus 5 µg of empty vector, either pBXG1 or pRTS)
was used. For reporter assays, 10 µg of CAT constructs plus 5 µg of
expression vector or pBXG1 or pRTS and 1 µg of pGL2 basic+HMP were used.
Reporter assays.
CAT and luciferase assays were performed as
described elsewhere (49). CAT results represent averages of
at least two separate transfections and are standardized for
transfection efficiency with luciferase activity.
Protein extracts and Western blots.
Cells were collected by
centrifugation, washed once in phosphate-buffered saline, and
resuspended in sodium dodecyl sulfate sample buffer at 106
cells/10 µl. Prior to separation on sodium dodecyl sulfate-12% polyacrylamide gels, samples (20 µl) were heated to 100°C for 5 min. Following electrophoresis, the proteins were transferred to
nitrocellulose membranes by electroblotting and blocked in 5% nonfat
dry milk overnight at 4°C. The blots were incubated with antisera,
diluted in 5% nonfat dry milk, at 25°C for 2 h, then washed
three times for 10 min each in TS (10 mM Tris [pH 7.5], 200 mM NaCl,
5% Tween 20), incubated with 125I-protein A for 1 h,
and washed again. The membranes were exposed overnight with
intensifying screens to Kodak XAR-5 film at 
70°C.
RNA isolation and Northern blots.
Samples of 2.5 × 106 cells were harvested 30 h following transfection.
Cellular RNA prepared by using an RNeasy Mini kit (Qiagen) was
electrophoresed through a 1% agarose-6% formaldehyde gel in 20 mM
MOPS (morpholinepropanesulfonic acid [pH 7.0]); the RNA was
transferred to Nytran and probed with 32P-radiolabeled
oligonucleotides. The BZLF1 oligonucleotide used spanned nucleotides 93 to 128 in exon I of the BZLF1 gene. A 531-bp excised EagI
fragment from EBV BamHI-M was used to detect the BMRF1 and
BaRF1 messages, while a 1.3-kb EcoRI/BamHI
fragment from BamHI-M was used to detect BMLF1 mRNA. The
BHRF1 probe consisted of a 2.477-kb SacI/BamHI
fragment from EBV BamHI-H. A radiolabeled 370-bp
NcoI-PstI fragment of H1 component of RNase P was
used as a probe to control for RNA loading (3).
Sequencing.
Genomic Rp and Zp sequences were PCR amplified
by using the high-fidelity Pfu DNA polymerase (Stratagene)
and primers I (5-CTC GTT AAC TGA GAG C), II (5-GCT CTA GAC TGC CTG ACT
GCG CTG A), III (5-GGA TAG CAG CGG TCC ACC), and IV (5-CAC TGG GAA CAG
CTG AGG). Primers I and II were used for Rp; primers III and IV were used for Zp. Resulting PCR products were gel purified and then sequenced by the HHMI Biopolymer Laboratory & W. M. Keck
Foundation Biotechnology Resource Laboratory at Yale University with
internal Rp and Zp primers. Each promoter segment was sequenced at
least two to three times from independent PCRs.
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RESULTS |
Cell background affects autostimulation of BRLF1 and BZLF1 in
HH514-16 and Raji cells.
In permissive cell lines, such as
HH514-16, transfection of a Rta expression vector leads to stimulation
of its own gene, BRLF1, activation of the other immediate-early gene,
BZLF1, and induction of downstream EBV early lytic cycle genes
(44). As shown in Fig. 1,
transfection of HH514-16 cells with either Rta or ZEBRA activated
transcription from the Rp and Zp promoters, yielding the characteristic
3.0-kb bicistronic and 1.0-kb monocistronic messages, which were also
induced by treating the cells with the chemicals TPA and sodium
butyrate. Although Rta induced expression of the BZLF1 and BRLF1 genes
less strongly than chemicals or ZEBRA, its effect in HH514-16 cells was
highly reproducible. In the BL cell line Raji, by contrast, Rta was
incapable of activating either gene. ZEBRA induced transcription solely
from Rp (Fig. 1B), as has been previously documented (29,
31). The 1.3-kb mRNA originates from the ZEBRA expression vector
(29). Only treatment with inducing chemicals led to the
stimulation of both the Rp and Zp promoters in Raji cells.
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FIG. 1.
Rta and ZEBRA stimulate the BRLF1 and BZLF1 genes to
different extents in HH514-16 and Raji cells. Northern blot analysis of
total cellular RNA isolated 30 h after chemical treatment or
transfection is shown. Cells were either untreated (lanes 1 and 7),
induced with TPA and sodium butyrate (lanes 2 and 8), or transfected
with empty vector (lanes 3, 5, 9, and 11) or Rta and ZEBRA expression
vectors (lanes 4, 6, 10, and 12). Northern blots were probed with a
30-base oligonucleotide from within exon I of BZLF1, which detects the
1.0-kb mRNA originating at Zp, the 3.0-kb bicistronic message from Rp,
and a 1.3-kb vector transcript from BXG1/genZ. A probe for the H1
component of RNase P was used to control for RNA loading. (A) HH514-16
cells; (B) Raji cells.
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Rta's inability to activate either BRLF1 or BZLF1 was not due to poor
Rta expression. In Raji cells, Rta was expressed to
similar levels
whether the cells had been transfected with RTS/Rta
or BXG1/genZ or
treated with inducing chemicals (Fig.
2).
The
transfected Rta protein was functional, as it induced the
expression
of the early antigen complex EA-D. Furthermore, Rta strongly
activated
the putative late gene product of BLRF2, a component of the
viral
capsid (
5,
48). However, in cells transfected with
ZEBRA
or induced by TPA and sodium butyrate, BLRF2 was minimally
induced.
ZEBRA efficiently induced the expression of endogenous Rta at
the protein level as well as at the RNA level, while Rta failed
to
induce detectable ZEBRA.
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FIG. 2.
Immunoblots comparing activation of lytic cycle genes
following the transfection of Rta and ZEBRA into Raji cells. Cells were
either untreated (lane 1), chemically induced with TPA and sodium
butyrate (T/B; lane 2), or transfected with 10 µg of plasmid DNA
(lanes 3 to 7). In lanes 4 and 6, cells received 5 µg of activator
(ZEBRA [Z] or Rta [R]) and 5 µg of empty vector. In lanes 3 and
5, cells received only vector pBXG1 (V1; lane 3) or pRTS (V2; lane 5).
In lane 7, vectors expressing both ZEBRA and Rta were transfected.
Immunoblots prepared 72 h following transfection were probed
sequentially with the indicated antisera for EBNA1, Rta, EA-D, ZEBRA,
BLRF2 (LR2), and BFRF3 (FR3).
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Rta does not detectably activate ZEBRA in Raji cells.
Although
Northern and Western analyses did not reveal any BZLF1 mRNA or ZEBRA
protein in Raji cells following transfection of Rta, we used a highly
sensitive reporter assay intended to detect even trace amounts of
biologically active ZEBRA. The Z3E4CAT reporter contains three copies
of the ZIIIB ZEBRA response element (ZRE) immediately upstream of the
adenovirus minimal E4 promoter fragment linked to the CAT gene in pCAT
(9). ZEBRA synergistically activates this reporter due to
the presence of oligomerized ZREs, which, as we will show, render the
reporter extremely responsive to even minute amounts of ZEBRA protein.
The responsiveness of Z3E4CAT to ZEBRA is shown in Fig.
3, illustrating a titration in which low
amounts of ZEBRA expression vector produced a dramatic stimulatory
effect on the CAT activity. For example, 1 ng of expression vector
yielded 8.7-fold stimulation of CAT activity over the level in cells
that had been transfected with E4CAT, the corresponding negative
control vector containing the minimal E4 promoter without ZREs. With
increasing amounts of BXG1/genZ, the stimulation index (the ratio of
Z3E4CAT activity over E4CAT activity) quickly rose to well above 100. By contrast, transfection of Raji cells with 5 µg of RTS/Rta resulted
in only a twofold increase in CAT activity over the negative control.
If Rta had induced biologically significant amounts of ZEBRA, we would
have expected a dramatic increase in CAT activity.
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FIG. 3.
Rta does not activate ZEBRA expression in Raji cells.
(A) Reporter assay. Raji cells were transfected with 16 µg of total
plasmid DNA consisting of the indicated amount of Rta or ZEBRA
expression vector (made up to 5 µg with pBXG1; U, only pBXG1), 10 µg of E4CAT or Z3E4CAT reporter vector, and 1 µg of luciferase
control vector. CAT and luciferase assays were performed 72 h
following transfection. CAT activity was standardized for transfection
efficiency on the basis of the luciferase data. Fold activation is the
ratio of CAT activity generated by the reporter E4CAT or Z3E4CAT in the
presence of Rta or ZEBRA divided by the activity in the presence of
empty expression vector. (B) Western blot. Protein extracts made from
the same experiment were probed on an immunoblot for EA-D and ZEBRA.
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To quantitate the sensitivity of this assay, we examined protein
extracts of the transfected Raji cells for ZEBRA and EA-D
expression in
the same experiment (Fig.
3B). Expression of EA-D
would indicate the
presence of sufficient quantities of either
Rta or ZEBRA to be
biologically active on downstream targets in
the viral genome. We could
not detect any ZEBRA in cells transfected
with 5 µg of Rta expression
vector, even after a prolonged (15
days) exposure of the blot to
autoradiography film. ZEBRA was
detectable in cells that had been
transfected with 50 and 100
ng of ZEBRA expression vector. At these two
BXG1/genZ concentrations
EA-D was also induced to appreciable levels.
The level of EA-D
induced by 5 µg of transfected Rta expression
vector was approximately
equivalent to the level induced by 10 to 50 ng
of ZEBRA expression
vector. Were this stimulation due indirectly to the
ability of
Rta to activate endogenous ZEBRA expression, rather than a
direct
effect of Rta, we would expect a stimulation index between 55-
and 111-fold in the CAT assay (Fig.
3A). However, only a twofold
stimulation by Rta was observed. In summary, three lines of evidence
favor the conclusion that Rta does not activate BZLF1 in Raji
cells:
(i) no BZLF1 mRNA is detectable by Northern blotting, (ii)
Rta does not
activate detectable expression of ZEBRA, and (iii)
Rta does not
activate a synthetic reporter that is exquisitely
sensitive to
ZEBRA.
Rta maximally activates some downstream targets in the absence of
ZEBRA in Raji cells.
Having established that Rta is unable to
induce the expression of ZEBRA in Raji cells, we next explored the
capacity of Rta to activate viral targets by itself. The level of lytic
cycle activation was compared in Raji cells transfected with Rta or ZEBRA expression vectors or treated with inducing chemicals. After a
30-h incubation at 37°C, total RNA isolated from the cells was analyzed by Northern blotting. The same blot was probed sequentially for messages from a representative group of viral lytic cycle genes.
These included BRLF1, the gene for Rta (23), BaRF1, the ribonucleotide reductase small subunit gene (19, 20), BMRF1, the viral polymerase processivity factor (28, 32), BMLF1, the early post transcriptional activator (8, 27), and BHRF1, the bcl2 homologue (38). For a loading control, the blot was probed for the RNA H1 component of the cellular RNase P (3). As shown in Fig. 4, all of the genes were
strongly activated by treating the cells with TPA and sodium butyrate
(lane 2) or by transfecting of BXG1/genZ (lane 6). Transfection of Raji
cells with RTS/Rta, on the other hand, did not lead to uniform
activation of the genes selected for study (lane 4). Rta did not
activate BRLF1, and it only weakly activated BMRF1 and BHRF1. However, the remaining two genes, BaRF1 and BMLF1, were activated by Rta at
least as strongly as by inducing chemicals or by ZEBRA (Fig. 4; compare
lanes 2, 4, and 10). These results suggested that BaRF1 and BMLF1 were
maximally stimulated by Rta in the absence of ZEBRA in Raji cells.
Their activation by transfected ZEBRA can be explained by the capacity
of ZEBRA to activate Rta from the endogenous virus.
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FIG. 4.
EBV lytic cycle genes activated by Rta alone or together
with Z(S186A). Cells were either untreated (lane 1), chemically induced
with TPA and sodium butyrate (lane 2), or transfected with 10 µg of
plasmid DNA (lanes 3 to 10). In lanes 4, 6, and 8, cells received 5 µg of activator and 5 µg of empty vector. In lanes 3, 5, and 7, cells received only vector pRTS (lane 3), pBXG1 (lane 5), or pCMV (lane
7). In lanes 9 and 10, Rta was transfected with ZEBRA and the mutant
Z(S186A), respectively. Total RNA prepared 30 h following
transfection was analyzed by Northern blotting using probes for the
indicated genes (see Materials and Methods). The blot was stripped
between probes. Classification of the genes according to primary
activator(s) is indicated to the right (see Discussion). The extra band
above the expected size of the BRLF1 mRNA in lane 10 is most likely the
result of an Rta-activated transcript from the Z(S186A) expression
vector.
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The ZEBRA mutant Z(S186A) allows the classification of genes that
respond in synergy to Rta and ZEBRA.
Genes such as BMRF1 and
BHRF1, that were only weakly activated by Rta, were maximally activated
by ZEBRA. This result could be explained if the genes were exclusively
controlled by ZEBRA or if they were controlled by a combination of
ZEBRA and Rta. To distinguish between these two possibilities, we
utilized the Z(S186A) mutant of ZEBRA. This mutant no longer initiates
the lytic cascade of EBV due to its inability to activate Rp (1, 17). Adding exogenous Rta rescues Z(S186A), and the lytic cycle is successfully triggered. Consequently, the mutant can still act in
synergy with Rta. Supplying Raji cells with either Rta, Z(S186A), or
both permitted us to determine whether target genes were activated in
synergy by Rta and ZEBRA. Raji cells were transfected with RTS/Rta,
CMV/genZ(S186A), and RTS/Rta plus CMV/genZ(S186A). Total RNA isolated
30 h following transfection was examined on the same Northern
blot. The ZEBRA mutant Z(S186A) was unable to activate any of the
examined genes (Fig. 4, lane 8). When Rta was supplied along with
Z(S186A), however, both BHRF1 and BMRF1 were activated to levels far
above those achieved by Rta alone. Moreover, the effects of Z(S186A)
together with Rta were indistinguishable from the effects of Rta
together with wild-type ZEBRA or ZEBRA by itself. BHRF1 and BMRF1
therefore represent genes that are activated in synergy by Rta and
ZEBRA. The signals for BaRF1 and BMLF1, on the other hand, were no
stronger when cells had been transfected with Rta and the Z(S186A)
mutant than with Rta alone, which indicates that these two genes are
exclusive targets of Rta and do not require ZEBRA. BRLF1 represents yet
another class of genes since it was not activated by Rta, by Z(S186A),
or by the two together. Rta did not compensate for the Z(S186A)
mutation in the activation of BRLF1. Thus, in Raji cells Rp is
regulated only by ZEBRA and not by Rta, and not by a combination of the ZEBRA and Rta proteins.
Rp and Zp do not differ significantly in HR1 and Raji cells.
The inability of Rta to activate either BRLF1 or BZLF1 in Raji cells
may be due to cell line differences or to inherent differences in the
viral genomes. The promoter sequences of BRLF1 and BZLF1 could contain
pertinent point mutations or deletions which might interfere with or
result in the loss of regulatory elements within the promoters. To
examine these possibilities, the nucleotide sequences of the Rp and Zp
regions from both Raji and HH514-16 cells were compared to the
sequences in the B95-8 prototype (2). The results of the
analysis are shown in Fig. 5A (Rp) and B
(Zp). Rp from Raji cells differs from the B95-8 sequence in four
locations within the region examined (965 to +140 relative to the
transcription start site). In three locations (840, 708, and +74),
a G has been changed to an A; in one (1), a C has been converted to
an A. The HH514-16 sequence deviates only in one location from B95-8, with the same C-to-A transversion at position 1. The 1 position is
the only location which falls into a known regulatory element of the
promoter, a YY1 binding site that overlaps the transcriptional start
site (53). This mutation within the YY1 site is shared by
the Raji and HH514-16 cell lines. The two point mutations at positions
708 and 840 of Raji Rp are likely to be too far upstream to have
any effect; the mutation at position +74 is significantly downstream of
the transcriptional start site and beyond the 5' splice site of the Rp
message.
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FIG. 5.
Comparison of Rp (A) and Zp (B) promoter sequences among
Raji, HH514-16, and B95-8 genomes. Promoters were amplified from total
Raji and HH514-16 cell DNA by PCR, and sequences were compared to that
of the B95-8 prototype. Deviations from B95-8 and their locations
relative to the transcriptional start site are indicated in bold; known
regulatory sites affected by the mutations are indicated on the right.
The diagrams represent the promoters with their known proximal
regulatory elements. ZRE, ZIIIA, and ZIIIB, ZEBRA response elements; Z1
and ZIA through -D, AT-rich sequences, reported to bind Sp1, Sp3, and
MEF2D (7, 33, 34); SRE, serum response element; OCT,
AP-1-like octamer; TRE, TPA response element.
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Within Zp, the HH514-16 sequence deviates at seven locations from the
B95-8 prototype. All are point mutations matching those
previously
reported by Jenson et al. (
25) and reside at least
99 bases
upstream of the transcriptional start. Five of the changes
are
transitions, while the other two are transversions. The Raji
genome
shares two of these mutations, at positions
364 and
459.
The former
falls within a ZIIIA ZEBRA response element and likely
destroys it
(
30). A mutation within the HH514-16 sequence that
is not
shared with Raji lies within another regulatory element,
the ZIC site
covering position
140. ZI sites have previously
been described as
AT-rich sequences involved in the TPA-mediated
induction of Zp. They
have been shown to bind Sp1, Sp3, and members
of the MEF2 family of
transcription factors (
7,
16,
33,
34). However, the
140
mutation in ZIC lies outside the Sp1/3
binding site (
33,
34). Although this mutation does fall within
the MEF2D
recognition sequence, ZIC is the only member of the
ZI sites that does
not bind MEF2D (
34). Consequently, the overall
function of
the variant ZI site is unlikely to be affected by
the transition. The
remaining four mutations in HH514-16 Zp do
not fall within any known
regulatory
sequences.
Rta activates RpCAT from B95-8, HR1, and Raji genomes equally well
in Raji cells.
We next determined whether the minor sequence
differences observed in Rp and Zp between the HR1 and Raji genomes
altered the response of the two promoters to activation by Rta in a
transient reporter assay in Raji cells. We have shown previously that
Rta not only activates Rp from the endogenous virus in HH514-16 cells but also efficiently activates an RpCAT reporter in these cells (44). The promoter sequences from the B95-8, Raji, and HR1
genomes were cloned into pCAT basic and transfected with or without Rta into Raji cells. As shown in Fig. 6, CAT
reporters bearing Rp sequences from the three different genomes were
activated equally well by Rta in Raji cells. These results suggest that
the differences in response of the endogenous Rp to Rta in Raji and
HH514-16 cells is not likely to be directly attributable to sequence
variations in the Rp promoter. The same comparison was performed using
cloned ZpCAT constructs cotransfected with Rta into Raji cells (data not shown). Again there was no difference in the response of the three
ZpCAT constructs to Rta, a result consistent with the conclusion that
the minor sequence differences do not significantly account for the
major differences in regulation observed in the two cell backgrounds.
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FIG. 6.
Activation of RpCAT containing sequences from B95-8,
HH514-16, and Raji genomes by Rta in Raji cells. See the legend to Fig.
3 for experimental details. Fold activation is the ratio of CAT
activity generated by a reporter in the presence of Rta divided by the
CAT activity in the presence of empty vector. pCAT, reporter lacking
promoter or enhancer sequences; RpCAT, Rp from the three respective
genomes cloned into pCAT.
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Rta bypasses the requirement for DNA replication in the activation
of a late gene, BLRF2, in Raji cells.
Since Raji cells are
deficient for expression of late genes (4, 55), the ability
of Rta to strongly activate expression of the specific mRNAs of BLRF2
(5) in Raji cells was unexpected (Fig. 2). BLRF2 has
previously been classified as a late gene whose expression is dependent
on viral DNA replication (5, 48). Identification of late
genes is achieved by treatment of cells with viral polymerase
inhibitors, such as PAA, along with the inducing stimulus. Analysis of
viral RNA will fail to detect late gene expression in PAA-treated cells
but will reveal the full lytic repertoire in cells not treated with PAA.
Using this definition, we first sought to reconfirm that BLRF2 is a
true late gene in HH514-16 cells. Expression of viral
genes was
examined both at the protein and RNA levels. Western
blots were probed
for the presence of Rta itself, BLRF2, and BFRF3,
another late gene
product (
52). EBNA1 was included as a loading
control (Fig.
7A). At the RNA level, BLRF2 expression
was analyzed
with an oligonucleotide probe on a Northern blot; the H1
component
of RNase P served as a loading control (Fig.
7B). Induction
by
TPA and sodium butyrate or by transfection of ZEBRA or Rta led
to
expression of BLRF2 (lanes 3, 7, and 11), yet under each induction
condition, BLRF2 expression was inhibited by PAA (lanes 4, 8,
and 12).
By contrast, the expression of Rta itself and EBNA1 were
unaffected by
PAA treatment. The results showed clearly that in
HH514-16 cells BLRF2
behaved as a late gene, similar to BFRF3.
View larger version (46K):
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|
FIG. 7.
Rta activates BLRF2, a late gene. (A and B) HH514-16
cells; (C and D) Raji cells. (A and C) Western analysis of protein
extracts from cells that were uninduced (lanes 1 and 2) or had been
chemically induced (lanes 3 and 4) or transfected with 5 µg of empty
vector (lanes 5, 6, 9, and 10) or 5 µg of expression vector (lanes 7, 8, 11, and 12). For each condition, the cells had also been either
untreated () or treated with the viral DNA polymerase inhibitor PAA
(+). Immunoblots were prepared 30 h following transfection and
probed sequentially with antisera to the indicated proteins (LR2,
BLRF2; FR3, BFRF3). (B and D) Northern analysis of RNA prepared from
cells of the same transfections as above. The blots were probed with a
32P-labeled oligonucleotide from within BLRF2 and a
fragment of the H1 component of RNase P as loading control.
|
|
The same analysis was performed in Raji cells with a different outcome
(Fig.
7C and D). The Raji genome has a deletion within
the
BamHI A fragment, containing among others the BALF2 gene,
which codes for a DNA binding protein essential for DNA replication.
Accordingly, treatment of Raji cells with TPA and sodium butyrate
induced the lytic cycle and the expression of Rta but not the
late gene
BLRF2 (Fig.
7C and D, lanes 3). The addition of PAA
had no effect on
the expression of either protein. Transfection
of Raji cells with Rta,
however, strongly induced BLRF2 at the
protein and RNA levels (Fig.
7C
and D, lanes 7). The addition
of PAA did not abrogate this effect. In
Raji cells, therefore,
exogenously expressed Rta can bypass the
requirement for DNA replication
and lead to the activation of BLRF2.
Expression of Rta did not
significantly induce BFRF3, however (data not
shown). BLRF2 may
thus be a specific target of Rta. The data implies
that Rta may
function in activation of late lytic
genes.
ZEBRA, on the other hand, had an inhibitory effect on the capacity of
endogenous Rta to activate the expression of BLRF2.
Following
transfection of ZEBRA, which activated the endogenous
Rta, only minute
levels of BLRF2 protein were synthesized (Fig.
7C and D, lanes 7 and
11) and the BLRF2 message was barely detectable.
This inhibition of
Rta's ability to activate BLRF2 was also seen
when Rta and ZEBRA were
cotransfected (Fig.
2, lane 7). Similarly,
following chemical
treatment, which induced both Rta and ZEBRA
(Fig.
2), only trace
amounts of BLRF2 protein were expressed.
Thus, in Raji cells ZEBRA
appeared to counteract the potential
stimulatory effect of both
endogenous and exogenous Rta on late
gene
expression.
| |
DISCUSSION |
In this report, we present three novel findings regarding the role
of Rta in activation of lytic cycle gene expression in the BL cell line
Raji. First, when transfected into Raji cells, Rta does not detectably
activate either Zp or Rp, and therefore no ZEBRA synthesis ensues.
Nonetheless, Rta activates lytic gene products. Second, among the genes
stimulated by Rta some are activated maximally by Rta alone, and others
are activated as the result of synergy between ZEBRA and Rta. The use
of the Z(S186A) mutant, which by itself lacks the capacity to activate
lytic cycle genes, permits the identification of genes which respond
synergistically to Rta and ZEBRA. Third, Rta can activate a subset of
late genes in Raji cells, bypassing the requirement for viral DNA
synthesis. ZEBRA suppresses this activity.
Extensive data presented here show that in the Raji cell line, Rta
fails to activate the Rp and Zp promoters. ZEBRA expression was not
detected at either the RNA or protein level. Moreover, a sensitive
reporter assay, performed in parallel with assays for endogenous viral
gene expression, strongly suggests that ZEBRA is not induced following
transfection of Rta. Yet in this cell background, Rta remains a
competent transactivator capable of activating several lytic genes such
as BaRF1 and BMFL1 to a maximal level and other genes, such as BMRF1
and BHRF1, to a lesser extent. Raji cells transfected with Rta
represent a de facto ZEBRA knockout system in which the role of Rta in
the activation of lytic genes can be explored. This system will
ultimately allow identification of all early genes that are responsive
to Rta, directly or indirectly, in the absence of ZEBRA and viral DNA replication.
Classes of EBV lytic cycle genes in Raji cells.
Using Raji
cells, we describe a method with which one can ascertain precisely
whether a gene is predominantly activated by Rta or by the combination
of Rta and ZEBRA. The ZEBRA mutant Z(S186A) is impaired in its ability
to activate all viral genes, including Rta (1, 17, 18).
Coexpression of Rta and Z(S186A), however, rescues the deficient mutant
phenotype and the usual program of gene activation ensues. The change
of a serine to an alanine in the DNA binding domain of ZEBRA may reduce
the affinity of the protein for its response elements within Rp, or it
may impair the ability of ZEBRA to be phosphorylated, a possible
prerequisite for interaction with other proteins involved in
stimulation of Rp (1, 17, 18). Nonetheless the mutation does
not significantly reduce the ability of ZEBRA to synergize with Rta on
downstream viral targets. Using this information, we classify early
lytic viral genes in Raji cells into three distinct groups. Class Z genes, represented by BRLF1, respond only to Z(S186A). Rta has no
effect on BRLF1 by itself or in combination with ZEBRA. Class R genes,
e.g., BaRF1 and BMLF1, are dominantly activated by Rta in Raji cells.
The level of stimulation of these genes by the combination of Z(S186A)
and Rta does not exceed the level observed with Rta alone. ZEBRA also
stimulates these genes maximally as a result of its ability to induce
endogenous Rta protein. Class RZ genes, including BMRF1 and BHRF1,
respond in synergy to Rta and ZEBRA. Rta by itself activates these
genes to low levels, but addition of the Z(S186A) mutant achieves
maximal stimulation. There are likely to be additional classes of lytic
cycle genes on which Rta by itself has no activity. One class is
represented by BZLF1, which in Raji cells appears under control of
factors other than Rta and ZEBRA. Another class would be genes other
than BRLF1 that are exclusively controlled by ZEBRA. Definition of this
class will require mutants that lack Rta expression even in the
presence of ZEBRA. Finally, there may be genes that are only stimulated
by the combination of Rta and ZEBRA.
Functional differences of Rta in Raji and HH514-16 cells.
Rta
is able to stimulate Rp and Zp in HH514-16 cells but not in Raji cells.
This difference in function can be attributed to cell background or
viral genome differences or both. Only a few point mutations
distinguish Rp and Zp in the EBV strains carried by Raji and HH514-16
cells. In general, these mutations do not fall within known regulatory
elements. Two mutations that may potentially affect such elements that
are present in the prototype B95-8 strain are shared by Raji and
HH514-16. Moreover, Rta is competent to activate RpCAT and ZpCAT from
all EBV strains when presented as plasmids in Raji cells. Although we
cannot discount the possibility that Rta activates the reporter
constructs from initiation sites different from those used in the
endogenous virus, we consider this possibility improbable. The low
background activation of the pCAT vector by Rta suggests an absence of
cryptic initiation sites within the vector. Since the RpCAT constructs
require the promoter TATA box for activation by Rta, transcription is
likely to initiate properly (44). For all of these reasons,
it seems unlikely that primary sequence differences in Rp and Zp
account for the dramatic differences in behavior. However, our
experiments have not explored the possible contributions of viral
sequence alterations elsewhere in the HR1 and Raji genomes. For
example, the HH514-16 cell line carries the HR1 strain, which lacks
EBNA2, and the Raji genome lacks EBNA3C, BARF1, BALF1, and BALF2
(15, 24, 40). The absence of these genes may directly alter
viral gene regulation or result in the creation of different cellular environments through their failures to activate or repress specific cellular genes. Alternatively, the different response to Rta may be a
consequence of cellular background effects on chromatinization, methylation, or the presence or absence of cellular coactivators or
repressors. These cofactors may also be specific for their viral
targets only in the context of the endogenous chromatinized viral
genome, which might explain why Rta could still activate the reporter
constructs in Raji cells.
Potential role of Rta in late gene activation.
Rta plays a
role in the activation of expression of a late gene, BLRF2, in Raji
cells. This was an unexpected finding because a defining defect of Raji
cells is the absence of DNA replication and late gene expression. Using
the standard assay to classify late viral genes, namely, inhibition by
viral polymerase inhibitors (52), we found that BLRF2
displays unambiguous late character in HR1 cells. It is expressed only
if viral DNA replication is allowed to proceed. In Raji cells, Rta can
circumvent this requirement. ZEBRA, on the other hand, appears to
downregulate Rta mediated activation of BLRF2. The late gene is only
stimulated by Rta in the absence of ZEBRA. In chemically treated or
ZEBRA-transfected cells which express both transactivators, BLRF2 is
expressed poorly or not at all. Two explanations could account for the
repressive effect of ZEBRA on late gene expression. In the early lytic
cycle, ZEBRA may bind to the late BLRF2 promoter and act as a
repressor. The BLRF2 promoter does contain several putative ZREs. Upon
DNA replication, ZEBRA is displaced or modified so that it loses its affinity for the promoter, allowing access to Rta and other putative late activators. In the absence of viral DNA replication, such as
occurs in Raji cells, ZEBRA remains bound to the BLRF2 promoter, thus
inhibiting its activation. The second scenario posits ZEBRA's ability
to sequester a factor that together with Rta is essential for late gene
expression. The capacity of ZEBRA to bind this essential factor is lost
after DNA replication. In Raji cells, in the absence of ZEBRA, the
factor is therefore available to work together with Rta in the
activation of BLRF2. While the expression of Rta unaccompanied by ZEBRA
expression is not likely to occur during the normal EBV life cycle,
this pattern of gene expression uniquely encountered in Raji cells
seems to provide attractive new insights into the function of the two
activators in regulation of late genes.
Implications of the findings.
Unlike regulation of prokaryotic
gene expression, the regulation of eukaryotic gene expression is rarely
controlled by a single activator or repressor (41). Instead,
the combinatorial effects of multiple activators and repressors
controls expression of eukaryotic genes. The switch from latency to the
lytic cycle of EBV is similarly under the control of an intricate
regulatory mechanism seeming to involve activators and repressors of
both cellular and viral origin. For a long while, ZEBRA was thought to
represent the single essential viral activator. However, it is now
clear that the cellular mechanisms that govern the lytic cycle switch
simultaneously promote expression of two viral proteins, ZEBRA and Rta,
that are involved in a complex interplay that is beginning to be
defined in this and other recent reports (1, 17, 44, 54). As
we show here, interactions between ZEBRA and Rta occur at the level of autostimulation and reciprocal stimulation of Rp and Zp. Furthermore, Rta and ZEBRA demonstrate complex interactions in their control of
different sets of downstream lytic cycle viral genes.
ZEBRA and Rta are both likely to possess multiple functions. ZEBRA's
capacity to activate transcription, to participate in
replication
(
46,
47), and to cause cell cycle arrest (
10,
11)
have all been well described. In earlier studies, ZEBRA
has been
implicated in interference with Rta's ability to activate
synthetic
Rta responsive promoters (
21). Here we demonstrate
that
ZEBRA possesses a potential role as a repressor of Rta-activated
late
gene expression under some conditions in the context of an
intact viral
genome (Fig.
7C and D). The ability of ZEBRA to prevent
late gene
expression activated by Rta may ensure the proper stage-specific
progression into the lytic cycle. The repressive effect of ZEBRA
on
late gene expression may be relieved by DNA replication. In
all
likelihood, Rta will also eventually be found to carry out
different
functions in the viral life cycle. Here we define its
capacity to
dominate expression of some genes and to facilitate
the activity of
ZEBRA on other genes (Fig.
4). Other functions
of Rta may be revealed
as the panel of genes known to be controlled
by ZEBRA, Rta, and cell
factors is
expanded.
In summary, detailed analysis of the roles of Rta and ZEBRA in the EBV
lytic cycle switch is beginning to reveal all the beauty
and complexity
of control of eukaryotic gene expression and should
continue to serve
as a model for this central process in
biology.
| |
ACKNOWLEDGMENTS |
This work was supported by grants CA12055 and CA16038 from the
NCI to G.M.
We thank T. Serio for helpful discussions and critically reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, Yale University School of Medicine, 333 Cedar St., New
Haven, CT 06520. Phone: (203) 785-4758. Fax: (203) 785-6961. E-mail: George.Miller{at}yale.edu.
| |
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Journal of Virology, December 1999, p. 9858-9866, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
