From the Section of Medical Oncology,
Department of Internal Medicine, and Genetic Therapy Program of the
Yale Cancer Center and the ¶ Department of Comparative Medicine,
Yale University School of Medicine, New Haven, Connecticut
06520-8032
Received for publication, August 7, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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The papillomavirus E2 protein plays an important
role in viral transcriptional regulation and replication. We chose to
study the cottontail rabbit papillomavirus (CRPV) E2 protein as a
transcriptional regulator because of the availability of an animal
model for papilloma formation, which may be relevant for human
papillomavirus (HPV) infection and replication. We studied the effect
of expression levels of E2 on the long control region, which contains
transcriptional promoter and enhancer elements, and synthetic
E2-dependent artificial promoters in which the E2 was the
dominant factor in the transcriptional activation. These experiments
indicated that high levels of E2 were inhibitory and low levels were
stimulatory for transactivation. In addition, we showed that the
complex formed between CRPV E2 and the cognate binding site was less
stable than the complex formed between HPV E2 and the same cognate
binding site. Furthermore, we showed that CRPV E2 binding to its
transcriptional regulatory sequence was stabilized by other proteins
such as E1, which produced increments in transcriptional activation of
E2-dependent genes. The data may be used to define
conditions in which the rabbit model can be used for the screening of
drugs which are inhibitory to the HPV and CRPV replication and gene expression.
The human papillomavirus
(HPV)1 is the etiologic agent
responsible for several benign epithelial proliferative lesions,
including plantar warts, genital warts, and respiratory laryngeal
papillomatosis (1-4). Chronic HPV infection is one of main risk
factors for the development of cancer (5). To characterize the
pathogenetic mechanisms involved in the development of virus-associated
cancers as well as premalignant lesions, and to develop effective
strategies for their prevention and treatment, it is imperative to have
a cell-free model or an animal model system to test new approaches to
the treatment of HPV infections. Although papillomavirus
infections are species-specific, common mechanisms of regulation of
transcription and replication may exist among different species
(6-15). The fact that many similarities have been identified
between cottontail rabbit papillomavirus (CRPV) and HPV
infections suggests that the CRPV infection may be relevant to
the understanding of human disease associated with human
papillomaviruses infection (16-18).
Recent studies have compared in depth the mechanisms governing the
bovine papillomavirus 1 (BPV-1) and HPV infections. Three BPV-1 E2
proteins have been reported. The largest protein, E2-TA, is a
transcriptional activator, and two small truncated proteins, E2-TR and
E8/E2, have been reported to be transcriptional repressors (14,
19-21). In contrast, there is as yet no definitive biochemical evidence that the HPV encodes truncated E2 repressor proteins. In both
species, the E2 protein regulates transcription of the viral early
genes and plays an auxiliary role in replication by recruiting the E1
protein onto the origin of replication (ori) prior to the binding of
the host cell DNA polymerase (22-27). E1 protein inhibits or modulates
transcription of other viral genes in the BPV-1 system (28, 29). Recent
studies have shown that E1 and E2 may interact not only in promoting
viral replication but also in modulating the expression of other viral
genes (30, 31). The E2 protein and its binding sites on the viral DNA
mediate attachment of the episomal HPV to the host cell chromosomes
(32, 33). The tethering may protect the HPV episomal DNA from
nucleolytic digestion. Thus, both the E2 and E1 proteins are important
for several steps of the viral life cycle.
Although the CRPV E2 protein has been reported as a transcriptional
activator previously (34), the mechanism of transcription of the CRPV
E2 protein has not been well studied in detail. We chose to study the
CRPV E2 protein as a model for papillomavirus transcriptional
regulatory proteins because the results in the CRPV animal model may be
relevant for the understanding of HPV infection. The results of our
studies suggested that high levels of CRPV E2 protein were inhibitory,
whereas low levels of CRPV E2 protein were stimulatory for
transactivation of reporter genes controlled by
E2-dependent promoters. Furthermore, E1 may promote the
CRPV E2-dependent gene expression by stabilizing E2 on its cognate recognition sites in CRPV infections. These data will be
relevant to defining conditions under which the rabbit model can be
used to isolate drugs which suppress HPV infection.
Cells and Transfections--
Sf1Ep cells derived from rabbit
epidermis and C33a cells derived from human cervical cancer were
obtained from the American Type Culture Collection. Sf1Ep cells and
C33a cells were grown in minimum essential medium and Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) containing 10% heat
inactivated calf serum, respectively.
Vector Construction--
To generate the pCMV-CRPVE2,
XbaI and BamHI sites were introduced upstream and
downstream of the CRPV E2 gene by PCR for subcloning, and PCR products
were digested with enzymes prior to their introduction into the pCG
vector driven by a CMV promoter (35). The primers for PCR were
5'-gctctagaatggaggctctcagccagcg-3' and
5'-cgggatccgcatacacgtcactaaagcc-3'. The CRPV-pLAII provided by Felix
Wettstein (UCLA, San Diego, CA) was used as template DNA. While
sequencing the resulting vector to ensure that PCR amplification did
not introduce any mutations into the E2 coding regions, we found that
the clone of the CRPV used as a template for PCR contained one
nucleotide transition from A to G at nucleotide position 3312 (silent
mutation) (34) and also contained other differences reported previously
(36). The pRSV-CRPVE2 was constructed by introducing CRPV E2 into
BstXI and XbaI sites of a pRSV vector
(Invitrogen). The pCMV-HPV16E2 (pCG-HPV16E2) was provided by Dr.
Lubomir Turek (University of Iowa College of Medicine). The pCMV-CRPVE1
contained CRPV nt 1362-3170. We found nucleotide transitions from C to
T at nt 1531, resulting in Leu instead of Pro, from T to C at nt 1535, resulting in no amino acid change and from T to C at nt 1951, resulting
in Ala instead of Val. The pCRPV-LCRWT was constructed by PCR. The
KpnI and XhoI sites were introduced into the CRPV
long control regions (LCR) by PCR, and the fragment was cloned into the
pGL3 basic vector (Promega) using the following primers:
5'-cggggtacctaattgtatattattggatgtgt-3'and 5'-gagccgctcgagctgctctaagtttcttgtctt-3'. We found that the clone of the
CRPV used as template for PCR contained nucleotide transitions from T
to C at nt 7354, from T to C at nt 7386, from C to T at nt 7430, from C
to T at nt 7473, from T to C at nt 7484, and an A insertion at nt 7607. To construct the 0pCRPV-LCRMut, a consensus E1 binding region was
abolished to introduce mutations from AACAAT to GGATCC starting at
position nt 4 (28) using a Quick Change (Stratagene). The pGL3tk
was constructed by PCR; HindIII and BglII sites
were introduced into the minimum thymidine kinase of the herpes simplex
virus type I (tk core promoter), and the fragment was cloned into a
pGL3 basic vector (Promega) using the following primers:
5'-gaagatctgtccacttcgcatattaaggtga-3'and
5'-cccaagcttagatctgcggcacgctgttac-3'. To create the 2E2luc
luciferase vector, synthetic oligonucleotides were annealed and
introduced into the KpnI and BglII sites of the
pGL3tk vector. Synthetic oligonucleotides of additional two E2 binding
sites were introduced into the KpnI site of the 2E2luc vector to create the 4E2luc vector. The 4//2E2luc and the 2//2E2luc vectors contained a 55-bp spacer between E2 binding sites, which was
derived from the multiple cloning sites in the pGL3 vector. To generate
the pRSET-HPV16E2 DNA binding domain (DBD) for production of the
peptide, XhoI sites were introduced into the DNA binding domain of HPV16E2 by PCR for subcloning, and that fragment was cloned
into a pRSET A vector (Invitrogen). The primers for PCR amplification
were 5'-cccgctcgagatgacacccatagtacatttaaag-3'and 5'-cccgctcgagtcatatagacataaatccagtaga-3'. The HPV-16 114/K clone provided by Dr. Matthias Dürst (Deutsches
Krebsforschungszentrum, Heidelberg, Germany) was used as the
template DNA. To generate the pRSET-CRPVE2DBD, XhoI sites
were introduced into the DNA binding domain of the CRPV E2 by PCR for
subcloning, and PCR products were introduced into a pRSET A vector. The
primers for PCR amplification were 5'-cccgctcgagatgccgcccgtgatttgc-3'
and 5'-cccgctcgaggcatacacgtcactaaagcc-3'. All constructs were confirmed
by sequencing (Yale Keck laboratories).
Luciferase Assay--
Cells were seeded onto six-well plates and
transfected with the reporter vector, the expression vector for E1
and/or E2 and the Ribonuclease Protection Assay--
The lysate ribonuclease
protection assay was performed according to the manufacturer's
instructions (Ambion). Briefly, to prepare the labeled RNA probes, the
CRPV genome from nt 3211 to nt 3361, which contains the E2 gene, was
amplified by PCR and introduced into the BamHI and
HindIII sites of a pspt 19 vector (Roche Molecular
Biochemicals). The primers for PCR amplification were
5'-taaatccagtgtgatatcttatgtgagtaggctttcaatgatc-3'and
5'-cgggccccaagctttgagtaggctttcaatgtac-3'. The vector with the E2 insert
was linearized by EcoRI digestion and a 150-nt riboprobe was
synthesized with the T7 RNA polymerase. To normalize the signal
strength for variations due to the amount of total RNA present in each
sample and variations in the amount of RNA loaded on the gel, the RNA
was simultaneously hybridized with a 32P-labeled riboprobe
complementary to 18 S RNA and subjected to RNase digestion and
protection (Ambion). The products were separated on the 5% acrylamide
nondenaturing gel and then exposed to x-ray films. The radioactive
protected bands in the gel were quantified by NIH image 1.60 software.
Preparation of Nuclear Extracts--
Sf1Ep cells in 100-mm
dishes were transfected with varying ratios of the E2 and the backbone
vector DNA. In each transfection mixture, the amount of the DNA vector
was constant. We modified the preparation of nuclear extracts as
described previously (37). Briefly, at 48-h posttransfection, cells
were scraped off the surface of the culture dish with a reporter lysis
buffer (Promega) containing 10 mM KCl, 0.1 mM
EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin. Cells
were incubated on ice for 30 min, then 25 µl of 10% Nonidet P-40 was added. After centrifugation, the nuclear extract was resuspended in 50 µl of buffer containing 50 mM HEPES (pH 7.6), 420 mM KCl, 0.1 mM EDTA, 5 mM
MgCl2, and 20% glycerol and incubated on ice for 30 min.
The nuclear extract was then centrifuged for 10 min at 4 °C, and the
supernatant was frozen in aliquots. The protein concentration was
determined by a BCA protein assay kit (Pierce).
Expression and Purification of His Tag Protein--
The
pRSET-CRPV E2DBD and the pRSET-HPV16DBD were transformed into
Escherichia coli BL21(DE3)(plysS) (Novagen). The
isopropyl- Electromobility Shift Assay--
A double-stranded
oligonucleotide DNA was end-labeled by Klenow fill-in with
[32P]dCTP and used as a probe. The binding reaction was
carried out in a final volume of 30 µl in a buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 1 mM
EDTA, 5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.7 mM
phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, 4 µg/ml
leupeptin, and 160 ng of dAdT. The reaction mixture containing 10 µg
of protein of total cell extracts was preincubated in the presence or
absence of the cold competitor DNA on ice for 10 min. After addition of
the radiolabeled DNA probe, samples were further incubated for 30 min
at 22 °C and loaded on the 5% polyacrylamide gel and run for 2 h at room temperature.
For affinity studies, purified E2 proteins were added to the reaction
mixture containing 0.15 mg/ml bovine serum albumin with various amounts
of cold competitor oligo DNAs. The dissociation constants
(Kd) were estimated by mixing
32P-labeled oligonucleotide probe (5 fmol) with increasing
concentration of unlabeled oligonucleotide in binding mixtures prior to
the addition of a 10-fold molar excess of E2 DBD protein as reported before (21, 38, 39). The Kd values were equal to the
unlabeled competitor concentrations that depleted the bound complex
formation by 50%. The off-rate ratio was determined by adding cold
competitor to each reaction mixture prior to running the gel after
binding purified proteins to the 32P-radiolabeled
oligonucleotide probe. Dissociation half-time ( t1/2) represents the time required to exchange 50%
of labeled, bound oligonucleotide DNA. The gels were dried and scanned
using a PhosphorImager TM (Molecular Dynamics).
Transient Replication Assays--
The pCRPV-LCRlucWT or the
pCRPV-LCRlucMut was cotransfected with both/either the pRSV-CRPVE2
and/or the pCMV-CRPVE1 vector in Sf1Ep cells. Sixty-eight h after
transfection, the cells were lysed by the alkaline lysis procedure,
followed by DpnI digestion (24). The pCRPV-LCRlucWT vector
was linearized with XbaI digestion, and the probe was
generated by a Prime-It II (Stratagene). Hybridizations were
carried out at 65 °C in 6 × SSC, 5 × Denhardt's
solution, 0.5% SDS, and 200 µg/ml salmon sperm DNA for 8 h. The
filters were washed in 2 × SSC and 0.1% SDS at 65 °C for 30 min, and then the filters were exposed to the Kodak X-Omat AR film at
Opposite Effects of the CRPV E2 Protein Driven by a CMV or a RSV
Promoter on the CRPV LCR--
Despite the fact that other workers had
reported that the CRPV genome was maintained as an episome in Sf1Ep
cells (40), we found no CRPV genome by PCR nor E6/E7/E2 transcript by
RT-PCR after six passages of Sf1Ep cells, which were used in our
laboratory for our experiments (data not shown). Thus we conclude that
the particular clone of the Sf1Ep cells, which is being used in our laboratory, is appropriate for the transactivation studies outlined below.
Since the level of E2 expression is known to be critical for
transactivation (41, 42) and has also been reported to be important for
BPV-1 replication (30), we compared the effects in the two systems of
varying the level of the E2 expression in cells in which the level of
the E2 dependent reporter gene expression was being studied. To
accomplish this (see Fig. 1), we used two different promoters to sustain the level of the expression of E2
protein: the RSV promoter, which is known to produce relatively low
levels of expression of a heterologous transgene, and the CMV promoter,
which is known to produce a high level of the transgene expression
(42). We measured the levels of luciferase activity expressed by a
luciferase transcriptional regulatory vector, in which the expression
of the luciferase gene was driven by the CRPV LCR, after the
cotransfection with the CRPV E2 expression vector. The activity as a
promoter of CRPV LCR depended on the level of the E2 protein. The CRPV
E2 protein was expressed from the expression vector driven by the CMV
or RSV promoter. Thus, we measured the influence of CRPV E2 protein on
its LCR activity. As shown in panel 1 of Fig.
2, no increase of luciferase activity above that seen in control experiments in the absence of any E2 expression vector was observed with E2 input vector DNA amounts less
than 0.1 ng (data not shown). Interestingly, at levels of the input E2
expression vector DNA greater than 1 ng, there was a decrease in the
level of expression of luciferase, which reached a minimum of the
luciferase activity at 1200 ng of DNA. We also obtained the same
results using C33a cells and 293 cells (data not shown).
In contrast, when we cotransfected the pRSV-CRPVE2 vector with the
pCRPV-LCRlucWT reporter vector into Sf1Ep cells, we found a pattern of
initial dose-dependent activation of the luciferase reporter gene expression (2.5-3-fold) as the amount of the E2 vector
DNA increased from 10 to 100 ng and then a decrement in luciferase
expression as the amount of the E2 vector DNA increased from 200 to
1200 ng. These results showed the opposite effects of CRPV E2 protein
driven by the CMV or the RSV promoter on the CRPV LCR.
Use of Simplified Promoters for Luciferase Assay to Study the
Function of E2--
As shown in Fig. 1B, there are
differences in the organization of the number of E2 binding sites in
the LCR of papillomaviruses among different species. The HPV16 has only
four E2 binding sites, whereas the BPV-1 has 12 E2 binding sites in the
LCR (43). Since many host and viral proteins interact to regulate
transcriptional activation in the LCR, we created simplified promoters
for the expression of luciferase gene which were E2 dependent. To
investigate how the number and specific distribution of the E2 binding
sites contribute to transcriptional activation, we constructed reporter gene promoters with different numbers of E2 binding sites and different
spacing among E2 binding sites (Fig. 1). The BPV-1 E2 is unable to
activate the expression of reporter genes driven by E2 dependent
promoters that contain only E2 binding sites and a TATA box. This can
be overcome by the addition of Sp1 binding sites to the E2 binding
sites and the TATA box transcriptional element (44). The Sp1 element is
known to be associated with the E2 protein in the native LCR element.
Thus, our vectors included Sp1 binding sites as well as E2 binding
sites next to the tk core promoter. Nevertheless, the use of the
simplified promoters will minimize the effects of other host factors on
reporter gene expression and make the results more dependent on the
binding of E2 to the reporter gene promoter.
When we cotransfected the pCMV-CRPVE2 expression vector with the
artificial reporter vectors, the 4//2 E2 luciferase, the 2//2 E2
luciferase, and the 4E2 luciferase vectors into Sf1Ep cells, we found a
pattern of initial dose-dependent activation of the
luciferase reporter gene expression (2-3.5-fold) at the 200 ng of the
input E2 expression vector DNA, and then a decrement in luciferase
expression as the amount of the E2 vector DNA increased from 200 to
1200 ng. In contrast, there was a 14-16-fold activation of all of the
luciferase reporter constructs (4//2 E2luc, 2//2 E2luc, and 4 E2luc) at
200 ng or greater of the pRSV-CRPVE2 expression vector (see Fig.
3, panel 2). Little or no
activation of the luciferase reporter gene was seen at any amount of E2
vector DNA with the 2 E2luc reporter construct. These data suggest that
the CRPV E2 is more stimulatory with the synthetic
E2-dependent promoters than with the native LCR. Some have
proposed that this suggests the presence of other proteins interacting
with E2 on the LCR, which are not present in the simplified promoters.
In addition, data suggested that the CRPV E2 has dramatically different
effects on the expression of reporter genes under the control of E2
dependent promoters, when the E2 expression vector was driven by
the CMV or the RSV promoter. These data also show that the number of E2 binding sites within the promoter of the reporter gene is critical and
the space separating these E2 binding sites are less important for
transactivation of CRPV E2.
Comparative Study of the Levels of the E2 Protein in Cells
Transfected with E2 Expression Vectors Governed by the CMV or the RSV
Promoters--
On the basis of the results described above, we assumed
that the different levels of luciferase expression were due to the different levels of the E2 expression driven by either the CMV or the
RSV promoter. To formally test if this were true, we used the mobility
shift assay to measure E2 protein levels in Sf1Ep cells that were
transfected with various concentrations of the E2 expression vector
driven by the CMV promoter as compared with those E2 protein levels in
cells transfected with the E2 expression vector driven by the RSV
promoter (see Fig. 4, panel
1). No complexes between proteins and the 32P-labeled
oligonucleotide carrying the E2 binding sites were detected with the
nuclear proteins in Sf1Ep cells transfected with the RSV E2 expression
vectors, whereas there were detectable complexes in the mobility shift
gel lanes on the right half of the gel, which were generated by
incubating nuclear extracts of Sf1Ep cells transfected with a CMV E2
expression vector with the 32P-labeled
oligonucleotide (see panel 1 of Fig. 4, lanes in the right half of the gel). Addition of an excess of unlabeled
oligonucleotide containing the E2 binding site to the protein extract
before the 32P-labeled oligonucleotide was added (see
lane C in Fig. 4, panel 1), suppressed the
formation of the detectable bands on the gel, suggesting that the
formation of protein/DNA complexes was sequence-specific.
We also examined the expression level of E2 mRNA in cells
transfected by E2 expression vectors driven by either the CMV or the
RSV promoters by the ribonuclease protection assay (see Fig. 4,
panel 2). The intensity of the signal generated by the
protected E2 mRNA extracted from Sf1Ep cells transfected with the
E2 expression vector driven by the RSV vector was much lower than found
in extracts of Sf1Ep cells transfected with equal amounts of the E2
driven by the CMV expression vector (lanes 6-10 of
panel 2). The amount of CRPV E2 mRNA reached a plateau
in the presence of 800-1200 ng of RSV E2 expression vectors
(lanes 4 and 5 in panel 3), which was
equal to the amount of mRNA level in the presence of 200 ng of the
CMV E2 expression vector (lanes 3, 4, and
7 in panel 3). It should be noted that the CRPV
E2 transactivated the luciferase gene driven by the artificial
promoters in Fig. 3 along with 800-1200 ng of the pRSV-CRPV E2 vector
or 200 ng of the pCMV-CRPV E2 vector.
Taken together, these results suggest that lower levels of the E2
expression were generated in Sf1Ep cells transfected with the
pRSV-CRPVE2 vector than was the case with the pCMV-CRPVE2 expression
vector. These results further support the contention that high doses of
CRPV E2 can be inhibitory to expression of reporter genes governed by
E2-dependent promoters (the artificial promoters presented
in Fig. 3 or the native rabbit LCR in Fig. 2), whereas low doses of
CRPV E2 resulted in transactivation.
Comparison between the CRPV E2 and the HPV16 E2 Proteins for
Transactivation--
We also conducted experiments on the luciferase
gene expression driven by either the tk core promoter or the SV40
promoter along with pCMV-HPV16E2 vector. Luciferase activity showed a
2- or 3-fold increase using the tk core promoter, whereas a 20-fold increase was seen using the SV40 promoter (p2 × 2xE2BS-luc),
which contains two pairs of two E2 binding sites separated by 35 base pairs, originally obtained from HPV6b as reported (kindly provided by
Dr. Robert Kovelman) (42). Interestingly, the luciferase gene driven by
the SV40 promoter showed much higher activity than the tk core promoter
independent of the number of E2 binding sites in our assay (data not
shown). It must be noted that the SV40 promoter contains six GC-rich
repeats that bind to Sp1, which might lead to enhancement or effacement
of the subtle fine tuning of transactivation by E2 protein.
As shown in Fig. 5, panel 2,
we found that the luciferase activity increased at all levels
(200-1200 ng) of the pCMV-HPV16E2 expression vector when the reporter
gene was driven by the following promoters: the 4//2 E2 luciferase or
the 2//2 E2 luciferase expression vectors. In contrast, no activation
of expression was seen when the luciferase gene was controlled with the
4E2 or the 2E2 luciferase expression vector. In contrast, when CRPV E2
expression vectors were cotransfected with the synthetic
E2-dependent luciferase reporter vectors, a
dose-dependent inhibition of the luciferase expression was
seen following transfection of 400 ng or greater of E2 expression
vector DNA, although an initial activation of the luciferase gene
expression was seen when 200 ng of the E2 expression vector DNA was
used for the transfection. Interestingly, the 4//2 E2 luciferase, the
2//2 E2 luciferase, and the 4E2 luciferase vectors all showed a similar
-fold increase between 200 and 800 ng of the CMV E2 expression vector,
whereas the 2 E2 luciferase reporter vector showed much lower levels of
expression. These data suggest that the number of E2 binding sites
within the promoter of the reporter gene is more important than the
space separating these E2 binding sites in terms of CRPV E2. In
addition, these data suggest that there might exist some intrinsic
differences between the HPV16 and the CRPV E2 protein.
Comparison of the Affinity and Stability of the CRPV and HPV16 E2
Proteins to the Cognate Sites--
We assumed the differences between
the result obtained with the E2 proteins of the two species (human and
rabbit) of E2 transcriptional activity was due to differences in the
levels of affinity of the E2 protein for the cognate E2 binding sites.
We therefore investigated the Kd value of E2 binding
to the cognate sites by mobility shift assay. The E2 binding site
presented here was reported as a high affinity binder previously (21)
and identical as those used in artificial promoters in Fig. 1. As shown
in panel 1 of Fig. 6, we
determined that Kd values of the HPV16 E2 and the
CRPV E2 protein for the E2 binding site was 13 and 20 nM,
respectively, suggesting that affinity of the E2 proteins derived from
both species was similar.
We then measured the stability of E2 proteins to the cognate
recognition site by increasing the amount of excess cold competitors following the formation of the complex between the E2 protein and the
oligonucleotide carrying the E2 binding site. As shown in panels
2a-2c of Fig. 6, the t1/2 for
dissociation of the CRPV E2 from the complex was 11 min and that of the
HPV E2 was greater than 90 min, indicating that the complex of the
HPV16 E2 protein with the E2 DNA binding site was more stable than that
of the CRPV E2 protein. We also tested the oligonucleotide DNA
sequence as a probe (ACCGAATTCGGT), which was located upstream of the
HPV16 LCR and reported as a high affinity binder (38, 39) for binding
studies. Its Kd value and t1/2
for dissociation showed similar results (data not shown). We conclude
from the results presented in Fig. 6, panel 1, that the
affinity of the HPV E2 and the CRPV E2 with their binding sites is
similar, but that of the stability of the complex once formed is
greater in the case of the HPV16 E2 than with the CRPV E2 (see Fig. 6,
panels 2b and 2c). Since the above results
suggested that the CRPV E2 does not by itself form a stable complex
with its binding site, we decided to test whether CRPV E1 contributed
the stability of the CRPV E2 with its DNA binding site in the LCR, and
enhanced the transcriptional activity. We showed that E2 protein driven by the RSV promoter activates the transcriptional activity of E2-dependent luciferase reporter constructs (see Fig. 2).
Therefore, we employed the pRSV-CRPV E2 expression vector together with
increasing amounts of the pCMV E1 expression vector cotransfected with
the CRPV LCR luciferase expression vector into Sf1Ep cells to study the
interaction of the E1 and E2 viral proteins. As shown by the data in
panel 1 of Fig. 7, all amounts
of the pCMV-CRPVE1 vectors increased the luciferase expression by
4-fold in the presence of 100 ng of the pRSV-CRPVE2 vector, whereas no
transcriptional activation of the luciferase vector was seen in the
absence of cotransfected pRSV-CRPVE2 vector DNA. Since the CRPV LCR
vector contains an origin of replication, it is possible that the
increased expression of luciferase seen in the presence of E1 (Fig. 7,
panel 1) is due to an increased copy number of the
luciferase-LCR vector. To test if this is the case, we compared the
luciferase activity in cells cotransfected with E1 and E2 expression
vectors with either the CRPV LCR-luciferase vector or the luciferase
vector in which the CRPV LCR was mutated so as to inactivate its
ability to promote the replication of vectors containing the LCR-CRPV (28). As shown in panel 2 of Fig. 7, there was no difference in the activation seen in the replication defective versus
the wild type LCR luciferase reporter vector in the presence of both E1
and E2, although the wild type LCR luciferase vector replicated in the
presence of E1 and E2, whereas the mutant CRPV LCR
(CRPV-LCRluc Mut) did not (panel 3, Fig. 7).
These data suggests that the CRPV E1 protein enhances the
E2-dependent transcriptional activation of reporter genes
governed by E2-dependent promoters, perhaps by stabilizing
the binding of the CRPV E2 protein on the CRPV E2 binding site of the
CRPV.
Importance of the Level of the E2 Protein as to whether Activation
or Inhibition Occurs in the Presence of E2--
According to the
previously proposed model for the binding of E2 to the papillomavirus
LCR, low levels of E2 bind to the higher affinity upstream E2 binding
sites, thereby activating transcription, whereas the lower
affinity-proximal E2 binding sites, which are proximal to the TATA box
of HPV16 P97 promoter, are occupied at high levels of E2 protein,
leading to transcriptional repression (38, 39). Since the number of
binding sites are quite different between CRPV and HPV, we investigated
how the CRPV E2 protein worked as a transcriptional regulator. In
addition to using the native LCR to drive the expression of a
luciferase reporter gene, we have utilized a series of artificial
promoters composed of two Sp1 sites, the tk core promoter, and a
varying number of CRPV E2 binding sites in which the number and spatial
organization of these sites have been varied. These synthetic promoters
were included in the experiment to study the interaction of the E1 and
E2 proteins in isolation of many other host cell proteins which may
interact with the native LCR regulatory elements.
We documented, by use of the mobility shift assay (for the protein
level) and the RNase protection assay (for the mRNA level), that
the experiments involving E2 expression vectors driven by the CMV
promoter generated much higher levels of E2 than was the case in
experiments with E2 expression vectors driven by the RSV promoter. The
dose-dependent decrement of the luciferase expression by
the authentic CRPV LCR was demonstrated by the cotransfection with the
CMV promoter-dependent CRPV E2 expression vector, whereas the activation of luciferase expression was demonstrated with the RSV
promoter-dependent CRPV E2 expression vector. Similar results were obtained by the use of E2 expression vectors cotransfected with the artificial E2 promoter-dependent luciferase gene.
This suggested that the CRPV E2 worked as a transcriptional activator when the concentration of expressed E2 proteins are low in cells and
high concentration of E2 results in switching off the transcription.
Kovelman et al. (42) reported that the space between cognate
binding sites was important for transactivation by the HPV16E2 protein.
The spacing between E2 binding sites did not appear to effect on the
outcome of the CRPV E2 protein, suggesting the spacing is less
important for transactivation in the rabbit E2 protein, perhaps because
of many binding sites present in the authentic CRPV LCR. Furthermore,
we recognized that transcriptional activation of the HPV-16 E2 reached
a plateau with the input DNA for transfection (see panel 1 of Fig. 5). Our experiments showed that the affinities of the DNA
binding domains of the human and rabbit E2 were similar, but that the
stability of the complexes differed markedly, with the CRPV E2 and the
HPV16 E2 having a t1/2 of dissociation of 11 min and
more than 90 min, respectively. This difference may be consistent with
the much greater number of E2 binding sites in the CRPV LCR than in are
present in the HPV LCR. One possible model that would explain the
observed activation of E2-dependent promoters at low levels
of the CRPV E2 expression and inhibition of E2-dependent
promoters at high levels of the CRPV E2, in view of the instability of
the complex between E2 proteins and its cognate recognition binding
sites, is that there are other proteins of host or viral origin which
bind to E2 and to the E2-dependent promoters, thereby
stabilizing the complex between the CRPV E2 protein and its promoter.
This action of the additional protein enhances the expression of the
E2-dependent promoters. Because of the greater stability of
the HPV E2 protein for its E2 binding sites, the role of the accessory
protein in transcriptional activation may not be as great as is the
case with the CRPV E2 protein. The greater number of E2 binding sites in the CRPV LCR may partially compensate at lower E2 concentrations for
the decreased binding stability of the CRPV E2 protein for cognate E2
binding sites. However, at higher levels of the E2 protein, the
interactions of the multiple E2 protein with each other and with other
proteins may alter the overall conformation of the LCR and its protein
complexes. It is possible that an excess amount of the CRPV E2 protein
results in the conformational change of the LCR (8, 45, 46) and thereby
eventually decreases the transactivation. Another possible model that
could explain the decreased transcriptional activation at higher levels
is that the E2 protein could have a squelching effect on the binding of other proteins which promote transcriptional activation by binding both
to the E2 protein and to the LCR. In other words, if there are proteins
other than E2, which directly promote the transcriptional activation by
binding to the LCR, but which also bind to E2, it is possible to
imagine that the binding of the other protein to the LCR is reduced.
This would only happen if the affinity of the E2 protein for the other
protein is as great or greater than the affinity of the other proteins
for the LCR. In contrast, the HPV16 E2 protein binds at a much higher
stability to cognate E2 binding sites in the LCR and therefore does not
need the other protein to bind to the LCR compared as the CRPV E2
protein. Therefore, there is no substantial squelching effect for the
HPV16 E2 protein at higher E2 concentrations in our experiment.
The Role of E1 Protein for Transactivation of
E2-dependent Promoters--
We have presented data in Fig.
7 that suggests that E1 is a protein that can interact with the E2
protein and the E2-dependent promoters, thereby enhancing
the expression of genes governed by E2-dependent promoters.
In our studies, the transfection of E1 expression vectors enhanced the
transactivation with the E2 expression vector in the luciferase assay.
The experiments with the CRPV with the mutant ori shows that increased
expression of the luciferase gene is not due to activation of
replication of the E2-dependent reporter construct as an
episome. Another possibility is that the E2 protein recruits the E1
protein to bind nonspecifically to DNA sequence, followed by a
conformational change in the transcription initiation complex as it is
being assembled. The results presented in this paper indicate that a
similar situation may also be the case for the HPV-16 (47) or the BPV-1
(28). Le Moal et al. (28) reported that low
concentrations of the E1 vector enhance the activity of
E2-dependent promoters, but that high concentrations of the
E1 vector showed repression. We did not find the repression at high
concentrations of the E1 vector in our system. It is difficult to
compare two systems, however, since any effect may be species-specific. It is also known that cellular transcriptional regulatory proteins such
as TFIID, TFIIB, USF, and SP1 are associated with the E2 protein and
modulate transcriptional activation (10, 44, 48-50). We believe that
not only host proteins, but also viral proteins, modulate
transcriptional activity in a species-specific manner. The
identification of the other transcriptional factors, which participate
along with E2 proteins in the regulation of the other HPV genes will
provide the information needed to understand more fully how the
papillomaviruses interact with the cellular proteins to replicate.
Here we show that different mechanisms of transcriptional regulation
may be involved in CRPV and HPV. These mechanisms may arise both from
the organization of E2 binding sites, which are different in the CRPV
and HPV LCR, and as well from the intrinsic differences in the
properties of the HPV and the CRPV E2 proteins. We have shown that the
CRPV E2 protein functions at low levels of expression of E2 protein as
a transactivator and at high levels of E2 expression as a repressor
substantially. These results may be useful for assembling a
comprehensive picture of the complex interactions occurring between
papillomavirus proteins and host proteins and will be useful as well
for the identification of compounds that can be used for the inhibition
of papillomavirus gene expression or replication.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal vector DNAs using the Superfect
(Qiagen) in triplicate. The amount of input DNA was equalized with the
corresponding backbone vectors. Cells were lysed at 48 h with a
reporter lysis buffer (Promega) and measured with a luminometer (Wallac
Inc.). Variations in the luciferase assay, which arise from
transfection efficiency, were normalized by dividing the values for the
luciferase activity by the
-gal activity. All transfection
experiments were performed at least three times to assess reproducibility.
-D-thiogalactopyranoside was added to a final
concentration of 1 mM at 28 °C for 3 h. Bacteria pellets were collected and washed in PBS and stored
80 °C. Pellets were resuspended in sonication buffer containing 20 mM
Tris-HCl (pH 8.0). After shearing with an 18-gauge needle, bacteria
were sonicated and subjected to ultracentrifugation at 90,000 × g at 4 °C for 1 h. The supernatant was recovered and
added to the Talon resin (CLONTECH). After
incubation at 4 °C for 3 h, the resin was washed three times
with sonication buffer prior to elution of the His tag proteins with
sonication buffer containing 10 mM imidazole. The eluted
His tag protein was dialyzed against dialyzed buffer containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 10% glycerol.
80 °C for 3 days.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
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Fig. 1.
A, construction of the luciferase
reporter vectors with E2-dependent promoters and E2
expression vectors for study of E2-dependent gene
expression. The nucleotide sequence shows the 2E2 and Sp1 cassette used
to assemble the E2-dependent promoters. The two E2 binding
sites are shown in bold letters, and the Sp1 binding sites
are underlined (21). The two E2 and two Sp1 cassette
(2E2luc) or the CRPV-LCR (pCRPV-LCRlucWT) was
cloned into the pGL3 basic vector. The 4E2luc contains four E2 binding
sites as indicated. The 2//2E2luc contains four E2 binding sites, which
were separated by 55 bp (//) from the original two E2 binding sites of
2E2luc vector. 4//2E2luc contains additional four E2 binding sites,
which were separated by 55 bp (//) from the original two E2 binding
sites of 2E2luc vector. The pCRPV-LCRlucMut contained the mutation in
the CRPV LCR ori to abolish the functional nature of the origin of
replication (28, 31). The designations for the E2 and E1 expression
vectors in which the CMV and RSV promoters are also shown.
B, comparison of E2 binding sites present in the LCR of
BPV-1 (43), HPV16 (38, 39), and putative CRPV E2 binding sites: the E2
(arrows) and E1 (circles) binding sites are
indicated.
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Fig. 2.
Transcriptional regulation of CRPV LCR
(pCRPV-LCRlucWT) reporter vectors by E2 proteins. Sf1Ep cells
(panels 1 and 2) in six-well plates were
transfected with the amounts, in nanograms, of E2 expression vector DNA
shown on the x axis; the pCMV-CRPVE2 (panel 1)
and the pRSV-CRPVE2 (panel 2) along with the CRPV-LCRlucWT
vector and the -gal vector. Luciferase activity was indicated as one
in the absence of the E2 expression vector (y axis).
Luciferase activity was normalized by a
-gal activity. This
experiment was performed at least three times to assess
reproducibility.
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Fig. 3.
Transcriptional regulation of a series of
artificial reporter vectors by E2 expression vectors. Sf1Ep cells
(panels 1 and 2) in six-well plates were
transfected with the amounts, in nanograms, of E2 expression vectors
DNA shown on the x axis; the pCMV-CRPV E2 (panel
1) and the pRSV-CRPVE2 (panel 2), along with the
-gal vector and a series of artificial luciferase vectors, are
indicated as shown by the symbol key at the bottom of the
figure. Luciferase activity was indicated as one in the absence
of the E2 expression vector along with each luciferase vector
(y axis). Luciferase activity was normalized by a
-gal
activity. This experiment was performed at least three times to assess
reproducibility.
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Fig. 4.
Expression of E2 proteins in transfected
cells. Panel 1, electromobility shift assay to detect
the levels of E2 expression with CMV E2 and RSV E2 expression vectors.
Sf1Ep cells were transfected with various concentrations of the E2
expression vectors driven either by the CMV promoter (right
lanes) or the RSV promoter (left lanes). The nuclear
proteins were extracted and subjected to electromobility shift assay.
The concentration of the E2 expression vector driven by the RSV
promoter in the left four lanes and by the CMV promoter in
the right four lanes was indicated by the
triangles above the lanes. The amount of input DNA for
transfection was normalized with each backbone vector. The
oligonucleotide DNAs, in which the E2 binding site was
underlined, were annealed and filled-in with 32P
dCTP (panel 4). The 32P-labeled oligonucleotide
in the absence of the extract is in the lane to the extreme left side
of the gel (F). The lane at the extreme right
side of the gel contains the nuclear extract to which unlabeled
oligonucleotide was added first before addition of the
32P-labeled oligonucleotides (C).
Bars on the right were E2 protein and
oligonucleotide DNA complex (top) and the free probe
(bottom). Panel 2, RNase protection assay to
estimate the level of the E2 mRNA in cells transfected with E2
expression vectors driven by the RSV promoter and by the CMV promoter.
Sf1Ep cells were transfected with the various concentration of the E2
expression vector. Lanes 1-5 contained RNA extracted from
the cells that were transfected with 0, 200, 400, 800, and 1200 ng of
the pRSV-CRPV E2 expression vector, respectively. Lanes
6-10 contained RNA extracted from the cells that were transfected
with 0, 200, 400, 800, and 1200 ng of the pCMV-CRPV E2 expression
vectors, respectively. The amount of DNA for transfection was
normalized with each backbone vector. The ratio of E2 and 18 S RNA
probe for protection was 100 to 1. Bars on the
left of the gel indicated the migration position of E2
mRNA (top) and 18 S ribosomal RNA (bottom).
Panel 3, the intensity of the protected E2 mRNA adjusted
with 18 S RNA was quantified by densitometric image analysis. The
numbers on the x axis correspond to those in panel
2. AU = arbitrary units of absorbance
(y axis).
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Fig. 5.
Transcriptional regulation of a series of
artificial reporter vectors by HPV16 and CRPV E2 expression vectors in
c33a cells. C33a cells (panels 1 and 2) in
six-well plates were transfected with the amounts of nanograms of E2
expression vectors DNA shown on the x axis; the pCMV-CRPVE2
(panel 1) and the pCMV-HPV16E2 (panel 2) along
with a -gal vector and a series of artificial reporter vectors
indicated as below. Luciferase activity was indicated as one in the
absence of the E2 expression vector (y axis). Luciferase
activity was normalized by a
-gal activity. This experiment was
performed at least three times to assess reproducibility.
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Fig. 6.
Panel 1, binding of the purified DBD of
the HPV16 E2 protein and that of CRPV proteins to the E2 binding site.
The HPV16 E2 (lanes 1-6) and the CRPV E2 (lanes
7-12) protein were examined by electromobility shift assay in the
presence of increasing concentrations of the unlabeled oligonucleotide
(cold competitor): 0 nM (lanes 1 and
7), 1.6 nM (lanes 2 and
8), 8 nM (lanes 3 and 9),
40 nM (lanes 4 and 10), 120 nM (lanes 5 and 11), and 600 nM (lanes 6 and 12). The top
bar corresponds to complex formed between the DNA binding domain
of HPV16 or CRPV E2 proteins and the 32P-labeled
oligonucleotide DNA containing one E2 binding site (see Fig. 4,
panel 1). The bottom bar corresponds to the free
probe. The Kd values represented the average of
triplicate assays. Panel 2a, the off rates of HPV16 or CRPV
E2 DBD proteins and the 32P-labeled oligonucleotide
DNA containing the E2 binding site in the presence of the excess cold
competitor: the HPV 16 E2 DBD (lanes 1-7), the CRPV E2 DBD
(lanes 8-14), and no protein (lane 15) were
first incubated with the 32P-labeled oligonucleotide probe
for 30 min at room temperature. These binding mixtures in lanes
2-7 and in lanes 9-14 were added to the sample well
of a running gel 3, 5, 10, 30, 60, and 90 min after addition of excess
amounts of unlabeled oligonucleotide (cold competitor) at a
concentration of 375 nM. The cold competitor was added
prior to addition of 32P-labeled probe in lanes
1 and 8. Panels 2b and 2c, the
intensity of the free probe and DNA-protein complex was measured by an
Image analyzer. The dissociation t1/2 of
HPV16 E2 (panel 2b) and CRPV E2 (panel 2c) was
measured at least three times and one of the representative experiments
is shown. The dissociation t1/2 was 11 min for CRPV
and >90 min for HPV 16, respectively. Arbitrary units of intensity was
indicated in the y axis. The excess amount of the cold
competitor (C) corresponds to lane 1 (HPV16) and lane 8 (CRPV) on
panel 2a and was incubated prior to addition of
32P-labeled probe.
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Fig. 7.
Role of E1 in the E2-dependent
transactivation of reporter genes driven by E2-dependent
promoters. Panel 1, E1 enhanced the E2 transcriptional
activity. Sf1Ep cells were transfected with a combination of the
pCMV-CRPVE1 expression vectors (0-1200 ng) and the pRSV-CRPVE2E2
expression vector (100 ng) along with the pCRPV-LCRlucWT and the
-gal vector. The amount of pRSV-CRPVE2 vector was constant at 100 ng
in all lanes, while the pCMV-CRPVE1 vector was varied from 0 to 1200 ng, indicated in x axis (solid black squares). In
another series of experiments, the CRPV-LCRluc reporter vector was
cotransfected with the pCMV-CRPVE1 vector and the
-gal vector (the
amount of the vector was varied from 0 to 1200 ng) in the absence of
the pRSV-CRPVE2 vector (open squares). Luciferase activity
was normalized by a
-gal activity. This experiment was performed at
least three times to assess reproducibility. Panel 2, both
the wild type CRPV LCR (pCRPV-LCRlucWT) or the mutant CRPV LCR
(pCRPV-LCRlucMut) was transactivated by combination with E1 and E2
expression vectors. The pCRPV-LCRlucMut (open squares) or
the pCRPV-LCRlucWT (solid squares) was transfected with both
0 ng of pCMV-CRPVE1 and pRSV-CRPVE2 expression vectors
(left), 0 ng of pCMV-CRPVE1 and 100 ng of pRSV-CRPVE2
vectors (middle), and 400 ng of E1 and 100 ng of E2
expression vectors (right) along with the
-gal vector in
Sf1Ep cells. Luciferase activity was normalized by a
-gal activity.
This experiment was performed at least three times to assess
reproducibility. Panel 3, the pCRPV-LCRlucWT (lanes
1-4) or the pCRPV-LCRlucMut (lanes 5-8) were tested
by transient replication assay. Sf1Ep cells were transfected with the
pCMV-CRPVE1 and/or pRSV-CRPVE2 vectors indicated as plus or
minus. Low molecular weight DNA was extracted from the
transfected cells followed by DpnI and XbaI
digestion. Southern blot analysis was performed as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Winship Herr, Cold Spring Harbor Laboratory, for his gift of the pCG vector; Matthias Dürst, Deutsches Krebsforschungszentrum, Heidelberg, Germany, for his gift of the 114/k; Felix Wettstein, UCLA, for his gift of the CRPV-pLAII; Lubomir Turek, University of Iowa College of Medicine, for his gift of the pCG-HPV16; and Robert Kovelman for his gift of p2 × 2xE2BS-luc. We also thank Daniel DiMaio, Yale University, Mohammad Nasseri, Wyth-Ayerst Research, and Yoshihiro Hara, Nippon Schering, for stimulating discussions.
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FOOTNOTES |
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* This work was supported by grants (to A. D.) from the Hull Development Fund, the George and Barbara Bush Leukemia Research Fund, the Ensign Professorship of Medicine, and the Yale Cancer Center Development Fund for interim developmental support.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
To whom correspondence should be addressed. Tel.:
203-737-5608; Fax: 203-737-5698; E-mail:
albert.deisseroth@yale.edu.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M007120200
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ABBREVIATIONS |
---|
The abbreviations used are:
HPV, human
papillomavirus;
CRPV, cottontail rabbit papillomavirus;
BPV-1, bovine
papillomavirus 1;
ori, origin of replication;
CMV, cytomegalovirus;
nt, nucleotide(s);
PCR, polymerase chain reaction;
LCR, long control region(s);
tk, thymidine kinase;
bp, base pair(s);
DBD, DNA binding
domain;
-gal,
-galactosidase;
RSV, Rous sarcoma virus;
E1, viral
helicase;
E2, viral transcriptional regulating protein.
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