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Journal of Virology, October 2000, p. 9755-9761, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Latent Membrane Protein 1 of Epstein-Barr Virus
Inhibits as Well as Stimulates Gene Expression
Mark L.
Sandberg,
Ajamete
Kaykas, and
Bill
Sugden*
McArdle Laboratory for Cancer Research,
University of Wisconsin Medical School, Madison, Wisconsin 53706
Received 3 March 2000/Accepted 24 July 2000
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ABSTRACT |
The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV)
functionally resembles a constitutively active, CD40-like receptor and
contributes to the maintenance of proliferation of EBV-infected primary
human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it
binds TRAF, TRADD, and JAK molecules to activate NF-
B-, AP-1-, and
STAT-dependent pathways as does CD40. Yet LMP-1 appears to lack a
ligand to regulate its signaling. We have found that LMP-1, when
expressed at physiologic levels, inhibits gene expression detectably.
Higher levels of LMP-1 expression eventually inhibit both the
steady-state level of RNA produced from a BamHI C promoter
reporter and general cellular protein synthesis. These findings
indicate that LMP-1 can limit its signaling and that this control is
manifest at two levels. The domain of LMP-1 that binds TRAF, TRADD, and
JAK/STAT molecules is not required for this regulation. A derivative of
LMP-1 that contains only its amino-terminal and membrane-spanning
domains is sufficient to inhibit reporter activity when the reporter
genes are expressed from the BamHI C and LMP-1 promoters.
This same derivative of LMP-1 in parallel assays is sufficient to
inhibit wild-type LMP-1's stimulation of NF-B-dependent gene
expression. We suggest that LMP-1 encodes stimulatory and inhibitory
activities; the latter could limit signaling in the apparent absence of
ligand-dependent down-regulation.
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TEXT |
Ligand-dependent surface receptors
can limit their signaling by requiring ligand for this function and, in
the presence of ligand, by being internalized and removed from their
signaling compartments. LMP-1 may have evolved from ligand-dependent
receptors of the tumor necrosis factor family (16), but it
fails to conform with this paradigm for receptor regulation because it
signals apparently in the absence of a ligand (29). LMP-1
contributes to the proliferation of cells infected by Epstein-Barr
virus (EBV) (24, 27) and can affect the growth properties of
some cell lines, identifying it as an oncoprotein (3, 8, 31, 34, 38). However, LMP-1 has been shown to limit the proliferation of
both epithelial and lymphoid cell lines, a phenotype which indicates
that LMP-1's expression or activities must be regulated for efficient
survival of the infected cell (12, 18, 25).
Several characteristics of LMP-1 are consistent with its functioning as
a ligand-independent, constitutively active growth factor receptor. It
is an integral membrane protein with an intracellular amino terminus of
25 amino acids, six hydrophobic membrane-spanning domains, and an
intracellular carboxy terminus that is known to bind cellular proteins
and activate signal transduction pathways as does CD40 (4, 5, 7,
9-11, 13, 15, 20, 22, 28, 32, 35). LMP-1 stimulates NF-B-,
AP-1-, and STAT-mediated transcription in cells (10, 15, 17, 21,
26, 30). LMP-1 stimulates these activities by aggregating, which
is dependent on its amino-terminal and membrane-spanning domains,
apparently independently of a ligand (16, 29).
Ligand-dependent stimulation of cellular receptors yields NF-B-,
AP-1-, and STAT-mediated transcription which is down-regulated in
normal cells following their stimulation. Such down-regulation is
important because, for example, both AP-1 and NF-B family members
are proto-oncogenes whose aberrant activation contributes to tumor
formation (2, 23). We have searched for mechanisms by which
LMP-1, in spite of its functioning like a constitutively active
receptor-like molecule, could limit its activation of these critical
signaling pathways consistent with survival of both the host cell and
the infected human being.
In this study we describe LMP-1's negative regulation both of gene
expression from EBV's BamHI C (Bam C) and LMP-1
promoters and of its own stimulation of NF-B-dependent gene
expression. For the purpose of this study, we define gene expression as
the sum of all cellular processes required to generate reporter
activity. EBNA-1 is a positive regulator of both the Bam C
and LMP-1 promoters (14, 33, 37). These two promoters drive
the expression of all EBV genes known to be required for
immortalization of B cells in culture. Here we show that increasing
levels of LMP-1 inhibit gene expression from the Bam C or
LMP-1 promoters to as much as 3% of uninhibited levels and that this
inhibition is first detectable at levels of LMP-1 normally expressed in
six different clones of EBV-infected B cells. This inhibition
correlates with a decrease in the steady-state levels of RNA
synthesized from the Bam C promoter and with a decrease in
cell protein synthesis. A derivative of LMP-1 that contains only its
amino-terminal and membrane-spanning domains, and thus cannot bind the
cellular signaling molecules known to bind to LMP-1, is
sufficient to mediate LMP-1's inhibitory activity. Finally, this
derivative of LMP-1 cannot stimulate NF-B-mediated transcription but can inhibit intact LMP-1's stimulation of
NF-B-mediated transcription. These combined observations support a
model in which LMP-1 signals independently of a ligand but dependent on its own concentration; beginning at physiological and at higher concentrations, it inhibits gene expression in a dose-dependent manner.
The levels of LMP-1 expressed in EBV-infected B cells likely represent
a balance between its stimulation and inhibition consistent with
survival of the infected host cell.
A more detailed description of protocols for methods used in this study
can be found in the website
http://mcardle2.oncology.wisc.edu/sugden-main.htm.
LMP-1 inhibits EBNA-1-mediated transactivation of the
Bam C and LMP-1 promoters.
We tested if LMP-1 could
regulate EBV's Bam C promoter, which can express
transcripts for all of EBV's nuclear antigens, with a vector which
contains all of the EBV DNA from oriP up to and including
the Bam C promoter fused to luciferase (oriP-Bam
Cp-luciferase) (Fig. 1).
oriP-Bam Cp-luciferase was cotransfected with an expression vector for EBNA-1 (oriP-EBNA-1) into EBV-negative BJAB cells
(Fig. 2A) and assayed as described
previously (35). All transfections in this study used the
same amount of DNA with empty vector as the filler. EBNA-1 stimulates
expression of luciferase (measured as relative light units [RLU])
from oriP-Bam Cp-luciferase 35-fold; this level of
stimulation was set to 100% activity. Cotransfection of increasing
amounts of an expression vector for LMP-1 (SVLMP-1) with a constant
amount of oriP-EBNA-1 and oriP-Bam Cp-luciferase yields a dose-dependent decrease in the detected luciferase activity. LMP-1 can therefore inhibit EBNA-1-mediated transactivation of the
Bam C promoter. The level of EBNA-1 was measured to vary by less than twofold in these experiments (data not shown). Parallel experiments performed in GG68 cells transfected with an expression vector for EBNA-2 (to complement this cell's deletion) yielded comparable results (data not shown).
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FIG. 1.
Diagrams of the B95-8 strain of EBV, oriP-Bam
Cp-luciferase, and oriP-LMP-1p-luciferase reporters. Shown
are the elements expressed and used by EBV in latent infection of
resting B cells in cell culture. Not shown are the more than 80 genes
used during the lytic phase of infection. Letters inside the circle
indicate the fragments of the B95-8 strain of EBV resulting from
digestion of the genome with BamHI. The promoters used in
latent infection are indicated by arrowheads, with the primary RNA
transcripts generated from these promoters indicated by dashed lines
and the open boxes representing exons of these transcripts. The origin
of DNA replication used in the latent life cycle (oriP), the
origin of DNA replication used in the lytic life cycle (ori
Lyt), and the site of DNA circularization after infection, the terminal
repeats (TR), are indicated by black boxes. Above the genome are
diagrams of the oriP-Bam Cp-luciferase and
oriP-LMP-1p-luciferase reporters used. oriP
consists of 20 EBNA-1 binding sites found in the family of repeats (FR)
and four binding sites for EBNA-1 in the dyad symmetry element (DS).
The luciferase open reading frame was inserted at the locations
indicated. The base pair numbers indicate locations of the
corresponding DNAs found in the B95-8 strain of EBV (1).
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FIG. 2.
LMP-1's effect on expression from oriP-Bam
Cp-luciferase and oriP-LMP-1p-luciferase. (A) Increasing
amounts of SVLMP-1 (0, 1, 3, 9, and 27 µg) were transfected as
indicated into BJAB cells along with 5 µg of oriP-EBNA-1
and 5 µg of oriP-Bam Cp-luciferase. In all transfections,
the same total amount of DNA was used with an empty vector added as a
filler. The average number of LMP-1 molecules per transfected cell was
calculated as described below. Relative activity of oriP-Bam
Cp-luciferase was determined 48 h posttransfection by setting the
luciferase detected in the presence oriP-EBNA-1 and absence
of SVLMP-1 to 100%. The average RLU detected at 100% of
oriP-Bam Cp-luciferase activity is 1.5 × 106. The data shown are averages of three independent
transfections. Error bars indicate 1 standard deviation from the mean;
where no error bar is indicated, the standard error was smaller than
the size of the symbol. Wild-type LMP-1 as it resides in the plasma
membrane is shown as an inset. (B) oriP-LMP-1p-luciferase
(10 µg) was cotransfected into BJAB cells with 0, 1, 3, or 9 µg of
SVLMP-1. The cells were assayed for luciferase activity 48 h
later. The average number of LMP-1 molecules per transfected cell was
calculated as described below. The data represent the averages of four
independent experiments, with 100% oriP-LMP-1p-luciferase
activity corresponding to the 5.5 × 103 RLU detected
in the absence of SVLMP-1. Error bars indicate 1 standard deviation
from the mean. (C) Measuring LMP-1 expression levels in the
EBV-immortalized B-cell line 721 and transfected BJAB cells. Luciferase
data are taken directly from the experiments represented in panel A. The average number of LMP-1 molecules per cell was calculated by the
following method. GST-LMP-1(181-386) was isolated from E. coli DH5 as described previously (35) and found to
be 50% pure. GST-LMP-1(181-386) extracts of transfected cells were
assayed for LMP-1 by quantitative Western blotting (35) with
LMP-1 signals corrected for the efficiency of transfection, which was
approximately 50%. All data represent the averages of three
independent experiments.
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We also tested if LMP-1 could regulate its own promoter. To determine
if LMP-1 can also inhibit EBNA-1-mediated expression
of
oriP-LMP-1p-luciferase (Fig.
1), increasing amounts of
SVLMP-1
were transfected into BJAB cells with
oriP-EBNA-1
and
oriP-LMP-1p-luciferase
(Fig.
2B). EBNA-1 positively
stimulates
oriP-LMP-1p-luciferase
fourfold in these cells;
this level of stimulation was set to
100% activity. LMP-1 when
expressed from 3 × 10
4 to 9 × 10
4
molecules per cell progressively inhibits this stimulation. We
used
BJAB cells in this experiment to be consistent with our other
experiments, although the EBNA-1-mediated stimulation of
oriP-LMP-1p-luciferase
in BJAB cells is less than that in
EBV-positive cells (
14).
The decrease in luciferase mediated
by 3 × 10
4 to 9 × 10
4 molecules of
LMP-1 per cell was statistically significant (
P = 0.02). LMP-1 has the ability to inhibit expression from its
own
promoter in the natural context of LMP-1's regulatory domain.
LMP-1
inhibited
oriP-Bam Cp-luciferase in a dose-dependent manner
in the absence of
oriP-EBNA-1 (data not shown). Parallel
experiments
performed in GG68 cells transfected with an expression
vector
for EBNA-2 yielded comparable results (data not
shown).
We determined whether LMP-1's ability to inhibit EBNA-1-mediated
transactivation occurs at levels of LMP-1 expression found
in
EBV-immortalized lymphoblastoid cells. Quantitative Western
blots were
used to measure the amount of LMP-1 expressed in six
clones of
EBV-immortalized B cells (721, RPMI-1788, JO-L-B1, 11/17-1,
11/17-3,
and 11/17-10) and in BJAB cells transfected with SVLMP-1.
A glutathione
S-transferase (GST)-LMP-1 derivative purified from
Escherichia coli was used as a standard for these
measurements.
These measurements, performed as described previously
(
25),
indicate that the average amount of LMP-1 required to
inhibit
EBNA-1-mediated expression of luciferase to 50% in BJAB cells
is equivalent to the average amount of LMP-1 detected in the
EBV-immortalized
cell clone 721 (Fig.
2C). The level of LMP-1 expressed
in 721
cells is the same (within twofold) as that in the five other
EBV-immortalized
cell clones studied (data not shown) (
36).
This finding supports
a model in which LMP-1 is expressed in
EBV-immortalized cells
at a level balanced by its positive and negative
activities that
is consistent with their continued
proliferation.
LMP-1 inhibits steady-state RNA levels produced from the
Bam C promoter.
S1 nuclease mapping was used to
determine if LMP-1 exerts its inhibition of expression of a reporter
gene by affecting steady-state levels of RNA encoded by the reporter.
Chloramphenicol acetyltransferase (CAT) was used as the reporter and
assayed as described elsewhere (37) because we found its RNA
to accumulate to higher levels than that of luciferase. As with
oriP-Bam Cp-luciferase, EBNA-1 positively stimulates
oriP-Bam Cp-CAT and LMP-1 inhibits EBNA-1's transactivation
of this reporter to 15% of uninhibited levels (Fig.
3A).
Cytoplasmic RNA was therefore isolated
from these cells and subjected to S1 nuclease mapping, as described
previously (14), using the S1 CAT oligonucleotide as a probe
for CAT RNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
oligonucleotide as a probe for GAPDH RNA. In the absence of cytoplasmic
RNA (Fig. 3B, lane 3), no specific GAPDH product was detected. RNA from untransfected and three transfected BJAB cell populations yielded an S1
nuclease-protected product for the GAPDH probe that migrated at the
expected size of 45 nucleotides (Fig. 3B, lanes 4 to 7). These signals
were quantified and used to normalize the input RNA for the signals
they yielded with the CAT probe. In the untransfected cell population,
no CAT RNA was detected (Fig. 3B, lane 9); however, in the transfected
BJAB cell populations, a protected CAT product of the expected size of
55 nucleotides was detected (Fig. 3B, lanes 10 to 12). The CAT RNA
increased in the presence of EBNA-1 and decreased when LMP-1 was
coexpressed (Fig. 3B, lanes 11 and 12). The level of GAPDH RNA was not
found to change significantly when SVLMP-1 was transfected into the
cells. This finding presumably reflects both the stability of GAPDH RNA
and the fact that only half of the cells received SVLMP-1. The
protected signals were verified to be dependent on RNA and not
contaminating DNA by their sensitivity to digestion with RNase A prior
to S1 mapping (data not shown). The protected RNAs were verified to
increase linearly within the range of RNA studied (data not shown).
LMP-1 decreases the steady-state levels of CAT RNA produced by
oriP-Bam Cp-CAT. The LMP-1-mediated decreases in both CAT
activity and steady-state CAT RNA levels as judged by the Wilcoxon rank
sum test are significant (P = 0.01). However, LMP-1
inhibited CAT activity by 87% and steady-state CAT RNA levels by only
40%. The decrease in steady-state CAT RNA levels is, therefore,
unlikely to account for the entire decrease in CAT activity. Although
LMP-1 inhibits the accumulation of CAT RNA, LMP-1 is also likely to
inhibit the accumulation of CAT activity by some other means.
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FIG. 3.
Measuring LMP-1's effect on oriP-Bam Cp-CAT
in BJAB cells by CAT assay and S1 nuclease mapping. (A) Extracts of
BJAB cells cotransfected with the indicated DNAs were assayed for CAT
activity as described previously (37); 5 µg of
oriP-Bam Cp-CAT, 5 µg of oriP-EBNA-1, and 9 µg of SVLMP-1 were cotransfected where indicated. The percentage of
chloramphenicol acetylated in each extract is indicated at the bottom.
(B) S1 nuclease mapping of GAPDH and CAT RNAs in the cell extracts
shown in panel A was performed as described previously (14).
The expected sizes of signals of undigested probes and digested probes
are indicated at the right. Lane 1 contains markers; their sizes are
indicated in nucleotides at the left. Above each lane is indicated the
probe used in the S1 reaction and the DNAs transfected in each set of
BJAB cells. Lane 3 is the GAPDH probe digested in the absence of any
cellular RNA. The fold induction of CAT activity was determined by
averaging the CAT activity observed in four independent experiments,
with the signal of oriP-Bam Cp-CAT set to 1. The difference
between the CAT activity observed for each point was determined by the
Wilcoxon rank sum test and has a P value of 0.01. The CAT
RNA levels were determined by PhosphorImager quantitation of signals in
the S1 nuclease mapping gels shown in panel B. The fold induction of
CAT RNA is the average of four independent S1 nuclease mapping
experiments after normalizing each for its detected GAPDH signals. The
statistical significance of the levels of CAT RNA in each point was
determined by the Wilcoxon rank sum test and has a P
value of 0.01. (C) Metabolic labeling of cells was performed by
incubating 107 BJAB cells with 100 µCi of
[35S]Met-Cys for 60 min at 37°C 48 h after
transfection. The cells were washed once with RPMI 1640 containing 10%
fetal bovine serum and then lysed in 1 ml of 0.2 N NaOH for 5 min at
25°C; 10 ml of 10% trichloroacetic acid containing unlabeled
methionine (30 µg/ml) was added, and the samples were bound to glass
fiber filters. The filters were washed twice with 10 ml of 10% trichloroacetic acid-unlabeled
methionine (30 µg/ml) and once with 10 ml of 100% ethanol. Filters
were measured for bound radioactivity in a liquid scintillation
counter. Normalized 35S incorporation was determined by
setting the radioactivity detected in labeled BJAB cells transfected
with the oriP-Bam Cp-CAT reporter alone to 1. 35S incorporation data are averages of three independent
experiments; ± indicates 1 standard deviation from the mean.
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LMP-1 inhibits cellular protein synthesis.
Metabolic labeling
of transfected BJAB cells with [35S]Met-Cys was used to
determine if LMP-1 can inhibit cellular protein synthesis. BJAB cells
were cotransfected with oriP-Bam Cp-CAT,
oriP-EBNA-1, and SVLMP-1. The CAT activity in these
transfected cells was measured along with their protein synthetic
capacity. oriP-EBNA-1 did not have a detectable effect on
protein synthesis; however, SVLMP-1 caused a decrease of protein
synthesis (Fig. 3C). In these experiments, 40 to 50% of the
transfected cells took up and expressed green fluorescent protein. In a
more sensitive assay, up to 60% of similarly transfected BJAB cells
take up and are killed by a Fas-expressing vector (data not shown). The
observed 40% reduction in protein synthesis at 48 h is therefore
distributed in up to 60% of the cells, indicating that in this
population protein synthesis is inhibited up to 68%. In addition to
inhibiting the steady-state levels of CAT RNA, LMP-1 inhibits the
majority of cellular protein synthesis when it is expressed at levels
twofold above that found in clones of EBV-immortalized B-cells.
The amino-terminal and membrane-spanning domains of LMP-1 are
sufficient to inhibit gene expression.
To determine if any portion
of the carboxy-terminal domain of LMP-1 is required for LMP-1's
inhibitory activity, we analyzed a derivative of LMP-1 that contains
only its amino-terminal and membrane-spanning domains (6MHALMP-1EE)
expressed from a cytomegalovirus immediate-early promoter. The
hemagglutinin (HA) epitope was placed between amino acids 2 and 3 of
the amino terminus, and two copies of an EE epitope were placed at
amino acid 194 to form the carboxy terminus of this truncated
derivative. The EE epitopes maintain the proper charge distribution of
the amino acids located adjacent to the last membrane-spanning domain
found in LMP-1, which have been proposed to be important for proper
protein insertion into the plasma membrane (40).
Unpermeabilized and permeabilized cells expressing
6MHALMP-1EE were stained with fluorescent antibodies to the two
epitope
tags to define the location within the cells of the amino and
carboxy termini of this derivative (Table
1). The unpermeabilized
cells were not
stained specifically with antibodies directed to
both the HA and EE
epitopes, while the permeabilized cells were,
indicating that the amino
and carboxy termini of 6MHALMP-1EE are
both inside the cell, as are
those of wild-type LMP-1 (
6).
6MHALMP-1EE was also verified
to be at the plasma membrane by
indirect immunofluorescence (data not
shown).
6MHALMP-1EE was cotransfected into BJAB and GG68 cells with
oriP-Bam Cp-luciferase and
oriP-EBNA-1 (BJAB
cells) or SVEBNA-2
(GG68 cells) (Fig.
4A). 6MHALMP-1EE inhibited luciferase
activity
from
oriP-Bam Cp-luciferase, indicating that the
amino-terminal
and membrane-spanning domains of LMP-1 are sufficient
for its
inhibition of gene expression from the
Bam C
promoter. The diffuse
migration of 6MHALMP-1EE in Western blots
precluded our measuring
the average number of 6MHALMP-1EE molecules
expressed per transfected
cell. Cotransfection of increasing amounts of
6MHALMP-1EE and
oriP-Bam Cp-luciferase into the
EBV-immortalized, EBNA-1-expressing
cell line 721 yielded a
dose-dependent inhibition of luciferase
activity (data not shown). The
inhibition of
oriP-Bam Cp-luciferase
in 721 cells
demonstrates that the amino-terminal and membrane-spanning
domains of
LMP-1 can inhibit signaling in an EBV-immortalized
cell line.
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FIG. 4.
6MHALMP-1EE inhibits gene expression. Shown in the inset
is 6MHALMP-1EE and its predicted secondary structure in the plasma
membrane. (A) The oriP-Bam Cp-luciferase reporter was not or
was cotransfected with 100 ng, 300 ng, 1 µg, 3 µg, or 9 µg of an
expression vector for 6MHALMP-1EE into BJAB (squares) and GG68 cells
(circles). After 48 h, the cells were assayed for luciferase
activity; 100% reporter activity corresponds to the 1.3 × 106 RLU in BJAB cells and 3 × 104 RLU in
GG68 cells detected in the absence of 6MHALMP-1EE.
oriP-EBNA-1 was not included in the experiments performed in
GG68 cells because these cells express EBNA-1 constitutively. The data
represent the averages of three independent experiments. Error bars
indicate 1 standard deviation from the mean. (B) BJAB cells were
cotransfected with a constant amount of a NF-B-responsive luciferase
reporter, the indicated amounts of 6MHALMP-1EE, and either 1 µg of
SVLMP-1 (circles) or 30 ng of an expression vector for a NF-B
p50/p65 fusion protein (squares); 100% reporter activity is calculated
from the luciferase activity detected in the absence of 6MHALMP-1EE and
in the presence of 1 µg of transfected SVLMP-1 (average of 2 × 105 RLU) or 30 ng of transfected p50/65 expression vector
(average of 1.5 × 105 RLU). Results shown are the
averages of three independent transfection experiments. Error bars
indicate 1 standard deviation from the mean. (C) A clone of BJAB cells
which expresses 6MHALMP-1EEGFP conditionally was tested for inhibition
of protein synthesis by this truncated derivative of LMP-1.
35S-incorporation was measured 48 h after addition of
the indicated amounts of tetracycline. Cells were labeled as described
in the legend to Fig. 3, with 35S-incorporation in the
uninduced cells set to 1. The percent 35S incorporated is
the average of four independent experiments, with ± indicating 1 standard deviation from the mean. The average number of 6MHALMP-1EEGFP
molecules per cell was determined by measuring the amount of
6MHALMP-1EEGFP expressed in one of the four experiments used to
determine the percent 35S incorporated.
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To determine if the amino-terminal and membrane-spanning domains of
LMP-1 can inhibit NF-
B-mediated transcription, we tested
the ability
of 6MHALMP-1EE to inhibit the NF-
B reporter, 4×NF-
B-luciferase,
when stimulated by LMP-1 or by NF-
B itself. BJAB cells were
cotransfected
with 4×NF-
B-luciferase and SVLMP-1 or an expression
vector for
a protein consisting of fused NF-
B members, p50/p65.
Levels of
the expression vectors were chosen to yield similar levels of
luciferase activity, and this luciferase activity was set equal
to
100%. Increasing levels of 6MHALMP-1EE were introduced with
either an
SVLMP-1 or p50/65 expression vector and 4×NF-
B-luciferase
(Fig.
4B). 6MHALMP-1EE inhibits LMP-1's stimulation both of
NF-
B-dependent
gene expression and of exogenously derived NF-
B.
This inhibition
of NF-
B-dependent gene expression is consistent with
LMP-1's
inhibition of RNA accumulation and protein
synthesis.
We tested directly if the amino-terminal and membrane-spanning domains
of LMP-1 are sufficient to inhibit protein synthesis
by expressing
6MHALMP-1EEGFP (green fluorescent protein at the
carboxy terminus of
6MHALMP-1EE) conditionally. A clone of BJAB
cells selected to
express 6MHALMP-1EEGFP from a modified cytomegalovirus
immediate-early
promoter which is inhibited on binding a fusion
of the tetracycline
repressor fused to KRAB was induced to express
the LMP-1 derivative by
adding increasing levels of tetracycline
(
25). The levels of
protein synthesis were measured for 1 h
48 h after addition of
tetracycline. The derivative of LMP-1 lacking
all of its
carboxy-terminal signaling domain inhibited protein
synthesis as
efficiently as did intact LMP-1 when it is expressed
efficiently (Fig.
4C).
In summary, we have found that at levels of expression of LMP-1 found
in clones of EBV-immortalized B cells, LMP-1 detectably
inhibits the
activity of reporters from three promoters. Expression
of LMP-1 at
levels greater than twofold that found in EBV-immortalized
cells can
inhibit expression of reporters to 10% of their uninhibited
levels.
This inhibition is reflected by both a decrease in the
steady-state RNA
levels and an inhibition of protein synthesis.
Levels of LMP-1 that
inhibit the accumulation of RNA by 40% inhibit
protein synthesis
similarly. These findings indicate that at the
level of expression of
LMP-1 measured in EBV-infected B-cells
and above, LMP-1 limits gene
expression. We propose that LMP-1's
ability to limit gene expression
represents a solution to the
problem inherent in its being
constitutively active while usually
supporting benign survival of the
infected cell. The region of
LMP-1 that encodes these functions may
have a parallel in fibroblast
growth factor receptor 3, for which the
extracellular and membrane-spanning
domain limits signaling of its
cytoplasmic domain (
39).
The inhibition of gene expression mediated by LMP-1 can be robust when
LMP-1 accumulates to more than 10
5 molecules per cell. This
inhibition is likely to underlie LMP-1's
inhibition of proliferation
of cells (
12,
19,
25). LMP-1
expressed in a variety of cells
at levels of 2 × 10
5 to 4 × 10
5
molecules per cell inhibits their proliferation; when expression
is
reduced, the cells resume proliferation (
25). A derivative
of LMP-1 which lacks the entire carboxy-terminal domain, and is
positioned in the plasma membrane as is wild-type LMP-1, inhibits
both
gene expression (Fig.
4) and cell proliferation (
25).
LMP-1's
inhibition of gene expression and cell proliferation may
therefore
be mediated by the same
activity.
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ACKNOWLEDGMENTS |
We thank Tim Bloss and Todd Hopkins for contributions to the
identification of LMP-1's inhibitory function, Ngan Lam for the 6MHALMP-1EEGFP-inducible cell line, and Susanna Mac for help with the
RNA work. We also thank Elizabeth Leight, Annette Pownell, Jun Komano,
Chris Bradfield, Dan Loeb, and Paul Ahlquist for critically reviewing
the manuscript.
This work was supported by Public Health Service grants CA-2243,
CA-07175, T32-CA-09135, and CA-70723. B.S. is an American Cancer
Society Research Professor.
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FOOTNOTES |
*
Corresponding author. Mailing address: McArdle
Laboratory for Cancer Research, University of Wisconsin Medical School,
Madison, WI 53706. Phone: (608) 262-6697. Fax: (608) 262-2824. E-mail: Sugden{at}oncology.wisc.edu.
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Journal of Virology, October 2000, p. 9755-9761, Vol. 74, No. 20
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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