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Journal of Virology, April 2000, p. 3752-3760, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mechanisms of Human Papillomavirus E2-Mediated
Repression of Viral Oncogene Expression and Cervical Cancer Cell
Growth Inhibition
Akiko
Nishimura,1
Takeshi
Ono,1
Akinori
Ishimoto,1
Jennifer J.
Dowhanick,2
Margaret A.
Frizzell,2
Peter M.
Howley,2 and
Hiroyuki
Sakai1,*
Laboratory of Gene Analysis, Department of
Viral Oncology, Institute for Virus Research, Kyoto University,
Sakyo-ku, Kyoto 606-8507, Japan,1 and
Department of Pathology, Harvard Medical School, Boston,
Massachusetts 021152
Received 11 October 1999/Accepted 11 January 2000
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ABSTRACT |
The papillomavirus E2 gene product plays a pivotal role
in viral replication. E2 has multiple functions, including (i)
transcriptional activation and repression of viral promoters and (ii)
the enhancement of viral DNA replication. It was previously reported
that E2 suppressed the growth of papillomavirus-positive cervical
carcinoma cell lines. In the present study, we investigated the
mechanisms of E2 growth inhibition. We found that the transcriptional
activation function of E2 is required for inhibition of the growth of
HeLa cells as well as for transcriptional repression of the viral
E6/E7 promoter. It had been previously postulated that
transcriptional repression of the E6/E7 promoter results
from E2 binding its cognate sites proximal to the E6/E7
promoter and displacing other cellular transcriptional factors. In this
study, we report a requirement for the transcription activation
function for the binding of E2 to transcriptionally active templates.
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INTRODUCTION |
The papillomavirus replication cycle
is regulated by the viral E2 protein, a sequence-specific DNA binding
protein (1, 35, 53). Depending on the promoter context, E2
can act either as a transcriptional activator or as a repressor of
viral gene expression. The promoters for E6/E7 gene
expression of human papillomavirus type 16 (HPV16) and HPV18 are
negatively regulated by E2. This repression is thought to be mediated
by the binding of E2 to its recognition sites within the promoter and
the displacement of cellular transcriptional factors from the promoter
(3, 12, 14, 15, 20, 23, 28, 41, 42, 54, 55, 58, 59, 61, 62).
E2 is also involved in the regulation of viral DNA replication through
its association with E1, the viral replication factor (36, 50, 63,
65, 66, 67, 68). The conserved N-terminal domain of E2 is
required for transactivation (TA), E1 binding, and DNA replication
functions. The conserved C-terminal domain forms a dimer and functions
as a DNA binding domain. Both conserved domains are linked by a hinged
region (reviewed in reference 24).
The loss of E2 expression has been also implicated in the development
of HPV-induced carcinoma. Most human cervical carcinoma cells contain
integrated HPV DNA and actively express E6/E7 genes (2,
52, 69). The E2 gene is frequently disrupted as a
consequence of the integration of the viral genome, and it has been
postulated that the loss of E2 somehow contributes to carcinogenic
progression (9, 40, 47, 64). E6/E7 genes are
invariably expressed in HPV-positive cancers and are considered to be
involved in the development of HPV-associated cancers. E6 targets the
ubiquitination and proteolysis of p53 through its association with the
ubiquitin protein ligase, E6AP (25, 45, 46). E7 binds pRB
and inactivates its tumor suppressor function (17, 38).
Although E6 and E7 may have additional functions and cellular targets,
it is believed that their inactivation of these important tumor
suppressor proteins is critical for HPV-associated carcinogenesis. As
mentioned above, E2 has the ability to suppress E6/E7
expression; thus, disruption of the E2 gene results in the
deregulated expression of E6 and E7 and thus contributes to
carcinogenic progression (24). This model is also supported
by the finding that mutations in the E2 gene increased the
immortalization activity of HPV16 DNA for human primary keratinocytes
(43).
The significance of the disruption of the E2 gene in
cervical carcinoma cells has been investigated by the reintroduction of
E2 into HPV-positive carcinoma cell lines such as HeLa cells, which
contain integrated HPV18 DNA and actively express E6/E7 (4, 49). The expression of E2 protein in HeLa cells markedly inhibits E6/E7 expression and inhibits cell proliferation
(16, 26, 27, 62). The suppression of cell growth was also
observed in other HPV-positive cancer cell lines, including SiHa and
Caski, but not in HPV-negative cervical carcinoma cells such as C33A cells (16). These results suggested that the growth
inhibition by E2 was specific for HPV-positive carcinoma cells and that
the suppression of E6/E7 expression contributed to this process.
To further investigate the functions of the E2 protein in the growth
inhibition of HPV-positive carcinoma cells, we tested the activities of
various E2 mutants derived from HPV16 E2. We found a clear correlation
between the TA and growth suppression activities of E2. The TA function
of E2 was required for efficient repression of the E6/E7
promoter. Furthermore, results of in vivo footprinting and other
functional assays strongly suggested that the TA function was required
for the efficient binding of E2 to a transcriptionally active DNA
target in vivo in order to affect the displacement of cellular factors
from the E6/E7 promoter. The involvement of a TA function of
some transcription factors in the binding to DNA forming chromatin
structures has been reported previously (5, 34). It is
possible that an intact E2 TA function may be required for interaction
with specific cellular factors leading to a remodeling of the chromatin
of transcriptionally active templates.
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MATERIALS AND METHODS |
Cell culture and transfection.
HeLa and CV1 cells were
maintained in Dulbecco's modified Eagle medium supplemented with 10%
heat-inactivated fetal bovine serum. DNA transfection was done by a
standard calcium phosphate precipitation method (31). The
cells (1.5 × 105) were seeded in a 6-cm-diameter dish
1 day before transfection. Plasmid and carrier DNA (total of 10 µg)
were incubated with 500 µl of HEPES-buffered saline transfection
buffer (140 mM NaCl, 0.75 mM Na2HPO4, 25 mM
HEPES, 110 mM CaCl2 [pH 6.90]) for 30 min at room
temperature and then added to a culture dish; 20 h after transfection, cells were washed once with phosphate-buffered saline (PBS) and fresh growth medium was added. All analyses except the growth
suppression assay were performed with the cells 2 days after transfection.
Plasmid preparations.
We have described construction of the
original HPV16 E2 expression plasmid in a previous report
(44). FLAG-tagged E2 expression plasmids were constructed
from those original E2 expression plasmids. The FLAG tag sequence was
added at the 5' end of the E2 gene and recloned into
pCMV4 expression vector (10). The reporter
plasmid p6xE2BStkCAT was described elsewhere (60). HPV16 and
HPV18 replication origin (ori)-containing plasmids were constructed by
inserting the DNA fragments amplified by a PCR into pCAT-basic (Promega Corp., Madison, Wis.). LCR16F contains the HPV16 long central region
(LCR) fragment spanning nucleotides (nt) 7003 to 100 (GenBank accession
no. K02718). LCR18F-CAT and LCR18S-CAT contain the HPV18 LCR fragments
from nt 7000 to 110 and 7810 to 110, respectively (GenBank accession
no. X05015). The sequences of the PCR-amplified portions of the final
plasmids were confirmed by direct sequencing not to have any mutation.
pSV
was purchased from the manufacturer (Clontech Laboratories,
Inc., Palo Alto, Calif.).
Gel-shift analysis of E2 DNA binding capacity.
Nuclear
extracts were prepared from transfected HeLa cells (48). The
transfected cells (ca. 5 × 105 cells/6-cm-diameter
plate) were washed once with phosphate-buffered saline (PBS), harvested
into 1.5-ml microtubes, resuspended in 400 µl of buffer A (10 mM
HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol [DTT], benzamidine HCl [16 µg/ml], phenanthroline
[10 µg/ml], aprotinin [10 µg/ml], leupeptin [10 µg/ml],
pepstatin A [10 µg/ml], 1 mM phenylmethylsulfonyl fluoride [PMSF]) for 20 min on ice, mixed with 25 µl of 10% NP-40, and centrifuged at 2,000 rpm for 5 min at 4°C. The nuclear pellets were
resuspended in 50 µl of buffer C (20 mM HEPES [pH 7.9], 0.4 mM KCl,
1 mM EDTA, 1 mM EGTA, 1 mM DTT, benzamidine HCl [16 µg/ml], phenanthroline [10 µg/ml], aprotinin [10 µg/ml], leupeptin [10 µg/ml], pepstatin A [10 µg/ml], 1 mM PMSF) for 20 min on ice and centrifuged at 12,000 rpm for 5 min at 4°C. The protein concentration of the supernatant was quantitated and used as the nuclear extract.
Ten micrograms of nuclear extract was incubated in 10 µl of binding
reaction mixture [20 mM HEPES (pH 7.9), 5 mM MgCl2, 100 mM
KCl, 0.2 mM EDTA, 10% glycerol, 5 mM DTT, d(I-C) [1 µg/µl], 32P-labeled probe (5 fmol/µl), benzamidine HCl (16 µg/ml), phenanthroline (10 µg/ml), aprotinin (10 µg/ml),
leupeptin (10 µg/ml), pepstatin A (10 µg/ml), 1 mM PMSF] for 30 min at room temperature. The 32P-labeled DNA probe was
obtained by annealing two oligonucleotides (5'-GGT AAC CGA AAC CGG
TTA-3' and 5'-GGT AAC CGG TTT CGG TTA-3'), and
[
-32P]dCTP was incorporated by fill-in reaction with
Klenow fragment DNA polymerase. The reaction mixture was then loaded on
8% acrylamide gel in 4
1× Tris-borate-EDTA buffer.
Electrophoresis was carried out at 200 V for 2 h. The image was
obtained by exposing the gel to X-ray film.
Northern blot analysis and immunoblotting.
The cytoplasmic
fraction of the transfected cells was obtained as a supernatant of
buffer A-treated samples as described above. Cytoplasmic RNA was
extracted by the acid guanidinium-phenol-chloroform method
(8), and poly(A)+ RNA was enriched with
oligo(dT)-cellulose beads (New England Biolabs Inc., Beverly, Mass.).
One microgram of poly(A)+ RNA was separated through a
formalin-containing 1.2% agarose gel and then transferred to a
Hybond-N+ nylon membrane (Amersham Pharmacia Biotech UK Ltd., Little
Chalfont, United Kingdom). Northern hybridization was performed
according to a standard protocol (51). The probes used in
this analysis were the HPV16 E2 DNA fragment, p53 cDNA, and the
E6/E7 coding region of HPV18 labeled by a random prime
labeling kit (TAKARA, Kusatsu, Japan).
The whole cell extract was prepared from transfected cells with
extraction buffer (250 mM NaCl, 20 mM sodium phosphate buffer
[pH
7.0], 30 mM sodium pyrophosphate, 5 mM EDTA, 10 mM NaF, 5
mM DTT,
0.1% NP-40, benzamidine HCl [16 µg/ml], phenanthroline
[10
µg/ml], aprotinin [10 µg/ml], leupeptin [10 µg/ml],
pepstatin
A [10 µg/ml], 1 mM PMSF); 400 µl of extraction buffer
was directly
added to the culture dish and incubated for 20 min at
4°C with
rocking. Then the cell lysate was transferred to a
microcentritube
and centrifuged at 15,000 rpm for 10 min at 4°C. The
supernatant
was used as the whole cell extract. Twenty micrograms of
cell
extract was applied to a polyacrylamide gel containing 10% sodium
dodecyl sulfate, transferred to a Hybond-P polyvinylidene difluoride
membrane, blocked in 5% dry fat-free milk-0.1% Tween 20-PBS
(PBS-T),
and incubated with a 1/1,000 dilution of anti-FLAG monoclonal
antibody M5 (Sigma, St. Louis, Mo.) in 2% dry fat-free milk-PBS-T
for
1 h. The filter was washed three times with PBS-T, incubated
for
1 h with a 1/3,000 dilution of horseradish peroxidase-labeled
goat
anti-mouse immunoglobulin G monoclonal antibody (Amersham
Pharmacia
Biotech UK), subjected to four more washes with PBS-T,
and visualized
with the enhanced chemiluminescence detection reagent
(Amersham
Pharmacia Biotech
UK).
TA and transrepression assays.
HeLa cells in 6-cm-diameter
dishes were transfected with 1 µg of pCMV-E2, 1 µg of p6xE2BStkCAT
or LCR18F-CAT, 1 µg of pSV, and 7 µg of herring sperm (HS) DNA.
Two days after transfection, the bacterial chloramphenicol
acetyltransferase (CAT) activity in the cell extract was measured by a
standard protocol. The -galactosidase activity from pSV was
monitored by o-nitrophenylglycoside assay and used for
normalizing the transfection efficiency of each sample. To obtain the
transfection efficiency, the transfected cells were fixed with 0.25%
glutaraldehyde-PBS and stained with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
by a standard protocol (6).
Growth suppression assays.
A total of 2 × 105 HeLa cells transfected with 2 µg of pCMV-E2, 0.5 µg
of pSV2neo, and 7.5 µg of HS DNA were cultured in growth medium
supplemented with G418 (0.5 mg/ml). The surviving colonies were
enumerated 14 days after transfection.
In vivo footprinting of the HeLa cell genome.
In vivo
footprinting in HeLa cells transfected with 1 µg of pCMV-E2, 1 µg
of pSV, and 8 µg of HS DNA was performed by following a standard
protocol (37). The HeLa cells were treated with 0.1% dimethyl sulfate (DMS)-growth medium for 2 min, and then the genomic DNA was harvested by the proteinase K-phenol extraction method. In a
parallel experiment, genomic DNA was obtained from mock-transfected HeLa cells without DMS treatment and then treated with 0.1% DMS in
vitro (the sample indicated as naked). The DMS-treated DNA was cleaved
with 1 M piperidine for 30 min at 90°C, purified, and used for
ligation-mediated PCR (LM-PCR). The primers used in the LM-PCR were P1
(5'-GCA GTG AAG TGT TCA GTT CCG TGC ACA-3'), P2 (5'-GGT AGC TTG TAG GGT
CGC CGT GTT GG-3'), and P3 (5'-GGT AGC TTG TGA GGT CGC CGT GTT GGA TCC
TC-3'), LM PCR-1 (5'-GCG GTG ACC CGG GAG ATC TGA ATT C-3'), and LM
PCR-2 (5'-GAA TTC AGA TC-3'). The annealing temperature was 60°C for
P1, 63°C for P2, and 65°C for P3. P3 primer was end labeled with
32P, and the products were visualized with a image analyzer
(BAS-2000; Fuji Film, Tokyo, Japan).
Transient DNA replication assays of HPV16 and HPV18
ori-containing plasmids.
A transient DNA replication assay was
performed as described previously (44, 67, 68). HPV16 ori-
and HPV18 ori-containing plasmids (1 µg of each) were used for
transfection into CV1 and HeLa cells, respectively, with 1 µg of
pCMV-E2, 3 µg of pCMV-E1, and 5 µg of HS DNA. The extrachromosomal
DNA was extracted from the transfected cells by Hirt's method,
digested with BamHI and DpnI, and then analyzed
by Southern blotting with a 32P-labeled CAT gene probe.
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RESULTS |
Relationship between TA and growth suppression activities of
E2.
We previously reported a structure-function analysis of the
HPV16 E2 N-terminal domain TA and DNA replication activities
(44). Since growth inhibition of HPV-positive carcinoma cell
lines also requires a function mapping to the N-terminal domain, we
used a subset of the E2 mutants described in our previous report to examine whether the TA and/or DNA replication functions were required for suppression of cell growth. Four E2 mutants that had clear phenotypic characteristics were used in this analysis: R37A and I73A,
defective in TA function; E39A, which maintains wild-type (wt) TA
activity but fails to support DNA replication because of insufficient
association with E1; and L79A, with wt TA and DNA replication
activities. The wt and mutant E2 proteins were tagged at the N terminus
with the FLAG epitope for convenience of detection. We confirmed that
the addition of the epitope tag did not affect the phenotypes of the
original E2 mutants. The tagged E2 mutants were expressed at comparable
levels in the transfected HeLa cells (Fig.
1A), and their nuclear localization was
confirmed by immunostaining (data not shown). To verify the DNA binding capacity of the FLAG-tagged E2 proteins, a gel shift assay with a probe
containing a single E2 binding site was performed with both in
vitro-translated E2 (data not shown) and the nuclear extracts of the
HeLa cells transfected with the E2 expression plasmids (Fig. 1B). The
results demonstrated that neither the N-terminal tag nor any of the
specific alanine substitution mutations significantly affected the
expression, stability, subcellular localization, or DNA binding
capacity of E2.
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FIG. 1.
Expression of epitope-tagged HPV16 E2 proteins. (A)
Total cell extracts and nuclear extracts were prepared from HeLa cells
2 days following transfection with the epitope-tagged E2 expression
plasmids. The E2 proteins were detected with anti-FLAG antibody M5. The
positions of E2 proteins and molecular weight markers are indicated
with open and closed arrowheads, respectively. (B) A gel shift assay
with the single E2 binding site probe was performed with the nuclear
extracts. The three left-hand lanes contain probe only (probe), probe
plus nuclear extract from mock-transfected cells (control), and the
extract from the cells transfected with 3 µg of wt E2 expression
plasmid plus probe with a 50-fold excess of cold probe as a competitor
(wt+competitor). Each of the other pairs of lanes contain the probe
plus the nuclear extract from HeLa cells transfected with either 1 or 3 µg of the indicated E2 expression plasmid. Black and white arrowheads
indicate the positions of E2-DNA complex and free probe,
respectively.
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The phenotype of the TA activity of each tagged E2 mutant was identical
to that previously described by us for the untagged
E2 proteins
(
44) (Fig.
2A). The levels of
TA activities were
comparable between the tagged and untagged E2 (data
not shown).
In addition, there was a strong correlation between the TA
and
growth suppression activities of the E2 mutants, suggesting that
the TA function may be required for efficient growth inhibition
of HeLa
cells (Fig.
2B). The number of colonies observed with
the I73A mutant
was nearly the same as that in the control plate,
but the colonies
transfected with I73A were smaller than those
on the control plates,
suggesting that the I73A mutant may have
retained some residual growth
inhibition activity. We also examined
the relationship between TA and
growth inhibition activities with
other mutant E2s described in our
previous report (
44) and found
a clear correlation (data not
shown). As shown previously (
16),
a requirement for an
activity mapping to the N-terminal domain
of E2 for growth inhibition
was demonstrated with bovine papillomavirus
type 1 (BPV1) E2TA and E2TR
proteins and with a chimeric protein
having the VP16 TA domain. Using
N-terminal domain mutants of
BPV E2, Goodwin et al. reported a similar
correlation (
21),
suggesting this is a conserved feature of
papillomavirus E2 proteins.
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FIG. 2.
Comparison of TA and growth suppression activities of E2
proteins. For the TA assay (A), HeLa cells were transfected with 2 µg
of effector expression plasmid and 1 µg of the p6xE2BS-CAT reporter
plasmid. CAT assays were performed 2 days after transfection. HeLa cell
growth suppression (B) was determined by the colony formation assay.
Percent suppression is expressed as (number of colonies on control
plate  number of colonies on sample plate)/(number of colonies
on control plate) × 100. In these experiments, the number of
colonies on the control plates ranged from 1.2 × 103
to 3.0 × 103. BPV-TA, BPV-TR, and VP16-E2 plasmids
have been described previously (see text) and were not FLAG tagged.
Data represent the average of three independent experiments, and
standard deviations are indicated as error bars.
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Repression of E6/E7 transcription by E2 in HeLa
cells.
E2 can suppress E6/E7 expression from the HPV18
P105 promoter and from the analogous promoter in HPV16,
P97 (3, 15, 20, 42, 61, 62). Since the HPV
E6 and E7 genes encode the major transforming
activities for the high-risk-type HPVs and are invariably expressed in
HPV-positive cancers, we tested whether there was a correlation between
the transcriptional repression of the E6/E7 promoter and the
growth inhibition by E2. HeLa cells contain approximately 10 copies of
HPV18 DNA integrated into host chromosomal DNA and actively express the
E6 and E7 genes. We investigated the
transcriptional repression of the P105 promoter of the
integrated HPV18 genome in HeLa cells by E2. HeLa cells were therefore
transfected with an E2 expression plasmid and analyzed for the
expression of E6/E7 mRNA. The transfection efficiency was
over 80%, as estimated by X-Gal staining (see Materials and Methods).
The levels of the E6/E7 mRNA were markedly diminished by the
TA-competent E2 mutants (E39A and L79A) but not the TA-defective E2
mutants (R37A and I73A) (Fig. 3). This
result indicated a strong correlation between the TA function of E2 and
its ability to efficiently repress E6/E7 gene expression
from the integrated HPV18 genomes in HeLa cells.
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FIG. 3.
Effects of HPV16 E2 mutants on endogenous HPV18
E6/E7 mRNA expression and on p53 protein levels. HPV18
E6/E7, p53, and E2 mRNAs in the
E2-transfected HeLa cells were measured by Northern blot analysis
48 h after transfection. The p53 protein was detected by Western
blotting using the DO-1 antibody. An extract from C33A cells was used
as a control. The TA capacity of each E2 mutant is indicated at the
bottom.
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We also examined the consequence of
E6/E7 mRNA repression by
E2 in HeLa cells by examining p53 mRNA and protein levels. Similar
to
what has been described for BPV E2 in HeLa cells (
16,
26),
the amount of p53 protein increased in the HeLa cells expressing
TA-competent HPV16 E2 proteins (Fig.
3). The amount of
p53
mRNA
was not affected by E2 expression, indicating that the
modification
of the expression level of p53 protein was a
posttranscriptional
effect. In addition, as previously described with
BPV E2, we observed
an increase of p21 protein and the downregulation
of Cdk activity
as downstream effects of p53 activation with the
TA-competent
E2 expression (data not shown). These results suggested
that the
TA function of E2 was connected to the growth inhibition of
HeLa
cells via transcriptional suppression of
E6/E7 expression.
An intact TA domain is required for E2 repression of the
P105 promoter in a DNA template-dependent manner.
We
analyzed the transrepression activities of E2 mutants with a CAT
reporter plasmid in order to investigate the role of TA function in
transcriptional repression. Two different reporter plasmids, LCR18F-CAT
and LCR18S-CAT, were used in this experiment. LCR18F-CAT contains the
entire LCR directing CAT expression from the P105 promoter,
but LCR18S-CAT has a deletion of all LCR sequences upstream of nt 7800, just upstream of the HPV18 ori region (Fig. 4A). The latter plasmid contains the core
P105 promoter elements but is devoid of the upstream
enhancer element, resulting in a 95 to 98% loss of transcriptional
activity compared to the LCR18F-CAT plasmid in HeLa cells (data not
shown).
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FIG. 4.
Transcriptional repression of LCR18F-CAT by E2. (A)
Schematic presentation of the structure of HPV18 LCR. Data for
transcription factors bound on the LCR are based on other reports
(11, 39). The LCR portions inserted in LCR18F-CAT and
LCR18S-CAT are indicated. (B) HeLa cells were transfected with 2 µg
of the indicated effector expression plasmid and 1 µg of LCR18F-CAT.
Mock-transfected cells were used as control. Two days after
transfection, CAT activity in the cells was determined as described in
Materials and Methods. The data represent the average of four
independent experiments, and standard deviations are indicated as error
bars.
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Wild-type E2 strongly suppressed CAT expression from the
P
105 promoter of LCR18F-CAT as did the TA-competent
mutants, E39A
and L79A. On the other hand, the TA-defective mutants,
R37A and
I73A, failed to suppress the P
105 promoter,
indicating a correlation
between an intact TA function with repression
of the P
105 promoter
as observed with the integrated HPV18
genome in HeLa cells (Fig.
4B). A requirement for an intact E2 TA
function was also noted
for repression of a CAT reporter plasmid
containing the entire
HPV16 LCR in which CAT is expressed from the
P
97 promoter that
directs expression of E6 and E7 of HPV16
(data not shown). No
repression was obtained in the case of VP16-E2
chimeric protein,
suggesting that a function specific to the E2 TA
domain is involved
in the
E6/E7 promoter
suppression.
We could demonstrate about twofold repression of the LCR18S-CAT
reporter plasmid by wt HPV18 E2 (Fig.
5).
Interestingly, each
of the E2 mutants assayed could also repress
expression from the
LCR18S-CAT reporter approximately twofold. This was
true for the
TA-competent E2 mutants (E39A and L79A) as well as for the
E2
mutants (R37A and I73A) which are impaired in TA function. Thus,
the
modest E2 repression of the HPV18 P
105 promoter devoid of
the LCR upstream elements does not require an intact TA domain.
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FIG. 5.
Transcriptional repression of LCR18S-CAT by E2. HeLa
cells were transfected with LCR18S-CAT and E2 expression plasmids. Two
days after transfection, cell extracts were prepared and CAT activity
was assayed by a standard method. The CAT activity of mock-transfected
cells was taken as 100% (control). The data represent the average of
three independent experiments, and standard deviations are indicated as
error bars.
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The E2 TA function is involved in E2 promoter binding in HeLa
cells.
It had been postulated that the negative regulation of
P105 activity by E2 was due to the binding of E2 to its
cognate sites and the displacement of cellular transcription factors
such as SP1 from the initiation complex. This model predicts that the DNA binding activity of E2 would then be sufficient for the
P105 suppression. Each of the E2 proteins tested in our
study maintained the DNA binding capacity by in vitro assay (Fig. 1B),
yet R37A and I73A failed to suppress P105 activity of the
integrated HPV18 genome and LCR18F-CAT plasmid (Fig. 3 and 4B). One
possible explanation for our findings is that an intact TA function may
be required for E2 binding in vivo, and the in vitro assay used may not
reflect the true situation in HeLa cells. Since the LCR18F-CAT plasmid is a transcriptionally active template in HeLa cells, it may more closely resemble the chromatin structure of the integrated HPV18 genomes.
To address the role of the intact TA function in the binding of E2 to
its cognate sites in the P
105 promoter, we performed
in
vivo footprinting of the integrated HPV18 LCR, using HeLa cells
transfected with E2 expression plasmids. As depicted in Fig.
6,
E2 binding sites proximal to
P
105 were protected by wt E2 and
the TA-competent E2
mutants E39A and L79A. In contrast, TA-defective
mutants R37A and I73A
were impaired in the ability to protect.
This experiment has been
repeated five times and is highly reproducible.
These results indicate
an intact E2 TA function is required for
the efficient binding to DNA
in the context of the chromosome
in vivo. It is noteworthy that even in
the in vivo situation,
the E2 TA function is not required for
transcriptional suppression
of the LCR18S-CAT reporter (Fig.
5),
suggesting that the TA requirement
for DNA binding is dependent on the
transcriptional activity of
the DNA template. LCR18S-CAT may reflect
the DNA status of in
vitro binding assay because it does not form a
complex structure
for active transcription.
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FIG. 6.
In vivo footprinting analysis with the integrated copies
of HPV18 genomes. In vivo footprinting was done with HeLa cells
transfected with E2 expression plasmids. Two days after transfection,
DNA was prepared and transfection efficiency was monitored in a
parallel experiment. Over 80% of cells appeared to be positive for
-galactosidase expression. The corresponding sequence of the
promoter region of HPV18 is shown at the top, and two promoter-proximal
E2 binding sites (E2BS-1 and E2BS-2) are indicated. Positions of G
residues protected by TA-competent E2 binding are indicated with dots.
Lanes: naked, purified genomic DNA of HeLa cells used for the
substrate; control, mock-transfected cells.
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Although the in vivo footprinting experiment was useful for revealing
the protein-DNA association in a more physiological
state, the changes
in the observed protection pattern could still
either be a direct or an
indirect consequence of E2 expression.
We therefore extended our
analysis to examine whether the TA activity
was required for the E2
binding to DNA in vivo, using a DNA replication
assay that employed an
origin in the context of the full-length
LCR.
The requirement for E2 TA function for DNA replication is dependent
on the template.
DNA replication of an HPV ori-containing plasmid
requires both E1 and E2 (7, 10, 63). Although E1 is the
major DNA replication factor for the papillomaviruses, it has little
affinity by itself for binding to the origin and must be recruited
through its protein-protein interaction with E2 (29, 65, 66,
68). Therefore, the critical role of E2 as an auxiliary factor in
transient ori-dependent DNA replication depends on its ability to bind
DNA as well as to form a complex with E1. We therefore used a DNA replication assay utilizing the LCR18F-CAT reporter plasmid to assess
the role of the TA function of E2 in augmenting E1-dependent DNA
replication. In this experiment, both wt and L79A mutant E2s supported
the DNA replication of the HPV18 (LCR18F) plasmid efficiently, whereas
the TA-defective R37A and I73A mutants had reduced activity for DNA
replication (Fig. 7, LCR18F). This
experiment revealed a role for the E2 TA function in E1-mediated
ori-dependent DNA replication, presumably by permitting more efficient
E2-DNA binding in vivo. The E2 mutants tested in this experiment are
known to have comparable levels of E1-E2 binding activity
(44). The E39A mutant of E2 that is defective in forming a
complex with E1 served as a negative control. The requirement for an
intact TA domain to support E2-dependent DNA replication efficiently
was also observed with a corresponding HPV16 ori-containing construct,
LCR16F-CAT (Fig. 7).
View larger version (53K):
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[in a new window]
|
FIG. 7.
Involvement of the TA activity of E2 in DNA replication.
Transient DNA replication was performed with an HPV18 ori-containing
plasmid, LCR18F-CAT or LCR18S-CAT, in HeLa cells. LCR16F-CAT was used
for the transient DNA replication assay in CV1 cells. Note that LCR16
is active for transcription in CV1 cells. Signals of replicated DNA are
indicated with black arrowheads, and those of input DNA are marked with
white arrowheads. Positions of DNA size markers are indicated at the
left.
|
|
In the transcriptional repression experiment with LCR18S-CAT, we found
TA function-independent suppression of P
105 promoter
activity, suggesting that TA function is not required for DNA
binding
with transcriptionally inactive templates (Fig.
5). We
tested the DNA
replication activity with the LCR18S-CAT reporter
plasmid. In contrast
to LCR18F-CAT, LCR18S-CAT could be replicated
equally with each of the
E2 mutants tested except E39A, which
served as the negative control
(Fig.
7). The result of the DNA
replication assay suggested the
requirement of the TA function
of E2 for DNA binding was dependent on
the transcriptional activity
of the DNA
template.
As we have previously reported, there is no requirement of TA function
for the E2-enhanced replication of a plasmid containing
only the HPV16
ori region without the upstream LCR enhancer sequences,
indicating that
E1 binding is the major function of the E2 TA
domain in DNA replication
and that the TA activity of E2 is dispensable
for ori-dependent DNA
replication (
44). Similar observations
have been reported by
other groups who studied DNA replication
analysis with papillomavirus
E2 mutants (
18,
22). These previous
reports are consistent
with the result shown here with LCR18S-CAT.
In contrast, the TA
function of E2 was required for the E2-enhanced
DNA replication of the
LCR18F-CAT template that contains the full-length
LCR and that is
active for transcription from the P
105 promoter
in HeLa
cells. The results obtained from the transient DNA replication
assay
showed that the TA function of E2 was vital for the binding
of E2 to
the transcriptionally active promoter
region.
| |
DISCUSSION |
Mechanisms of E2-mediated growth inhibition.
In this study, we
investigated the molecular mechanisms by which HPV16 E2 protein
inhibits the growth of HeLa cells. We have previously reported that
growth inhibition by E2 is specific for HPV-positive carcinoma cell
lines (16). The continuous expression of E6 and
E7 genes from integrated HPV DNA is a hallmark in the majority of HPV-positive carcinomas and in cell lines derived from
them. E6 and E7 are considered to play central
roles in the progression to and maintenance of cancers. There is
considerable information on the mechanisms of the carcinogenesis
induced by the expression of high-risk-type E6 and E7 proteins
(24). Both E6 and E7 disturb the G1 and
G2 checkpoints, resulting in the accumulation of genetic
mutations, which can culminate eventually in the progression to full malignancy.
HPV16 E2 suppressed the expression of
E6 and
E7
in HeLa cells, and we demonstrate here a clear correlation between the
downregulation
of
E6/E7 expression and cell growth arrest.
Our results are in
agreement with previous studies that have also
suggested that
the expression of
E6 and/or
E7 is
indispensable for the continued
proliferation of carcinoma cells. The
repression of
E6/E7 expression
by E2 reactivates the cell
cycle checkpoints controlled by p53
and pRB. We have not yet determined
the relative contributions
of the p53 and pRB pathways to growth arrest
in HeLa cells. In
addition to their well-characterized roles in
regulating the pRB
and p53 pathways, E6 and E7 each likely have other
important functions
in cellular transformation and immortalization. It
is anticipated
that E2 repression of
E6 and
E7
expression in HPV-positive cancer
cells could also lead to the
activation of the pathways governed
by these additional targets. For
instance, E6 has been shown to
upregulate cellular telomerase activity
(
32). The downregulation
of E6 by E2 would presumably
suppress the telomerase activity,
and the subsequent detection of
critical short telomeres might
then signal cell growth arrest and/or
senescence. There have been
several reports describing the associations
of E6 and E7 with
cellular factors, and it will be interesting to
explore the functional
correlation between these associations and
growth inhibition.
It is also possible that functions of E2 independent
of its ability
to repress expression of
E6/E7 could be
involved in the growth
inhibition of the cervical carcinoma cells. For
instance, there
may be cellular genes whose expression is either
activated or
repressed by E2, or there may be cellular factors whose
activities
are somehow modulated by E2. Additional experiments will be
necessary
to determine whether repression of
E6/E7 by E2 is
indeed sufficient
to induce growth inhibition in HeLa
cells.
Correlation between the TA and growth inhibition functions of E2
protein.
It has previously been reported that both the TA and DNA
binding domains of BPV1 E2 protein are required for the growth
inhibition effect (16). A detailed analysis using a panel of
E2 proteins with mutations in their transactivation domains was
performed with BPV1 E2 by Goodwin et al., who found that the TA
function was required for growth inhibition (21). Our
results confirm their data and extend them to the HPV16 E2 protein.
Transcriptional repression of the
E6/E7 promoter is believed
to occur via the binding of E2 to its recognition sites proximal
to the
E6/E7 promoter and displacement of cellular transcriptional
factors necessary for assembly or activation of the initiation
complex.
It has also been thought that the DNA binding activity
of E2 was
sufficient for this transcriptional repression (
13).
We
found here that the TA function is also required for the
transcriptional
repression of P
105 by E2, in accordance
with the published results
of Goodwin et al. (
21). Desaintes
et al. reported that E2TR
of BPV1, which lacks the N-terminal
transactivation domain, could
suppress the HPV18 P
105
promoter as efficiently as E2TA (
13).
In our experiments
using LCR18F-CAT, we observed a requirement
for the N-terminal
transactivation domain of BPV1 E2 for repression
of the
P
105 promoter (Fig.
4). The results obtained using BPV1
E2
by Goodwin et al. (
21) also support an involvement of TA
function in P
105 repression. We must point out that our
data are
in disagreement with those of Desaintes et al., but a recent
study
by Francis et al. also could not reproduce the observations by
Desaintes et al. that the BPV E2TR can repress the endogenous
HPV18
E6/E7 promoter in HeLa cells (
19).
Involvement of the TA activity in the repression of the
E6/E7 promoter.
The requirement of TA function in
transcriptional repression of E6/E7 expression in HeLa cells
raised the possibility that the TA function of E2 is involved in DNA
binding in vivo. To test this hypothesis, in vivo footprinting was used
with the integrated HPV18 genome in HeLa cells. Although HeLa cells
harbor approximately 10 copies of HPV18 DNA in the genome, protection
of the E2 binding sites was observed with wt and TA-competent E2
mutants. No such protection was seen with TA-defective mutants,
indicating that the TA function of E2 was required for its efficient
binding to the integrated P105 promoter sequence in HeLa
cells. All of the E2 mutants tested here exhibited similar DNA binding
activities in vitro. Thus, the requirement of the TA activity for DNA
binding was specific for the in vivo situation of the integrated HPV18 genomes in HeLa cells. These results support our model, although they
do not rule out the possible importance of cellular factors interacting
with a TA-competent E2 protein to affect the repression of the
P105 promoter.
E2 is known to participate in papillomavirus DNA replication in
association with viral replication factor E1. The function
of E2 in DNA
replication is thought to be the recruitment of E1
to the ori through
the E2 binding sites located adjacent to the
ori. We and others have
reported that the TA activity of HPV16
E2 is dispensable for transient
DNA replication; however, these
previous studies used a minimal ori in
the replication assay (
18,
44). Utilizing a replication
template that includes the entire
HPV18 LCR (or the entire HPV16 LCR),
we now see a requirement
for an intact TA function for efficient DNA
replication. Thus,
there are elements within the LCRs of the HPV16 and
HPV18 genomes
that require the TA function of E2 in addition to its
ability
to complex E1 to support ori-dependent DNA replication. It
seems
likely that these elements are the enhancer sequences within the
LCRs that confer strong transcriptional activity to the templates
themselves. In the transient DNA replication assay, the amplification
of transcriptionally active templates was dependent on the TA
activity
of E2, but the amplification of templates with low transcriptional
activities (LCR18S and LCR16S) was not. Therefore, the TA function
of
E2 was not required for DNA binding in the case of templates
of low
transcriptional activity even in vivo (Fig.
7). There was
a report that
the TA function of E2 was dispensable for the episomal
DNA replication
of HPV31 in human foreskin keratinocytes (
56).
Although we
do not know why our result conflicts with this finding,
some assay
conditions might be critical for TA dependency of HPV
DNA replication.
The correlation between TA function and DNA binding
activity of E2 can
be made with repression of the HPV18 P
105 promoter.
Using a
P
105 reporter plasmid with a low level of transcriptional
activity and devoid of the LCR enhancer elements, all of the HPV16
E2
mutants (regardless of their TA function status) could repress
transcription approximately twofold, indicating that the TA function
of
E2 was not required for the binding to E2 binding sites on
the
transcriptionally silent template. Both the DNA replication
assay and
the P
105 repression assay indicated that the requirement
for an intact E2 TA function was dependent on the transcriptional
status of the DNA templates (Fig.
8).
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 8.
TA activity of E2 is involved in DNA binding to a
transcriptionally active template (model). The requirement of E2 TA
activity for DNA binding is dependent on the state of DNA template.
With transcriptionally active DNA template (left), the TA activity is
required for DNA binding of E2. With transcriptionally silent template
(right), the DNA binding domain may be sufficient for DNA binding of
E2.
|
|
The TA domains of several transcription factors have been reported to
enhance sequence-specific binding to chromatin (
5,
34).
Physical interaction between the TA domain of transcription
factors and
chromatin remodeling factor(s) is supposed to play
an important role in
enhancing this DNA binding (
30,
33).
It is possible that
elements within the LCR that affect the transcriptional
activity of the
HPV16 and HPV18 templates used in our study may
also affect chromatin
structure. The chromatin organization of
the HPV LCR is supposed to
have an important regulatory function
in the viral life cycle
(
57). Although transiently transfected
DNA is not thought to
form well-organized chromatin structures,
it is possible that the
TA-competent E2 mutants could associate
with some cellular factors that
are involved in the chromatin
remodeling activity. The short LCR
constructs containing the ori
and flanking E2 binding sites and core
promoter sequences may
more closely resemble the naked DNA template
used in in vitro
assays, but the more transcriptionally active
templates containing
the full LCR might form a more complex structure
with cellular
DNA binding proteins including histones and
transcriptional factors.
The cellular factor(s) associated with E2 TA
activity might then
be required for the remodeling of the complex DNA
template to
facilitate the DNA binding of E2. This model would account
for
our results demonstrating a requirement for a functional E2 TA
domain for binding to the E2 binding sites of the integrated HPV18
promoter sequences in HeLa cells (Fig.
6) and for supporting
E2-dependent
DNA replication or E2 repression of the P
105
promoter in assays
that use the full LCR-containing templates and
reporters (Fig.
3,
4, and
7).
As Dowhanick et al. (
16) and Goodwin et al. (
21)
reported, heterologous TA domains such as that of VP16, p300, or Spi
can not functionally replace the E2 TA domain in growth inhibition
of
cervical carcinoma cell lines or transcriptional suppression
of
E6/E7 genes. These results indicate that a function specific
for E2 is therefore necessary for these activities. We do not
yet know
the basis of this specificity, but possibly it is mediated
through
enhanced DNA binding exerted through a specific interaction
between the
E2 TA domain and some cellular factor(s). It is also
possible that
cellular factors bound to the HPV LCR participate
in the recruitment of
E2 to its binding site. More detailed analyses
will be required to
elucidate the molecular mechanism of this
E2-specific inhibition of
cell growth and
transcription.
| |
ACKNOWLEDGMENTS |
We thank Yasumasa Iwatani (Yamanashi Medical School) for many
helpful discussions and Atsue Ueda for technical assistance and
manuscript preparation. We are grateful to Susanne Schmid and John
Benson for critical reviews of the manuscript.
This research was supported in part by grants to H.S. from the Japanese
Ministry of Education, Science and Culture and the Japanese Ministry of
Health and Welfare and by a grant from the National Institutes of
Health (RO1 CA77385) to P.M.H.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Gene Analysis, Department of Viral Oncology, Institute for Virus
Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Phone:
81-75-751-4010. Fax: 81-75-751-3995. E-mail:
hsakai{at}virus.kyoto-u.ac.jp.
| |
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Journal of Virology, April 2000, p. 3752-3760, Vol. 74, No. 8
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Abstract
Introduction
