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Transcriptional Activity among High and Low Risk Human
Papillomavirus E2 Proteins Correlates with E2 DNA Binding*
Received for publication, July 9, 2002, and in revised form, August 28, 2002
Published, JBC Papers in Press, September 17, 2002, DOI 10.1074/jbc.M206829200
Samuel Y. Hou, Shwu-Yuan Wu, and Cheng-Ming Chiang‡
From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935
The full-length E2 protein, encoded by human papil-
lomaviruses (HPVs), is a sequence-specific transcription
factor found in all HPVs, including cancer-causing high
risk HPV types 16 and 18 and wart-inducing low risk
HPV types 6 and 11. To investigate whether E2 proteins
encoded by high risk HPVs may function differentially
from E2 proteins encoded by low risk HPVs and animal
papillomaviruses, we conducted comparative DNA-
binding and transcription studies using electrophoretic
mobility shift assays and cell-free transcription systems
reconstituted with purified general transcription fac-
tors, cofactor, RNA polymerase II, and with E2 proteins
encoded by HPV-16, HPV-18, HPV-11, and bovine papil-
lomavirus type 1 (BPV-1). We found that although differ-
ent types of E2 proteins all exhibited transactivation
and repression activities, depending on the sequence
context of the E2-binding sites, HPV-16 E2 shows stron-
ger transcription activity and greater DNA-binding af-
finity than those displayed by the other E2 proteins.
Surprisingly, HPV-18 E2 behaves more similarly to
BPV-1 E2 than HPV-16 E2 in its functional properties.
Our studies thus categorize HPV-18 E2 and BPV-1 E2 in
the same protein family, a finding consistent with the
available E2 structural data that separate the closely
related HPV-16 and HPV-18 E2 proteins but classify to-
gether the more divergent BPV-1 and HPV-18 E2
proteins.
Human papillomaviruses (HPVs)
1
are a family of small DNA
viruses that cause a wide variety of human diseases ranging
from benign epithelial lesions, such as warts, to invasive can-
cers, such as cervical carcinoma. So far, more than 100 HPV
types have been identified and fully sequenced, whereas more
than 120 putative novel types have been partially character-
ized (1, 2). HPV types frequently found in invasive cancers
include HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -58, and -59.
These are classified as high risk HPVs. In contrast, HPV types
that are rarely found in cancers but are associated with genital
warts, such as HPV-6 and HPV-11, are considered low risk
HPVs (1–3). Because the genomic structures of HPVs are
highly conserved, it is important to determine the functional
differences among individual HPV gene products which lead to
etiologically high and low risk phenotypes. Previous compara-
tive studies have primarily focused on HPV-encoded E6- and
E7-transforming proteins. These studies found that E6 and E7,
from high risk HPVs, lead to cellular transformation much
more readily than low risk E6 and E7 proteins (for review, see
Refs. 4 and 5). In both high and low risk HPVs, expression of E6
and E7 is transcriptionally regulated via the E6 promoter by
many cellular and viral proteins. The full-length viral E2
protein is a sequence-specific transcription factor that func-
tions as an activator or repressor to regulate tightly the E6
promoter through four consensus E2-binding sites (E2-BSs),
ACCGN
4
CGGT (6, 7), whose locations within the upstream
regulatory region (URR) are highly conserved among genital
HPVs. Efficient activation of the E6 promoter requires binding
of E2 protein to the promoter-distal E2-BSs in conjunction with
binding of cellular factors to the enhancer elements also located
within the URR (8 –11). Activation directly mediated through
E2 protein may occur via different mechanisms. First, E2 may
recruit the general transcription machinery to the promoter
through direct interactions with TATA-binding protein (TBP),
TBP-associated factors in TFIID, TFIIB, as well as RNA po-
lymerase II (12–17). Second, E2 interacts with nuclear factors
Sp1, Gps2/AMF-1, or TopBP1, which may then serve as coregu-
lators for transcription (18 –20). Third, E2 may potentiate tran-
scription by remodeling DNA structure at the chromatin level
(21). Indeed, studies have shown E2 interacts with proteins
(CREB-binding protein, p300, and p300/CBP-associated factor)
possessing histone acetyltransferase activity (22–24). It is
likely that different types of E2 proteins function differentially
in regulating preinitiation complex assembly and show varied
cooperativity with cellular enhancer-binding factors that rec-
ognize constitutive, inducible, or cell type-specific enhancer
elements located in the URR, as well as with general or gene-
specific transcription cofactors that modulate E2 activity on
HPV chromatin.
Although low levels of E2 occupy promoter-distal binding
sites, it is postulated that transcriptional repression occurs
with high levels of E2 leading to occupancy at promoter-prox-
imal E2-BSs thus displacing cellular factors critical for E6
promoter activity (9, 10, 25–27). We have shown previously
that E2 protein can also actively repress transcription by pre-
venting preinitiation complex formation (28). This active re-
pression was demonstrated to be independent of cellular en-
hancer-binding factors, Sp1, and general cofactors such as
TBP-associated factors, TFIIA, mediator and PC4 (28). Con-
versely, cellular proteins may bind to DNA and hinder access of
* This work was supported by Grants CA81017 and GM59643 from
the National Institutes of Health and Grant RPG-97-135-04-MBC from
the American Cancer Society. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ Mt. Sinai Health Care Foundation scholar. To whom correspond-
ence should be addressed: Dept. of Biochemistry, W-409, Case Western
Reserve University School of Medicine, 10900 Euclid Ave., Cleveland,
OH 44106-4935. Tel.: 216-368-8550; Fax: 216-368-3419; E-mail:
c-chiang@biochemistry.cwru.edu.
1
The abbreviations used are: HPV(s), human papillomavirus(es);
BE2, BPV-1 E2 protein; BPV-1, bovine papillomavirus type 1; CREB,
cAMP-response element-binding protein; CMV, cytomegalovirus; E2-
BS(s), E2-binding site(s); EMSA, electrophoretic mobility shift assay;
HPV-11, HPV type 11; HPV-16, HPV type 16; HPV-18, HPV type 18;
11E2, HPV-11 E2 protein; 16E2, HPV-16 E2 protein; 18E2, HPV-18 E2
protein; PC4, positive cofactor 4; pol, polymerase; TBP, TATA-binding
protein; TF, transcription factor; URR, upstream regulatory region.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 47, Issue of November 22, pp. 45619–45629, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 45619
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E2 protein, thus inhibiting E2 function (29 –31). Papillomavi-
ruses may also use its own proteins to modulate E2 activation
function. These include various forms of E2 repressor proteins
created through alternative splicing to generate gene products
that contain the same DNA-binding domain linked to different
N-terminal regions (32–34) or the use of viral E1 protein to
abrogate E2 transactivation function through direct protein-
protein interaction (35, 36). Because E2 only weakly activates
(⬃2-fold) but strongly represses (up to 100-fold) the native HPV
promoter in both transfected cells (9) and in currently available
cell-free transcription systems (for review, see Ref. 28), our
initial effort was focused on dissecting the mechanisms of tran-
scriptional repression by various E2 proteins on their homolo-
gous E6 promoter (28).
Interaction between viral E1 and E2 proteins also regulates
papillomavirus DNA replication and viral episome mainte-
nance (37–40). In addition to binding to viral DNA, E2 ensures
proper segregation of viral genomes during cellular replication
which is independent of the E2 DNA-binding domain (41–43).
Analogous to abrogation of E2 transactivation function, it has
been suggested that E1 also regulates E2 binding to mitotic
chromosomes (44). Besides regulating viral transcription, rep-
lication, and viral episome maintenance, E2 has been impli-
cated in several cellular processes relevant to carcinogenesis.
E2 transfected into HPV DNA-containing cell lines can lead to
growth arrest, apoptosis, or abrogation of mitotic checkpoints
(45–47). E2 repression of E6 and E7 oncogene expression lead-
ing to reactivation of p53 and pRb tumor suppressor pathways
has been the proposed mechanism for growth arrest in HPV-
positive cell lines (48, 49). Surprisingly, growth arrest and
repression of E6 and E7 expression require an intact E2 trans-
activation domain (50 –52). Expression of E2 protein in a trans-
formed cell line devoid of HPV DNA also displayed growth
arrest through an undefined mechanism (45, 51). E2-triggered
apoptosis can occur through both p53-independent (53) and
p53-dependent pathways (54). Similar to E2-induced growth
arrest, E2 caused apoptosis in HPV-negative cell lines through
unknown pathways (54).
Although many functions have been attributed to papilloma-
viral E2 proteins, very few studies have been conducted to
compare directly the transcription activities among HPV and
BPV-1 E2 proteins (11, 55, 56). Experiments directly compar-
ing high risk HPV-16 and HPV-18 E2 proteins with low risk
HPV-6 E2 protein by transient transfection assays found that
high risk E2 proteins have higher activation activities than low
risk E2 protein, but this activity did not correlate with either
higher steady-state levels of protein in vivo or with DNA-
binding properties of the two classes of E2 proteins (56). There-
fore, it was hypothesized that high risk E2 proteins were in-
trinsically better transcriptional activators than low risk E2
proteins, which in turn regulate the levels of E6 and E7 onco-
proteins in transformed cells and thereby control the processes
leading to carcinogenesis. In a more recent study, high risk E2
was found to have higher affinity for transcriptional coactiva-
tor CREB-binding protein than that of low risk E2 (23). These
studies on E2 transcriptional activity were assayed primarily
in transiently transfected cells. Therefore, interpretation of the
results may be complicated by interference of other cellular
factors, potentially masking the true activity of E2 protein.
To address the differences in transcriptional activities be-
tween high and low risk HPV E2 proteins, we employed in vitro
transcription systems reconstituted either with individually
purified recombinant general transcription factors (TFIIB,
TFIIE, and TFIIF), epitope-tagged protein complexes (TFIID,
TFIIH, and pol II) and general cofactor PC4, or with a preas-
sembled RNA polymerase II (pol II) holoenzyme complex and
TBP (17, 28). Using this highly purified in vitro transcription
system along with an HPV responsive G-less cassette template
containing two E2-BSs (17), high risk HPV-16 E2 protein was
found to possess greater transactivation activity than those
exhibited by E2 proteins encoded by HPV-18, HPV-11, and
BPV-1. For repression assays, we employed an in vitro tran-
scription system reconstituted with pol II holoenzyme and TBP
with HPV template containing either homologous or heterolo-
gous URR linked to a G-less cassette. HPV-16 E2 (16E2) was
determined to be the strongest transcriptional repressor,
followed by BPV-1 E2 (BE2), HPV-18 E2 (18E2) and HPV-11
E2 (11E2). The transcriptional activities of these E2 proteins
correlated well with their corresponding binding affinities to
E2-BSs derived from the promoter-proximal regions of natu-
rally occurring HPV-11, HPV-16, and HPV 18 E6 promoters, as
well as BPV-1. Although previous in vivo studies revealed little
correlation between E2 transcriptional activities and DNA-
binding affinities, our in vitro studies indicate that higher
transcriptional activation and repression activities of various
E2 proteins correlate directly with their relative DNA-binding
affinities for E2-BSs.
EXPERIMENTAL PROCEDURES
Plasmid Constructions—Bacterial expression plasmids pF:E2-11d
(28), pF:16E2-11d, pF:18E2-11d, and p:BE2-11d were used to express
HPV-11, HPV-16, HPV-18, and BPV-1 E2 proteins, respectively. The
plasmid pF:16E2-11d was constructed by cloning the HPV-16 E2 open
reading frame, generated by PCR amplification using pCMV-16E2 (56)
as template with an NdeI site-containing sense primer (5⬘-AAGTCGA-
CATATGGAGACTCTTTGCCAA-3⬘)andaBamHI site-containing an-
tisense primer (5⬘-AACTCGAGGATCCTCATATAGACATAAATCC-3⬘),
into pF:E4-11d (28), after swapping the inserts between NdeI and
BamHI sites. The plasmid pF:18E2-11d was created similarly by using
pCMV-18E2 (56) as template with sense primer (5⬘-AAGTCGACATA-
TGCAGACACCGAAGGAAA-3⬘) and antisense primer (5⬘-AACTCGAG-
GATCCTTACATTGTCATGTATCCC-3⬘) for PCR amplification. Like-
wise, the plasmid pF:BE2-11d was cloned using pdBPV-1(142–6) (57)
as template with sense primer (5⬘-AAGTCGACATATGGAGACAGCA-
TGCGAA-3⬘) and antisense primer (5⬘-TTCTCGAGGATCCTATTGAT-
GCAAGC-3⬘).
The G-less cassette template p18URR-GLess, containing the HPV-18
URR spanning nucleotides 6929 –7857/1–81, used for in vitro transcrip-
tion was constructed by cloning the PCR product, generated from
genomic HPV-18 DNA (gift from L. T. Chow) using an EcoRI site-
containing sense primer (5⬘-CTGGGTACCGAATTCGGATCCCTAT-
GATAAG-3⬘) and an SacI site-containing antisense primer (5⬘-CT-
GAGATCTGAGCTCTTTTATATACACCG-3⬘) into the EcoRI-SacI-
linearized G-less cassette vector pGL (58). The p16URR-GLess G-less
cassette template, spanning HPV-16 URR nucleotides 7007–7904/1–72,
was constructed similarly by cloning the PCR product generated from
genomic HPV-16 DNA (gift from P. M. Howley) using sense primer
(5⬘-CTGGGTACCGAATTCAGACCTAGATCAGTTTC-3⬘) and antisense
primer (5⬘-CTGAGATCTGAGCTCTTTTATACTAACCG-3⬘) into pGL.
The other transcription templates, p11URR-GLess (originally named
p7072–70GLess/I
⫺
, see Ref. 28), p7862–70(23M)GLess/I
⫺
, pML⌬53,
p2E2(IR)⌬53, and pG
5
MLT, have already been described (17, 28).
Protein Purification—Bacterially expressed FLAG-tagged HPV-11
E2, HPV-16 E2, HPV-18 E2, and BPV-1 E2 were purified from
BL21(DE3)pLysS strain harboring pF:E2-11d, pF:16E2-11d, pF:18E2-
11d, and pF:BE2-11d, respectively, according to the protocol published
previously (17, 28). Isolation of recombinant FLAG-tagged human gen-
eral transcription factors (TFIIB, TBP, TFIIE
␣
, and TFIIE

), FLAG-
tagged multiprotein complexes (TFIID, TFIIH, and pol II), six histidine-
tagged TFIIF subunits (RAP30 and RAP74), recombinant PC4, and
TFIID-deficient pol II holoenzyme was conducted as described previ-
ously (17, 28, 59 –61).
In Vitro Transcription—The transcription repression assays were
performed in a two-component transcription system reconstituted with
TFIID-deficient pol II holoenzyme and TBP using 50 ng of p11URR-
GLess, p16URR-GLess, p18URR-GLess, or p7862–70(23M)GLess/I
⫺
G-less cassette template, 50 ng of pML⌬53, 1 ng of TBP, and 3
l(⬃90
ng) of pol II holoenzyme, in the absence or presence of 11E2, 16E2,
18E2, or BE2 protein as specified in the figures. Reactions were then
conducted and analyzed as described previously (28, 60).
Transcription and DNA Binding Activities of HPV and BPV E245620
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The E2 transactivation assay was carried out in a highly purified in
vitro transcription system in a 30-
l reaction containing 50 ng of
pG
5
MLT, 50 ng of p2E2(IR)⌬53, 10 ng of TFIIB, 20 ng of TFIID, 2.5 ng
each of TFIIE
␣
and TFIIE

, 2 ng of TFIIF, 22.5 ng of TFIIH, and 5 ng
of pol II, in the presence or absence of 150 ng of PC4 and an increasing
amount of 11E2, 16E2, 18E2, or BE2 as specified, following the two-step
incubation protocol (17). Reactions were then performed and analyzed
as described previously (17, 60). Transcription signals were quantified
by PhosphorImager analysis (Molecular Dynamics).
Electrophoretic Mobility Shift Assay (EMSA)—DNA probes used for
EMSA were generated by PCR amplification of respective HPV E6
promoter-proximal fragments and BPV-1 URR. The HPV-11 E6 pro-
moter-proximal fragments, HPV-11(7902–92), HPV-11(7902–92)3M,
HPV-11(7902–92)4M, and HPV-11(7902–92)34M, which span HPV-11
nucleotides 7902–7933/1–92 containing wild-type 3 and 4, mutated 3,
mutated 4, or mutated 3 and 4 E2-BSs, were created by PCR amplifi-
cation using sense primer (5⬘-AACCCGGGTACCTACCCACACCCTA-
CATA-3⬘) and antisense primer (5⬘-GGACACAGATCTGAGCTCT-
GCTAATTTTTTGGG-3⬘) with DNA templates pGL7072–161, p24-N-
3M, p24-N-4M, and p24-N-34M (9, 28), respectively. The wild-type
HPV-16 E6 promoter-proximal DNA fragment HPV-16(7868 –96), span-
ning HPV-16 nucleotides 7868 –7904/1–96 and containing two wild-type
E2-BSs, was generated by PCR amplification of HPV-16 genomic DNA
(gift from P. M. Howley) using sense primer (5⬘-CTGGGTACCGAAT-
TCTTACACATTTACAAGCA-3⬘) and antisense primer (5⬘-GGACACA-
GATCTGAGCTCTTTTGGTGCATAAA-3⬘). The wild-type HPV-18 E6
promoter-proximal DNA fragment HPV-18(7834 –101), spanning
HPV-18 nucleotides 7834 –7857/1–101 and containing two wild-type
E2-BSs, was generated by PCR amplification of HPV-18 genomic DNA
(gift from L. T. Chow) using sense primer (5⬘-CTGGGTACCGAATTC-
TGGGCAGCACATACTAT-3⬘) and antisense primer (5⬘-GGACACAGA-
TCTGAGCTCATTGTGGTGTGTTTCTC-3⬘). The BPV-1 DNA fragment
BPV-1(7874 –85), spanning BPV-1 nucleotides 7874 –7945/1–85 (num-
bering based on GenBank accession number NC_001522) and contain-
ing two wild-type E2-BSs, was generated by PCR amplification using
pdBPV-1(142–6) as template (57) with sense primer (5⬘-CTGGGTAC-
CGAATTCGCAGCATTATATTTTAAG-3⬘) and antisense primer (5⬘-G-
GACACAGATCTGAGCTCAACCGGGGTCTGTCAGC-3⬘). These PCR-
amplified DNA fragments were then purified by gel electrophoresis
using a 2% agarose gel and used for end-labeling reactions with T4
polynucleotide kinase and [
␥
-
32
P]ATP. DNA probes were purified fur-
ther by NICK column (Amersham Biosciences).
EMSA was conducted in a 10-
l reaction containing ⬃0.5 fmol of
32
P-labeled DNA probe in the presence of 10 mMHEPES-Na (pH 7.9), 5
mMdithiothreitol, 0.2 mMEDTA, 4 mMMgCl
2
, 10% glycerol, 0.1 mg/ml
bovine serum albumin, 10 ng/
l poly(dI-dC), and increasing amounts of
respective purified E2 proteins (adjusted to 2
l in BC300). Reactions
were incubated at 30 °C for 1 h and then loaded onto a 5% polyacryl-
amide gel in 0.5⫻TBE and run at 175 V for ⬃2.5hat4°C. The gels
were then dried and exposed to x-ray films. The signals were quantified
by PhosphorImager analysis.
Calculation of Equilibrium Binding Constants—The intensity of the
remaining probe in each lane after EMSA was quantified using Image-
Quant (Molecular Dynamics) without (
) or with (
⑀
for each lane) an
increasing amount of 11E2, 16E2, 18E2, or BE2 protein. Fractional
occupancy (
) was then determined by Equation 1.
⫽100% 共1⫺关
⑀
/
兴兲 (Eq. 1)
Binding isotherms were obtained by plotting fractional occupancy as a
function of protein concentrations and analyzed by nonlinear least
squares analyses. The equilibrium binding constant, K
d
, was deter-
mined by analysis of the titration curves against Equation 2,
⫽关protein兴/共关protein兴⫹Kd兲(Eq. 2)
where
⫽fractional occupancy, [protein] ⫽protein concentration, and
K
d
⫽the equilibrium binding constant (62).
RESULTS
Transcriptional Repression Mediated by High and Low Risk
HPV and BPV-1 E2 Proteins—To compare the transcriptional
activities between high risk (HPV-16 and HPV-18) and low risk
(HPV-11) HPV and animal papillomavirus E2 proteins, we first
expressed full-length HPV-11, HPV-16, HPV-18, and BPV-1 E2
FIG.1. Comparison of HPV and
BPV-1 E2 proteins and the sequences
of different E2-BSs. A, structural fea-
tures of E2 proteins. The N-terminal
activation domain (AD) is linked to the
C-terminal DNA binding/dimerization do-
main (DBD) of each E2 protein via the
hinge (H) region. Numbers refer to bound-
aries of amino acids depicting the protein
domains found in 11E2, 16E2, 18E2, and
BE2 proteins, based upon reports pub-
lished previously (72–75). The apparent
molecular mass (in kDa) of each FLAG-
tagged E2 protein, estimated by SDS-
PAGE, is listed on the right.B, Coomassie
Blue-stained gel of purified E2 proteins.
Recombinant FLAG-tagged BE2 (lane 1),
11E2 (lane 2), 16E2 (lane 3), and 18E2
(lane 4) were expressed in and purified
from bacteria, resolved by 10% SDS-
PAGE, and visualized after Coomassie
Blue staining. Positions of prestained pro-
tein size markers (in kDa) are indicated
on the left.C, transcriptional templates
containing natural HPV URR used for in
vitro transcription assays. Constructions
of various G-less cassettes driven by
HPV-11 URR (p11URR-GLess), HPV-16
URR (p16URR-GLess), or HPV-18 URR
(p18URR-GLess) were as described under
“Experimental Procedures.”Each tem-
plate contains the naturally occurring
HPV URR and the TATA box of the E6
promoter preceding a 377-nucleotide
G-less cassette. Nucleotide numbering for
boundaries of E2-BSs is based upon pre-
vious studies (9, 28) and HPV nucleotide
accession numbers AF125673 (for HPV-
16) and X05015 (for HPV-18).
Transcription and DNA Binding Activities of HPV and BPV E2 45621
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proteins in bacteria. All E2 proteins share similar structural
features with an N-terminal activation domain linked to a
C-terminal DNA-binding/dimerization domain by a flexible
hinge region (Fig. 1A). These bacterially expressed E2 proteins
were purified to near homogeneity and clearly migrated at
positions corresponding to their predicted molecular sizes (Fig.
1B). The amounts of various purified E2 proteins were then
normalized by Western blotting with anti-FLAG monoclonal
antibody, which recognizes the same epitope sequence intro-
duced at the N terminus of individual E2 proteins (data not
shown). For functional studies, we also created three transcrip-
tion templates, containing enhancer elements and the E6 pro-
moter derived from each type of HPV, by cloning respective
URR into a G-less cassette of 377 nucleotides (Fig. 1C). We
demonstrated previously that correct initiation from the HPV
E6 promoter whose transcription is regulated by different
amounts of E2 proteins, similar to the in vivo observations, was
recapitulated in our cell-free transcription systems using the
G-less cassette templates (28).
When examined in a cell-free transcription system reconsti-
tuted with TBP and human pol II holoenzyme, which provides
pol II and general transcription factors TFIIB, TFIIE, TFIIF,
and TFIIH (60, 61), transcription from these URR-driven
G-less templates was inhibited by BE2, 11E2, 16E2, and 18E2,
in a dose-dependent manner (Fig. 2). Repression of the E6
promoter could be observed on both homologous and heterolo-
gous URR-driven templates, irrespective of the types of E2
proteins used (compare left panels of Fig. 2, A–C). In contrast,
the internal control template pML⌬53, which contains the ad-
enovirus major late core promoter linked to a G-less cassette of
⬃280 nucleotides, did not respond to increasing amounts of E2
proteins, indicating a URR-specific repression mediated by E2
proteins. We had illustrated in our previous studies (28) that
repression of the E6 promoter from HPV-11 URR-containing
G-less cassette templates was mediated mainly through E2
binding to the promoter-proximal #3 and #4 E2-BSs, which are
immediately adjacent to the TATA box of the E6 promoter (see
Fig. 1C).
These comparative studies also revealed several interesting
findings. First, 16E2 is apparently a better repressor compared
with 18E2, 11E2, and BE2, because 10 ng of 16E2 almost
completely inhibited HPV transcription, whereas 10 ng of the
other E2 proteins only led to less than 50% of inhibition (com-
pare Fig. 2, A–C,lanes 3,8,13, and 18, and the diagrams shown
on the right of each panel). Obviously, ⬃50 ng (i.e. a 5-fold
higher amount) of 18E2, 11E2, and BE2 is required to reach a
level of repression similar to that achieved by 10 ng of 16E2
(see the diagrams in Fig. 2, A–C). Again, stronger repression of
the E6 promoter by 16E2, relative to the other E2 proteins, was
observed on both homologous (Fig. 2B) and heterologous (Fig.
2, Aand C) HPV templates. Second, animal papillomavirus E2
protein (BE2) could also inhibit HPV E6 promoters as effi-
ciently as HPV E2 proteins (Fig. 2), consistent with many
previous studies using the BPV-1 E2 expression plasmid to
study HPV promoter regulation by transfection assays (11, 55,
56). Third, the repression activity of 18E2 is more similar to
that exhibited by 11E2 and BE2, but not to its closely related
family member (i.e. high risk 16E2). This observation could be
best explained by the available C-terminal DNA-binding/
dimerization domain structures of 16E2, 18E2, and BE2 (see
“Discussion”).
Transcriptional Activation Mediated by High and Low Risk
HPV and BPV-1 E2 Proteins—To compare the transactivation
activity of E2 proteins encoded by HPV and BPV-1, we em-
ployed a highly purified in vitro transcription system reconsti-
tuted with only recombinant general transcription factors
(TFIIB, TFIIE, and TFIIF), general cofactor PC4, and epitope-
tagged multiprotein complexes (TFIID, TFIIH, and pol II). It
has been shown previously (17) that 11E2 can activate tran-
scription from p2E2(IR)⌬53 DNA template, which contains two
copies of the HPV-11 #2 E2-BS linked to a major late core
promoter-driven G-less cassette of ⬃280 nucleotides, but not
from an internal control template containing five copies of
Gal4-binding sites connected to a similar major late core pro-
moter construct with a longer G-less cassette (Fig. 3A,bottom
drawings). When we compared HPV and BPV-1 E2 proteins
directly in this reconstituted transcription system, we found
that 16E2 was the strongest activator achieving a maximum
level of activation at only 5 ng of protein compared with the
other E2 proteins (Fig. 3A, compare lanes 5,13,23, and 31, and
Fig. 3B). BE2 and 18E2 displayed similar levels of activation
(Fig. 3A,lane 7 versus lane 34, 11.6- and 13.7-fold activation,
respectively), but BE2 reached maximum activation at a lower
dose-dependent amount than 18E2 (Fig. 3A, compare lanes 7
and 34,50ngofBE2versus 150 ng of 18E2). 11E2 attained its
maximum level of activation at the same dosage as BE2 (Fig.
3A,lane 15, 50 ng of 11E2), but with only 7.4-fold activation.
After the maximum level of activation is achieved for each E2
protein, a general decline in fold activation, representing tran-
scriptional squelching, is observed (Fig. 3B). It is clear from
both experiments for activation and repression that 16E2 is the
dominant activator and repressor compared with 18E2, 11E2,
and BE2 in these comparative studies.
DNA-binding Activities of High and Low Risk HPV and
BPV-1 E2 Proteins—Because E2 is a sequence-specific DNA-
binding protein, we speculated that the transcription activity of
E2 protein may correlate with its DNA-binding activity,
thereby accounting for the functional differences in transacti-
vation and repression between 16E2 and the other E2 proteins.
To explore this possibility, we performed EMSA using purified
E2 proteins and DNA probes derived from promoter-proximal
#3 and #4 E2-BSs of respective HPV E6 promoters and a URR
region containing #11 and #12 E2-BSs from BPV-1 (63). As
shown in Fig. 4A, two protein䡠DNA complexes (C1 and C2) were
detected with each E2 protein when a DNA probe containing
E2-BSs #3 and #4 of HPV-11 was used for EMSA. The C1
complex represents an E2 dimer binding to one E2-BS first
appearing at low concentrations of E2, whereas the C2 complex
is formed between two E2 dimers binding to both E2-BSs and
observed at increasing concentrations of E2 protein. At higher
concentrations of 16E2 and 18E2, we also observed additional
protein䡠DNA complexes migrating slower than the C2 complex
(Fig. 4A,lanes 30 and 40), presumably generated by oligomer-
ization of E2䡠DNA complexes. An appearance of a band (indi-
cated by an asterisk) below the C1 complex of BE2 (Fig. 4A,
lanes 11–20) is likely formed between minor degradation prod-
ucts of BE2 and the DNA probe. Consistent with BE2 being the
largest E2 protein in this assay, BE2䡠DNA complexes migrated
slower than HPV E2䡠DNA complexes. Similar patterns of
E2䡠DNA complexes were also detected with DNA probes de-
rived from HPV-16 (Fig. 4B) and HPV-18 (Fig. 4C). When
BPV-1 probe was used, 16E2 and 18E2 showed migration pat-
terns similar to those observed with HPV probes (Fig. 4D,lanes
21–40). The C1 and C2 complexes formed with BE2 apparently
dissociated during electrophoresis, causing two clusters of
large smears on the gel (Fig. 4D,lanes 11–20). The affinity of
11E2 for the BPV probe is extremely low because no clear
complexes could be detected even with the use of 200 ng of
protein (Fig. 4D,lanes 1–10), which accidentally led to the
formation of large protein䡠DNA aggregates unable to enter the
gel. It is apparent from these EMSAs that the C1 complex was
formed at much lower concentrations of 16E2 and 18E2 than
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11E2 and BE2 (compare lanes 21–40 with lanes 1–20 of Fig. 4,
A–C), indicating that high risk E2 has higher affinity for E2-
BSs than 11E2 and BE2.
To compare and quantify accurately the binding affinities
among various E2 proteins for each DNA probe, we calculated
the equilibrium binding constant (K
d
) estimated from three or
more gel shift assays by measuring the reduction of free probe
(Table I). Calculations of the equilibrium binding constant
clearly indicate that 16E2 has the highest affinity to both
homologous and heterologous HPV E6 promoter-proximal E2-
BSs, followed in order by 18E2, BE2, and 11E2 (see Table I, K
d
of 16E2:18E2:BE2:11E2 ⫽0.719:1.597:3.239:5.190 for HPV-11
FIG.2.Transcriptional repression mediated by high and low risk HPV and BPV-1 E2 proteins. A, transcriptional repression of the
HPV-11 E6 promoter mediated by different E2 proteins. In vitro transcription was performed in a two-component system comprising pol II
holoenzyme and TBP using p11URR-GLess template, which contains the HPV-11 URR spanning nucleotides 7072–7933/1–70, and the internal
control template pML⌬53, in the absence (⫺) or presence of increasing amounts (2, 10, 50, and 200 ng) of BE2 (lanes 1–5, respectively), 11E2 (lanes
6–10), 16E2 (lanes 11–15), or 18E2 (lanes 16–20). Protein concentrations of different E2 proteins were normalized by Western blotting with
anti-FLAG M2 monoclonal antibody (Sigma). The line graph shows the relative transcription intensity of HPV-11 template with different amounts
of E2 protein. Relative intensity is defined as the signal intensity, quantified by PhosphorImager analysis, from p11URR-GLess relative to that
from the same DNA template performed in the absence of E2 (i.e. the first lane of each reaction set). B, transcriptional repression of the HPV-16
E6 promoter mediated by different E2 proteins. In vitro transcription was performed as described in A, except that a transcriptional template
p16URR-GLess containing the HPV-16 E6 promoter spanning HPV-16 nucleotides 7007–7904/1–72 was used. The line graph shows relative
transcriptional intensity for the HPV-16 E6 promoter at increasing concentrations for each E2 protein titration. C, transcriptional repression of
the HPV-18 E6 promoter mediated by different E2 proteins. In vitro transcription reactions were performed as described in A, except that a
transcriptional template p18URR-GLess containing the HPV-18 E6 promoter spanning HPV-18 nucleotides 6929 –7857/1–81 was used. The line
graph shows relative transcriptional intensity for each HPV-18 E6 promoter signal derived from the titration of each E2 protein.
Transcription and DNA Binding Activities of HPV and BPV E2 45623
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probe, and similar comparisons for HPV-16 and HPV-18
probes). In the case of BPV-1 DNA-derived probe containing
two E2-BSs, homologous BE2 bound with the highest affinity
(K
d
⫽1.079) followed by 16E2 (K
d
⫽1.567), 18E2 (K
d
⫽8.422),
and 11E2 (K
d
⫽25.360), consistent with results published
previously (11). This indicates that there may be some species
specificity between the DNA-binding activity of BPV and HPV
E2 proteins, which allows BE2 to bind better to BPV-1 DNA
and HPV E2 to bind better to HPV DNA. This phenomenon
likely reflects inherent variation between HPV and BPV E2-BS
sequences with HPV preferring A/T spacer nucleotides
(ACCGN
4
CGGT, see Fig. 1C), whereas BPV contains more G/C
spacer nucleotides within their respective E2-BSs. Differences
among E2 recognition of DNA (see “Discussion”) and decreased
stability of BE2 protein may also contribute to the variation in
results from EMSA performed with HPV DNA compared with
BPV-1 DNA (7, 64).
Binding Properties of Individual E2-BSs #3 and #4 in the
HPV-11 E6 Promoter-proximal Region—We and others have
demonstrated previously that E2-mediated repression of the
E6 promoter via inhibition of preinitiation complex formation
functions mainly through E2 binding to the promoter-proximal
#4 E2-BS (9, 10, 25, 28). To explore the possibility that high
risk HPV E2 protein may bind with higher affinity to the
promoter-proximal #4 E2-BS than low risk 11E2 and BE2 to
mediate transcriptional repression, we performed EMSA using
an HPV-11 E6 promoter-proximal DNA probe with mutated #3
but wild-type #4 E2-BS (Fig. 5A). As expected, only one pre-
dominant complex (C1), formed via E2 binding to the #4 E2-BS,
was detected with HPV-11(7902–92)3M probe by different E2
proteins (Fig. 5A). Surprisingly, additional E2䡠DNA complexes
(C2 and oligomers) were also observed with #3 E2-BS-mutated
DNA probe at higher concentrations of 16E2 and 18E2 (Fig. 5A,
lanes 10 and 17–20), indicating that either high risk HPV E2
proteins do have higher DNA-binding activities able to over-
come half-site mutations, or the flanking sequences surround-
FIG.3. Transcriptional activation
mediated by different E2 proteins. A,
E2-dependent activation assays. Recon-
stituted in vitro transcription reactions
were performed with purified TFIIB,
TFIID, TFIIE, TFIIF, TFIIH, and pol II,
in the absence (⫺) or presence (⫹)ofPC4
and increasing amounts of different E2
proteins using p2E2(IR)⌬53 and pG
5
MLT
templates, as described under “Experi-
mental Procedures.”MLP, major core late
promoter. B, line graph displaying fold
activation for each E2 protein. Fold acti-
vation is defined as the signal intensity,
quantified by PhosphorImager, from
p2E2(IR)⌬53 relative to that from the
same DNA template performed in the ab-
sence of E2 and PC4 (i.e. the first lane).
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ing E2 half-site mutations may contribute to E2䡠DNA recogni-
tion and binding. Comparison of the equilibrium binding
constants (K
d
, Table II) revealed that 16E2 indeed bound with
the highest affinity (K
d
⫽0.871) to HPV-11(7902–92)3M probe,
followed by 18E2 (K
d
⫽2.163), BE2 (K
d
⫽5.026), and 11E2
(K
d
⫽9.577). To test whether E2-BSs #3 and #4 in the HPV-11
E6 promoter-proximal region are equivalent for E2 binding
because they have the same consensus sequence, AC-
CGAAAACGGT (see Fig. 1C), we also performed EMSA with
DNA probe containing mutated #4 E2-BS (Fig. 5B). The results
were nearly identical to the mutated #3 probe except that
higher order HPV-16 E2䡠DNA complexes (C2 and oligomer)
were clearly observed (Fig. 5B,lanes 8–10), again suggesting
that flanking sequences surrounding the E2-BS mutations in-
deed contribute to E2䡠DNA binding, which is consistent with
previous studies (65). Although higher order E2䡠DNA com-
plexes appeared with high risk E2 proteins when individual
E2-BSs were mutated, comparison of binding constants for
each E2 protein binding to either HPV-11(7902–92)3M or HPV-
11(7902–92)4M probe indicates that the binding properties of
each E2 protein to #3 and #4 E2-BSs are very similar (Table II,
compare 11E2 K
d
9.577 versus 10.261, 16E2 K
d
0.871 versus
0.674, 18E2 K
d
2.163 versus 1.807, and BE2 K
d
5.026 versus
7.569). As a control, additional EMSA was performed with a
DNA probe containing mutations at both #3 and #4 E2-BSs
(Fig. 5C). As expected, 11E2 was unable to bind to HPV-
11(7902–92)34M probe (Fig. 5C,lanes 1–10). Surprisingly,
protein䡠DNA complexes were detected with BE2, 16E2, and
18E2 (Fig. 5C,lanes 18 –20,26 –30, and 35–40), albeit at sig-
nificantly lower affinities than binding to wild-type E2-BSs.
These studies further confirmed that high risk HPV E2 pro-
teins indeed have higher DNA-binding activity than low risk
E2 protein even to overcome mutations in the half-E2-BS,
consistent with previous studies reporting that the C-terminal
FIG.4. EMSA performed with E2
binding to DNA probes containing
two E2-BSs derived from different
types of HPVs and BPV-1. A, binding of
11E2, BE2, 16E2, and 18E2 to a wild-type
HPV-11 E6 promoter-proximal DNA frag-
ment containing two E2-BSs. EMSA was
performed in the absence (⫺) or presence
of increasing amounts of E2 protein (in
ng), as indicated above each lane, with a
32
P-labeled HPV-11 E6 promoter proxi-
mal-probe spanning HPV-11 nucleotides
7902–7933/1–92. The identities of the
shifted complexes, as indicated by C1,C2,
Oligomer, and *, are described under “Re-
sults.”B, binding of 11E2, BE2, 16E2, and
18E2 to a wild-type HPV-16 E6 promoter-
proximal DNA fragment containing two
E2-BSs. EMSA was performed as de-
scribed in A, except that an HPV-16 E6
promoter probe spanning HPV-16 nucleo-
tides 7868 –7904/1–96 was used. C, bind-
ing of 11E2, BE2, 16E2, and 18E2 to a
wild-type HPV-18 E6 promoter-proximal
DNA fragment containing two E2-BSs.
EMSA was performed as described in A,
except that an HPV-18 E6 promoter probe
spanning HPV-18 nucleotides 7834 –
7857/1–101 was used. D, binding of 11E2,
BE2, 16E2, and 18E2 to a wild-type
BPV-1 DNA fragment containing two E2-
BSs. EMSA was performed as described
in A, except that a BPV-1 DNA probe
spanning BPV-1 nucleotides 7874 –7945/
1–85 was used.
TABLE I
Equilibrium binding constant (K
d
) for E2 binding to DNA probe with two E2-BSs
K
d
is in nMand was estimated from three or more gel shift assays.
Protein URR-derived DNA probe
HPV-11(7902–92) HPV-16(7868–96) HPV-18(7834–101) BPV-1(7874–85)
11E2 5.190 ⫾0.680 4.740 ⫾0.617 4.969 ⫾0.690 25.360 ⫾4.279
16E2 0.719 ⫾0.061 0.721 ⫾0.050 0.701 ⫾0.062 1.567 ⫾0.118
18E2 1.597 ⫾0.231 1.610 ⫾0.102 1.706 ⫾0.175 8.422 ⫾0.638
BE2 3.239 ⫾0.414 3.306 ⫾0.301 4.518 ⫾0.437 1.079 ⫾0.187
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domain of 16E2 has 180-fold higher affinity for nonspecific
DNA than the C-terminal domain of BE2 (66).
Correlation of Transcriptional Repression Activity with DNA-
binding Activity between 18E2 and 11E2—There is a clear
distinction between repression activity of 16E2 and both 18E2
and 11E2 proteins (see Fig. 2). This activity correlated well
with the binding affinities among HPV E2 proteins to DNA
probes derived from the HPV E6 promoter-proximal region, in
that 16E2 had the highest affinity followed by 18E2 and then
11E2 (Tables I and II). If there is indeed a direct correlation
between binding affinities and E2 repressor activity, 18E2
should be a better transcriptional repressor than 11E2 because
it has a higher affinity for promoter-proximal E2-BSs than
11E2 (Table I and Fig. 4). However, this was not clearly ob-
served (Fig. 2). If the mechanism of transcriptional repression
at the E6 promoter is caused by disruption of preinitiation
complex formation through promoter-proximal DNA binding
(9, 10, 25, 28), then one possibility that may account for this
lack of difference is that there were not enough titration points
to measure the differences in activity between 18E2 and 11E2
adequately. The other possibility is that promoter-distal
E2-BSs may sequester E2 protein away from promoter-
proximal E2-BSs.
To address these issues, we performed an in vitro transcrip-
tion assay using the two-component transcription system with
an HPV-11 URR-containing G-less cassette template where
E2-BSs #2 and #3 were mutated (28) while carefully titrating
both 11E2 and 18E2 (Fig. 6A). At low concentrations of 11E2
and 18E2 (e.g. 1 ng), the activity from the HPV-11 E6 promoter
is almost reduced by half with 18E2, whereas 11E2 showed
negligible reduction in E6 promoter activity (Fig. 6A, compare
lane 15 with lane 5). Plotting relative intensity determined by
PhosphorImager analysis revealed that there is a significant
difference between the repression activities of 18E2 and 11E2
FIG.5. EMSA performed with E2
binding to DNA probes containing
mutated promoter-proximal E2-BSs
derived from HPV-11. EMSAs were
performed as described in Fig. 4, except
that
32
P-labeled HPV-11(7902–92)3M (A),
HPV-11(7902–92)4M (B), and HPV-
11(7902–92)34M (C) probes spanning
HPV-11 nucleotides 7902–7933/1–92 with
mutated #3, mutated #4, and mutated #3
and #4 E2-BSs were used.
TABLE II
Equilibrium binding constant (K
d
) for E2 binding to E2-BS-mutated HPV-11 probe
K
d
is in nMand was estimated from three or more gel shift assays.
Protein Mutated HPV-11 E6 promoter-proximal #3 and #4 E2-BSs
HPV-11(7902–92)3M HPV-11(7902–92)4M HPV-11(7902–92)34M
11E2 9.577 ⫾2.607 10.261 ⫾2.582 No binding
16E2 0.871 ⫾0.077 0.674 ⫾0.054 31.479 ⫾4.963
18E2 2.163 ⫾0.246 1.807 ⫾0.284 28.221 ⫾2.068
BE2 5.026 ⫾0.597 7.569 ⫾1.585 376.874 ⫾67.853
Transcription and DNA Binding Activities of HPV and BPV E245626
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at low amounts of E2 proteins, which indeed correlates with
their relative DNA-binding affinities for E2-BSs (Fig. 6B).
DISCUSSION
In this report, we described the first comparative analysis of
transcription and DNA-binding activities of the full-length
high risk and low risk HPV and BPV-1 E2 proteins in well
defined cell-free environments devoid of other cellular proteins
whose presence in the cell may complicate the studies of intrin-
sic activities of E2 proteins. We uncovered a direct correlation
of transcriptional activity with the DNA-binding activity in
which high risk 16E2 is the strongest transcriptional activator/
repressor, compared with 18E2, 11E2, and BE2, and shows the
highest affinity for both homologous and heterologous HPV E6
promoter-proximal E2-BSs. Our studies thus provide unequiv-
ocal comparison of functional properties of E2 proteins encoded
by high risk and low risk HPVs as well as BPV-1, which play an
important role in virus-induced pathogenesis in both humans
and animals.
High Risk E2 Proteins Display More Transcriptional Activity
than Low Risk HPV and BPV-1 E2 Proteins—The full-length
E2 protein can function as a transcriptional repressor to inhibit
the expression of virus-encoded gene products mainly through
promoter-proximal E2-BSs (25, 27, 28, 67–69). We found that,
similar to high versus low risk HPV E6 and E7 oncoproteins,
there are also distinct differences in the repression activities
between high and low risk HPV E2 proteins. Using an in vitro
transcription system reconstituted with human pol II holoen-
zyme and TBP, we defined a strict order of transcriptional
repression activity with 16E2 acting as the strongest repressor,
followed by 18E2 and BE2, and lastly 11E2 (Figs. 2 and 6). The
potency of transcriptional repression activity exhibited by dif-
ferent E2 proteins strongly correlates with their DNA-binding
affinities for E2-BSs (Fig. 4 and Table I). 16E2 bound with the
highest affinity for the E6 promoter-proximal DNA fragments
derived from HPV-11, HPV-16, and HPV-18 and was followed
closely, again, by 18E2, BE2, and finally 11E2. The strong
DNA-binding activity observed with 16E2 and 18E2 even al-
lows these high risk E2 proteins to bind to DNA probes con-
taining half-site mutations in both promoter-proximal #3 and
#4 E2-BSs (Fig. 5), whose binding could not occur with low risk
11E2 protein.
Although comparison of transactivation activity among some
high and low risk HPV E2 proteins has been described in
previous studies using either transient transfection or in vitro
transcription performed with HeLa nuclear extract (11, 55, 56),
the presence of numerous unidentified cellular proteins in their
assays often complicates the interpretation. Therefore we have
developed a highly purified E2-dependent in vitro transactiva-
tion system (17) comprised only of essential recombinant gen-
eral transcription factors (TFIIB, TFIIE, and TFIIF), recombi-
nant general cofactor PC4, and epitope-tagged multiprotein
complexes (TFIID, TFIIH, and pol II). Using this reconstituted
cell-free transcription system, we found that 16E2 is the most
responsive and strongest transactivator compared with 18E2,
BE2, and 11E2 (Fig. 3), suggesting that 16E2 may be more
effective at recruiting components of the general transcription
machinery to the promoter region. This interesting possibility
remains to be investigated further.
Significance for the Order of Transcriptional Activity between
High and Low Risk HPV E2 Proteins—In our studies, we found
a strict order of both transcriptional activation and repression
activity among various E2 proteins. High risk 16E2 displays
the highest activation and repression activity, followed by high
risk 18E2 and then low risk 11E2. In a recent epidemiological
FIG.6. Transcriptional repression
of the HPV-11 E6 promoter mediated
by 11E2 and 18E2. A, transcriptional re-
pression of the HPV-11 E6 promoter by
different amounts of E2 proteins. In vitro
transcription was performed in a two-
component system as described in Fig.
2A, except that p7862–70(23M)GLess/I
⫺
(28), a transcriptional template contain-
ing HPV-11 URR spanning nucleotides
7862–7933/1–70 with mutations at #2
and #3 E2-BSs, was used. B, line graph of
relative intensity for 11E2 and 18E2 pro-
teins. Relative intensity is defined as the
signal intensity, quantified by Phospho-
rImager, from p7862–70(23M)GLess/I
⫺
relative to that from the same DNA tem-
plate performed in the absence of E2 (i.e.
the first lane of each reaction set).
Transcription and DNA Binding Activities of HPV and BPV E2 45627
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study, 99.7% of 1,000 cases of invasive cervical cancer were
HPV DNA-positive with HPV-16 DNA (53%) being the most
prevalent followed by HPV-18 DNA (15%) (2, 3). Previous stud-
ies have also shown that high risk HPV E6 and E7 are more
active in inducing cellular transformation than low risk E6 and
E7 (for review, see Refs. 4 and 5). A common hallmark of
cervical cancer is viral integration, which often disrupts the E2
open reading frame, leading to the loss of E6 promoter regula-
tion and thus increased the expression of HPV E6 and E7
oncoproteins. We speculate that HPV-16 developed a more
transcriptionally active E2 protein to regulate its highly active
E6 and E7 oncoproteins tightly and govern the viral life cycle
more stringently than low risk HPVs, which express less potent
E6 and E7 proteins. Once regulation of the E6 promoter by high
risk E2 proteins is lost because of viral integration, cancer may
then develop.
Differences in DNA Target Site Discrimination by Papilloma-
virus E2 Proteins—Although previous studies of binding site
affinities between HPV and BPV-1 E2 proteins revealed little
variation (11, 56), we found a distinct order of E2 binding to
both wild-type and mutated HPV E6 promoter-proximal E2BSs
among 16E2, 18E2, BE2, and 11E2 (Figs. 4 and 5 and Tables I
and II). One possible explanation for the variation between our
results and previously published studies might be attributed to
the DNA probes from which the equilibrium binding constants
were calculated. Although we used a much larger DNA probe
containing two E2-BSs derived from the natural HPV URRs,
others have used much smaller DNA probes containing only
one or two E2-BSs with minimal flanking regions (11, 56).
Smaller DNA probes would naturally have more inherent DNA
flexibility than larger DNA probes, which would favor BE2
binding to more flexible DNA, whereas HPV E2 has a predis-
position to bind to pre-bent DNA (7). Variations in Mg
2⫹
salt
concentrations between DNA-binding experiments may also
play a role in the differences in calculating equilibrium binding
constants because there is evidence that Mg
2⫹
enhances HPV
E2 recognition of specific E2-BSs (70). Furthermore, the four-
nucleotide spacers within our E2-BSs also vary from previous
DNA probes (11), consistent with previous studies identifying
that HPV E2 favors spacer nucleotides that are A/T-rich, spe-
cifically AATT, whereas BPV E2 does not display an ability to
discriminate between spacer nucleotides (7, 71). This may ex-
plain our results in which 16E2 favors binding to DNA probes
derived from HPV URR, whereas BE2 binds better to BPV-
derived E2-BSs that are less A/T-rich (Fig. 4 and Table I), a
conclusion in agreement with previous studies (11).
It should not be surprising that E2 proteins vary in binding
affinities, considering that there are distinct differences in the
quaternary structures of the DNA-binding domains between
16E2 and 18E2 (7). These differences are critical because they
dictate the spatial arrangement of side chains presented to the
major grooves for DNA sequence recognition (64). Further-
more, structural and biochemical studies of E2 proteins from
BPV-1 and HPV-16 have suggested they use different mecha-
nisms to discriminate their DNA targets (71) even though they
are highly homologous proteins. Recent E2䡠DNA-binding stud-
ies have also reported that there are significant differences in
the binding affinities between the DNA-binding domains of
HPV-16 and BPV-1 (66). In fact, BE2 shares more structural
similarities to 18E2 than 16E2, whereas 16E2 is structurally
more similar to HPV-31 E2 protein (7). This may explain why
the transcriptional and DNA-binding activity of 18E2 is more
equivalent to BE2 than to its closely related human homolog
16E2. Although BE2 and 18E2 are structurally similar, and
they both induce the same global DNA deformation upon bind-
ing, their mechanisms of deformation are different. BE2 has a
positive charge localized near the C terminus of the recognition
helix, whereas 18E2 has a positive charge in the center of its
DNA interaction surface, and 16E2 is even less charged all
along the DNA interaction surface (7). This would lead to
differences in E2䡠DNA contacts, with 16E2 making fewer con-
tacts with the DNA phosphate backbone than either BE2 or
18E2 (7), which would be consistent with increased DNA-bind-
ing specificity. Although the DNA-binding domain sequences of
high risk 16E2 and 18E2 share 54% identity and 77% homol-
ogy, they still differ in DNA binding. Thus it is likely that the
DNA-binding domain of low risk 11E2 may vary further in
structure from both 16E2 and 18E2, thus contributing to vari-
ations in DNA-binding affinity among high and low risk HPV
E2 proteins. This remains to be elucidated once the structure of
11E2 becomes available.
Acknowledgments—We thank L. T. Chow for genomic HPV-18 DNA,
R. Kovelman for pCMV-16E2 and pCMV-18E2 plasmids, P. M. Howley
for genomic HPV-16 DNA and pdBPV-1 (142–6) plasmid, and K. Chiu
along with J. Watson for assistance in plasmid constructions. We are
also grateful to Mary C. Thomas for insightful discussion and critical
reading of the manuscript.
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Transcription and DNA Binding Activities of HPV and BPV E2 45629
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Samuel Y. Hou, Shwu-Yuan Wu and Cheng-Ming Chiang
Proteins Correlates with E2 DNA Binding
Transcriptional Activity among High and Low Risk Human Papillomavirus E2
doi: 10.1074/jbc.M206829200 originally published online September 17, 2002
2002, 277:45619-45629.J. Biol. Chem.
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