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Journal of Virology, October 1998, p. 8166-8173, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Fifteen-Amino-Acid Peptide Inhibits Human
Papillomavirus E1-E2 Interaction and Human Papillomavirus DNA
Replication In Vitro
Hiroaki
Kasukawa,
Peter M.
Howley, and
John D.
Benson*
Department of Pathology, Harvard Medical
School, Boston, Massachusetts
Received 3 June 1998/Accepted 10 July 1998
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ABSTRACT |
Mutation of the conserved glutamic acid residue at position 39 of
human papillomavirus type 16 (HPV-16) E2 to alanine (E39A) disrupts its
E1 interaction activity and its replication function in transient
replication assays but does not affect E2 transcriptional activation.
This E39A mutation also disrupts replication activity of HPV-16 E2 in
HPV-16 in vitro DNA replication. On this basis, we designed 23- and
15-amino-acid peptides derived from HPV-16 E2 sequences flanking the
E39 residue and tested the ability of these peptides to inhibit
interaction between HPV-16 E1 and E2 in vitro. The inhibitory activity
of these peptides was specific, since analogous peptides in which
alanine was substituted for the E39 residue did not inhibit
interaction. The 15-amino-acid peptide E2N-WP15 was the smallest
peptide tested that effectively inhibited HPV-16 E1-E2 interaction.
This peptide also inhibited in vitro replication of HPV-16 DNA. The
efficacy of E2N-WP15 was not exclusive to HPV-16: this peptide also
inhibited interaction of HPV-11 E1 with the E2 proteins of both HPV-11
and HPV-16 and inhibited in vitro replication with these same
combinations of E1 and E2 proteins. These results provide further
evidence that E1-E2 interaction is required for papillomavirus DNA
replication and constitute the first demonstration that inhibition of
this interaction is sufficient to prevent HPV DNA replication in vitro.
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INTRODUCTION |
The papillomaviruses are small,
circular double-stranded DNA viruses that cause epithelial lesions. Of
the greater than 70 known human papillomaviruses (HPVs), those
associated with anogenital lesions have been classified into two
general categories, based on the relative tendencies of their
associated lesions to remain dysplastic but benign (termed low-risk
HPVs) or to progress to high-grade intraepithelial neoplasias and to
invasive malignant tumors (high-risk HPVs). At least 90% of human
cervical cancers contain viral sequences of a high-risk HPV subtype
(i.e., HPV-16, HPV-18, HPV-31, or HPV-33). Of the high-risk HPVs,
HPV-16 is the predominant subtype and is found in 50 to 70% of human
cervical cancers (48). Our laboratory has focused on HPV-16
as a particularly important target for the study of functions
associated with papillomavirus oncogenesis and viral pathogenesis.
The papillomaviruses, like simian virus 40 (SV40), have served as a
model system for the study of mammalian DNA replication. In vitro
replication studies have shown that papillomavirus and SV40 resemble
each other in their utilization of a number of cellular replication
proteins, including DNA polymerase 
primase (4, 30), DNA
polymerase 
, RPA, PCNA, and topoisomerases (18, 26, 29,
43), although papillomavirus DNA replication may also require
some additional cellular factors (26). Two virus-encoded proteins, E1 and E2, are required for the initiation of papillomavirus DNA replication (40). The papillomavirus E1 protein is the
replication enzyme that initiates viral DNA replication. The E1
proteins encoded by the various papillomaviruses are well conserved and
bear significant homology to the large T antigen replication protein of
SV40 (8, 23). Both the papillomavirus E1 proteins and SV40
large T antigen bind to a region of dyad symmetry within their
respective replication origins (14, 15, 27, 41) and possess
ATP-dependent DNA helicase activities (16, 17, 36, 37, 44).
In comparison to T-antigen-dependent SV40 replication, however, very
little is known about the biochemical events and cellular activities involved in papillomavirus replication.
Unlike SV40 large T antigen, E1 does not bind the viral replication
origin autonomously. Instead, the papillomavirus E2 protein is required
as a cofactor for E1 binding to the origin (3, 28, 32). The
papillomavirus E2 proteins are composed of two functional domains: an
amino-terminal transcriptional activation domain and a carboxy-terminal
DNA binding domain that binds as a dimer to the ACCN6GGT
recognition sequence (2, 24, 25). These domains of E2 are
separated by a less-conserved, dispensable hinge region. The
amino-terminal domain of E2 serves two functions in the viral life
cycle, both as a transcriptional regulator of viral gene expression and
as an E1 interaction domain that is required for recruitment of the E1
replication protein to the papillomavirus origin of replication. E2
binding sites flank the replication origin. By binding to these sites
and through interaction with E1, E2 directs E1 to the papillomavirus
replication origin (11, 21, 22, 34-36, 39, 43). This
interaction is thought to facilitate recognition of the viral
replication origin by E1 and subsequent assembly of the replication
initiation complex, including recruitment of the DNA polymerase priming enzyme (4, 30), and is essential for efficient viral
DNA replication in transient transfection assays (31, 32).
The results presented here show that we have achieved replication of
HPV-16 DNA in vitro. This replication mimics that seen in transient in
vivo replication assays in that it requires interaction of E2 with E1
(31, 32). Furthermore, we demonstrate the ability of a 23- or 15-amino-acid peptide derived from the HPV-16 E2 amino terminus to
disrupt both interaction between E1 and E2 and papillomavirus DNA
replication in vitro. The activity of such a peptide as a functional
inhibitor in these assays has several implications: (i) it identifies a
discrete region of E2 that is necessary and sufficient for specific
interaction with E1, (ii) it provides proof of the concept that a
relatively small molecule may be used to inhibit E1-E2 interaction and
papillomavirus DNA replication, (iii) it provides a starting point for
the design or discovery of biologically active molecules capable of
inhibiting papillomavirus DNA replication, and (iv) it provides a
reagent that may facilitate functional dissection of the dynamic
processes that result in the stepwise assembly of the papillomavirus
DNA replication initiation complex. These observations may aid in the
identification of effective bioactive antiviral compounds that would
inhibit HPV DNA replication.
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MATERIALS AND METHODS |
Baculovirus expression of HPV E1 and E2 proteins.
An
NcoI-SmaI fragment from pUC-E116
(9) containing the HPV-16 E1 protein coding region was made
blunt by using T4 polymerase and inserted into the blunt-ended
BamHI site of pBS-EE to generate pBS-EE-E116.
[pBS-EE was generated by cloning the XbaI-BamHI
fragment encoding the polyomavirus middle T antigen EE epitope
(18) from pUC-EE into pBluescript-SK(+) (Stratagene).] The
EcoRI-NotI fragment from pBS-EE-E116
was recloned into the pVL1392 baculovirus transfer vector (Pharmingen).
HPV-16 E2 and HPV-16 E2 E39A were cloned as
BamHI-EcoRI fragments into pVL1392.
Recombinant baculovirus expressing HPV-11 EE-E1 and HPV-11 E2 were
generously provided by Louise Chow. Hi-Five cells were infected at a
multiplicity of 5 to 10 PFU per cell and incubated at 27°C for
48 h. Cells were scraped into ice-cold phosphate-buffered saline,
pelleted by centrifugation in a Sorvall RT6000D tabletop centrifuge at
1,000 rpm for 5 min, washed once in ice-cold phosphate-buffered saline,
and repelleted. The cell pellet was then resuspended in hypotonic
buffer (buffer A) containing 10 mM HEPES-K+ (pH 7.5), 10 mM
KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), and
protease inhibitor cocktail (PIC; 1 mM phenylmethylsulfonyl fluoride,
10-µ-g/ml aprotinin, 10-µg/ml leupeptin, 10-µg/ml pepstatin A,
10-µg/ml phenanthroline, 16-µg/ml benzamidine). Cells were pelleted
as before, resuspended in buffer A containing 0.5% Nonidet P-40
(NP-40), and incubated on ice for 10 min. Lysates were then spun at
4°C in a Sorvall tabletop microcentrifuge (3,000 rpm). The
cytoplasmic extract supernatant was removed, and an equal volume of
50% (vol/vol) glycerol was added. Lysates were quick-frozen on dry ice
and stored at 
70°C until use. The nuclear pellet was washed once in
buffer A without NP-40, resuspended in buffer C (20 mM
HEPES-K+ [pH 7.9], 429 mM NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 25% [vol/vol] glycerol, 0.5 mM DTT containing PIC), and
incubated on ice for 30 to 40 min with intermittent vortexing. After
clarification by centrifugation at full speed in a Sorvall
microcentrifuge for 10 min at 4°C, the nuclear extract was
quick-frozen on dry ice and stored at 70°C until use.
In vitro HPV E1-E2 binding assay.
Protein (300 to 500 ng)
from HPV-16 or HPV-11 EE-E1-expressing recombinant baculovirus-infected
cell nuclear extracts and 80 to 120 ng from HPV E2-expressing
baculovirus-infected cell nuclear extracts were incubated in 500 µl
of NET-gel buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP-40,
1 mM EDTA, 0.25% gelatin) at 4°C for 1 h. Anti-EE monoclonal
antibody (1 to 2 µg) was added, and the mixture was incubated with
shaking at 4°C for 1 h. Immune complexes were precipitated with
protein G-agarose beads and washed twice for 30 min with ice-cold
NET-gel buffer. Precipitated proteins were detected by Western blotting
with appropriate primary antibodies and anti-rabbit immunoglobulin
G-horseradish peroxidase or anti-mouse immunoglobulin G-horseradish
peroxidase (Amersham). Membranes were blocked for 1 h in TNET (10 mM Tris [pH 7.5], 2.5 mM EDTA, 50 mM NaCl, 0.1% Tween 20) containing
4% (wt/vol) dry milk. Washes and antibody reactions were carried out
in TNET, and proteins were visualized by chemiluminescence (Renaissance
ECL; NEN) and autoradiography. Bands were quantitated by using the NIH
image program.
Purification of HPV EE-E1 and E2 proteins.
EE-E1-containing,
baculovirus-infected cell nuclear extract (3 to 5 mg of total protein)
containing 1% NP-40 was passed over a mono-Q column (Econo-Pac Q
cartridge, Bio-Rad no. 732-0025). The flowthrough was incubated at
4°C for 1 h with 200 µl of anti-EE monoclonal antibody that
had been covalently cross-linked to protein G-Sepharose beads. Beads
were pelleted by centrifugation and washed once with 800 µl of (1%)
NP-40 lysis buffer with PIC. Beads were then washed three times for 10 min each time at 4°C with 800 µl of high-salt buffer (20 mM Tris
[pH 7.0], 0.5 M NaCl, 0.5 mM DTT, 0.1% NP-40, PIC), followed by
three 10-min washes with 800 µl of sodium phosphate buffer (10 mM
NaPO4 [pH 8.0], 0.5 mM DTT, 0.1% NP-40, PIC). EE-E1 was
eluted from the pelleted beads in 100 mM triethylamine (pH 11.5). The
beads were pelleted by centrifugation at 4°C, and the supernatant was
dialyzed for 3 h at 4°C against 1 liter of dialysis buffer
(HEPES-K+ [pH 7.5], 1 mM DTT, 10% glycerol, 150 mM
NaCl). The dialysis buffer was then changed, and dialysis was continued
overnight at 4°C. This procedure yielded approximately 100 to 150 ng
of EE-E1 (>95% pure) per µl. Twenty-microliter aliquots of purified EE-E1 were quick-frozen on dry ice and stored at 70°C until use.
Baculovirus-infected cell nuclear extract containing E2 in buffer C (3 mg/ml) was applied to an Econo-Pac heparin column cartridge
(Bio-Rad
no. 732-0075) at 4°C. After washing with 5 column volumes
of buffer C
containing PIC at a flow rate of 1 ml/min, E2 was
eluted in 5 column
volumes of buffer C containing 0.7 M NaCl and
0.5-ml column fractions
were collected. Fractions containing E2
were determined by Coomassie
staining of sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
gels loaded with aliquots of eluted fractions.
This procedure yielded
300 to 500 ng of E2 per µl, which was stored
at
70°C until use.
In vitro replication reactions.
In vitro replication
reactions were prepared essentially as described by Kuo et al.
(18), in 25-µl reaction mixtures at 37°C for different
times. Standard reactions lasted 2 h and were terminated by
addition of 200 µl of stop solution (20 mM Tris-HCl [pH 7.5], 10 mM
EDTA, 0.1% sodium dodecyl sulfate, 20-µg/ml RNase A). After
incubation at 37°C for 15 min, proteinase K was added (200 µg/ml)
and incubation was continued for 30 min. Samples were extracted with
250 µl of phenol-chloroform-isoamyl alcohol (25:24:1), ethanol
precipitated, washed with 70% ethanol, dried, resuspended in 20 to 30 µl of TE (10 mM Tris [pH 8.0], 1 mM EDTA), and analyzed on a 0.8%
agarose gel. The agarose gel was dried onto a Hybond N+
membrane (Amersham), and replicated DNA was visualized by
autoradiography. Bands were quantitated by PhosphorImager analysis.
Replication templates were pKS7838-7905 (nucleotides 7838 to 7905 and 1 to 130) containing the HPV-16 replication origin and pUC7874-99
containing the HPV-11 origin of replication (nucleotides 7874 to 7933 and 1 to 99). pUC7874-99 was generously provided by Louise Chow.
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RESULTS |
In vitro replication of HPV-16 DNA.
Transient replication
assays have demonstrated that interaction between HPV-16 E1 and E2 is
required for efficient origin-dependent papillomavirus replication in
vivo (31). E2 stimulation of bovine papillomavirus (BPV) and
HPV-11 replication has been previously demonstrated in vivo (9,
19, 38, 40) and in vitro (18, 19, 43). We sought to
establish a system for the study of HPV-16 DNA replication in vitro and
to determine whether specific interaction between HPV-16 E1 and E2 is
required for HPV-16 DNA replication in this system. Fig.
1A shows replication of an HPV-16 origin-containing plasmid in vitro using 293 cell extracts, along with
partially purified HPV-16 E2 and increasing amounts of
affinity-purified HPV-16 EE-E1. As has been documented for other
papillomavirus E1 proteins, some replication was observed when
relatively large amounts of E1 were used in the absence of E2 (lanes 2 through 7) (5, 6, 16, 22, 33, 40, 48). Addition of 6 ng of
E2 greatly stimulated in vitro replication (lanes 8 to 13), whereas no
replication was observed in the absence of EE-E1 (lane 1). Replication
in this assay is origin dependent, since the small amount of
replication of the pKS plasmid lacking the HPV-16 origin detected at
high concentrations of EE-E1 (Fig. 1B, lanes 1 to 6) was not stimulated
by E2 (Fig. 1B, lanes 7 to 12). Quantitation of these results is shown
in Fig. 1C.
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FIG. 1.
E2 stimulates replication of HPV-16 in vitro. (A)
Replication of HPV-16 origin-containing plasmid p16ori, a Bluescript
KS+ plasmid containing HPV-16 genomic sequences (7838-139), by using
293 cell extract and increasing amounts of EE-16E1 (10 to 50 ng) in the
presence (lanes 8 to 13) or absence (lanes 2 to 7) of HPV-16 E2. The
reaction mixture in lane 1 contained no HPV-16 E1 or E2. The positions
of replicative intermediates (R.I.) and form I and II DNAs analogous to
those observed by Kuo et al. (18) are indicated. (B) In
vitro HPV-16 DNA replication is origin dependent. Replication of
Bluescript KS+ that does not contain HPV-16 origin sequences under
conditions identical to those described for panel A is shown. (C)
PhosphorImager quantitation of the replication experiments shown in
panels A and B.
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E1 interaction is required for E2 stimulation of HPV-16 replication
in vitro.
We have previously demonstrated that substitution of
alanine for the conserved glutamic acid residue at amino acid residue 39 of HPV-16 E2 (E39A) disrupts both HPV-16 E1 interaction and transient HPV-16 DNA replication in transient cotransfection assays. E39A is, however, fully competent as a transcriptional activator (31). To test the capacity of this mutant E2 protein to
support HPV-16 DNA replication in vitro, increasing amounts of
wild-type HPV-16 E2 or the E39A HPV-16 E2 mutant protein were tested
for in vitro replication activity in the presence of 20 ng of EE-E1 (Fig. 2A). Wild-type E2 greatly
stimulated in vitro replication in a dose-responsive manner, whereas
the E39A mutant of HPV-16 E2, which does not interact with E1, had only
a slight stimulatory effect, even at relatively high concentrations.
This result demonstrated the dependence of E2 amino-terminally mediated
interaction with E1 in in vitro replication. Quantitation of these
results is shown in Fig. 2B.
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FIG. 2.
Interaction of the E2 amino terminus with E1 is required
for HPV-16 DNA replication in vitro. (A) Replication of p16ori (40 ng)
was determined in the presence of 293 cell extract, 20 ng of EE-E1, and
increasing amounts of wild-type HPV-16 E2 (lanes 2 to 9) or E1
interaction-defective E39A HPV-16 E2 (lanes 10 to 16). (B)
PhosphorImager quantitation of the replication experiments shown in
panel A.
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Inhibition of interaction between HPV-16 E1 and E2 in vitro.
The results described above suggested that a region of HPV-16 E2
including the E39 residue might encompass a domain sufficient for
interaction with E1. Therefore, we reasoned that a peptide encompassing
this domain of HPV-16 E2 might inhibit interaction between HPV-16 E1
and E2, thereby inhibiting HPV-16 DNA replication (Fig.
3). To test this possibility, HPV-16 E2
and epitope-tagged full-length HPV-16 E1 (EE16-E1) proteins were
expressed separately, using baculovirus vectors. EE-E1 and associated
E2 proteins were immunoprecipitated by using an anti-EE epitope
monoclonal antibody. Precipitated E2 was detected by Western blotting
with polyclonal antiserum raised to the DNA binding domain of HPV-16 E2
(35). As shown in lane 2 of Fig.
4A, E2 coimmunoprecipitated with EE16-E1 but was not precipitated by the EE epitope antibody in the absence of
EE16-E1 (lane 1). Peptides encompassing HPV-16 E2 amino acids 29 to 51 (E2N-WP23) or residues 33 to 47 (E2N-WP15) of HPV-16 E2 were then
tested for inhibition of E1-E2 interaction. These peptide sequences
were derived from the sequence of HPV-16 E2 and were based on an
alignment of amino-terminal residues that are conserved among the
papillomavirus E2 proteins (Fig. 3). Lanes 3 to 9 of Fig. 4A (top) show
the inhibitory effects of increasing concentrations of the E2N-WP23
peptide. The smaller E2N-WP15 peptide displayed comparable inhibition
of E1-E2 interaction (Fig. 4A, bottom, lanes 3 to 9). Both the HPV-16
E2 protein itself and the E2-derived peptides act specifically in these
assays, since the E39A replication-defective HPV-16 E2 mutant bound
only slightly to EE16-E1 (lanes 14). Furthermore, 23-mer and 15-mer
peptides containing an amino acid substitution analogous to that of the HPV-16 E2 E39A mutant did not inhibit E1-E2 interaction at
concentrations at which the wild-type peptide could do so (lanes 10 to
13). These peptides are named E2N-MP23 and E2N-MP15, respectively (Fig.
3). Quantitation of these results is shown in Fig. 4B. The 50%
inhibitory concentrations (IC50) of E2N-WP23 and E2N-WP15
for inhibition of E1-E2 interaction in these assays were 5 and 7.5 µM, respectively. In addition, when inhibition of E1-E2 interaction
by a peptide was plotted as a function of the substrate concentration,
this analysis indicated that the E2N-WP23 and E2N-WP15 peptides act through competitive inhibition (data not shown). We have previously shown that a domain spanning amino acids 421 to 647 of the HPV-16 E1
carboxy terminus is necessary and sufficient for E2 interaction (45). Specific and comparable inhibition of E1-E2
interaction was also observed in analogous experiments examining the
ability of these peptides to inhibit interaction between the HPV-16 E2 protein and the epitope-tagged HPV-16 E1 C terminus (amino acids 421 to
647; data not shown).
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FIG. 3.
Derivation of candidate HPV-16 E1-E1
interaction-inhibitory peptides based on conserved residues within the
E2 amino terminus. Mutagenesis studies identified residue E39 of HPV-16
E2 as critical for interaction with E1. (A) Alignment of conserved
proximal sequences in other papillomavirus E2 proteins. A deduced
consensus sequence is shown below in panel A. (B) Sequences of the
synthetic peptides derived from this portion of HPV-16 E2. WP peptides
retain the conserved glutamic acid at the position analogous to E39 of
HPV-16 E2, whereas MP peptides contain a substituted alanine at this
position.
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FIG. 4.
Peptides derived from the HPV-16 E2 amino terminus
inhibit in vitro interaction between HPV-16 E1 and E2. (A) Indicated
amounts of epitope-tagged HPV-16 E1 (EE-E1) and HPV-16 E2 were
incubated in NET-gel buffer in the presence of increasing
concentrations of the 23-mer (E2N-WP23; top, lanes 2 to 9) and the
15-mer (E2N-WP15; bottom, lanes 2 to 9). Binding of EE-E1 to E2 was
determined by immunoprecipitation with an anti-EE epitope monoclonal
antibody, and E2 was detected by using anti-HPV-16 E2C serum
(31). Precipitation of E1 in each reaction was confirmed by
probing the same blot with polyclonal antiserum recognizing HPV-16 E1.
Increasing amounts of the E2N-MP23 (top) and E2N-MP15 (bottom) peptides
were used in lanes 10 to 13. Lane 14 represents the level of
interaction observed between the HPV-16 E39A mutant protein and EE-E1.
Input wild-type HPV-16 E2 and E39A mutant HPV-16 E2 proteins are shown
in lanes 15 and 16. (B) PhosphorImager quantitation of the E2-reactive
bands from the experiments shown in panel A.
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Since the E2N-WP23 and E2N-WP15 peptides could inhibit interaction
between HPV-6 E1 and E2, their ability to inhibit in vitro
HPV-16 DNA
replication was also tested (Fig.
5A and
B). Figure
5C shows the compiled quantitative results of several
independent
experiments. Both the 23-mer and 15-mer forms of the
wild-type
peptide could effectively inhibit in vitro replication. The
inhibitory
effects of E2N-MP23 and E2N-MP15 were also tested as
specificity
controls. Each of the wild-type peptides inhibited
replication
much more effectively than the respective E39A-substituted
analog,
a result consistent with the importance of the E39 residue of
HPV-16 E2 in E1 interaction. The replication-inhibitory effects
of the
peptides are a result of inhibition of E1-E2 interaction,
since the
peptides had no effect on the low levels of replication
observed with
E1 alone (data not shown). Smaller peptides derived
from sequences
within E2N-WP15 did not inhibit HPV-16 E1-E2 interaction
or replication
in vitro (data not shown).
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FIG. 5.
Peptides derived from the HPV-16 E2 amino terminus
inhibit HPV-16 replication in vitro. (A and B) In vitro inhibition of
HPV-16 DNA replication was determined by using 40 ng of EE-E1 and 6 ng
of HPV-16 E2 in the presence of increasing amounts of E2N-WP23 (A) or
E2N-WP15 (B). Negative controls included reaction mixtures with only
293 cell extract and p16ori (lanes 1 in panels A and B), EE-E1 in the
absence of E2 (lanes 2 in panels A and B), or complete replication
reaction mixtures containing increasing concentrations of E2N-WP23 and
E2N-WP15 (panels A and B, lanes 11 to 14). (C) PhosphorImager
quantitation of the replication experiments shown in panels A and B. The amount of replication relative to that in lanes 3, in the absence
of an inhibitory peptide, is plotted logarithmically as a function of
the peptide concentration.
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Peptide inhibition of in vitro E1-E2 interaction and replication is
not limited to HPV-16.
Very little is known about the physical
properties of domains within the papillomavirus E1 and E2 proteins that
are involved in intermolecular recognition and association. Within the
sequence of the HPV16-E2-derived 15-amino-acid minimal peptide that
inhibits HPV-16 E1-E2 interaction, there are seven well-conserved
residues (W-X-X-M/V/I-R-X-E-X-X-I/L-X-X-X-X-A-R)
(see also Fig. 3). We reasoned that if these residues adequately
represented a conserved E1 interaction domain, the HPV-16 E2-derived
peptide might also inhibit interaction between other HPV E1 and E2
proteins. We therefore tested the inhibitory effects of the E2N-WP15
peptide by using the HPV-11 E1 and E2 proteins. Epitope-tagged versions
of the HPV-11 (HPV-11 EE-E1) and HPV-16 E1 (HPV-16 EE-E1) proteins were tested for binding to HPV-11 and HPV-16 E2. The effect of the E2N-WP15
peptide on these interactions was also assessed. As shown in Fig.
6A, E2N-WP15 effectively inhibited
interaction between HPV-11 E1 and HPV-16 E2 (lanes 10 to 13), along
with the HPV-11 E1-E2 interaction (lanes 20 to 24), with an
IC50 of approximately 10 µM. The E2N-WP15 peptide also
inhibited interaction between HPV-11 E1 and E2, although this effect
was weaker than that observed when other combinations of E1 and E2 were
used. (We estimate the IC50 of this inhibition to be
approximately 20 µM.) As was the case for interaction between HPV-16
E2 and HPV-16 E1, the HPV-16 E2 E39A mutant did not bind HPV-11 E1
(lanes 8 and 16). The E2N-MP15 peptide did not inhibit HPV-11 E1-E2
interaction or interaction between HPV-11 E1 and HPV-16 E2. This
suggests that the domain of HPV-16 E2 that interacts with its natural
counterpart in HPV-16 E1 can also interact specifically with HPV-11 E1.
Interaction between HPV-16 E1 and HPV-11 E2 was not observed (data not
shown). This is consistent with the inability of HPV-16 E1 and HPV-11 E2 to support transient replication in cotransfection assays
(47). Thus, although many elements of E1-E2 interaction are
conserved among the HPV subtypes, these interactions are not completely interchangeable. Further examination of the nature of this
nonreciprocal functionality may reveal important determinants in the
specificity of E1-E2 interaction.
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FIG. 6.
E2N-WP15 inhibits E1-E2 interaction and in vitro DNA
replication of HPV-11. (A) Inhibition of E1-E2 interaction was compared
between HPV-16 E1 and HPV-16 E2 (lanes 1 to 8), between HPV-11 E1 and
HPV-16 E2 (lanes 9 to 18), and between HPV-11 E1 and HPV-11 E2 (lanes
19 to 26). Epitope-tagged E1 proteins were precipitated by using an
anti-EE epitope monoclonal antibody, and associated E2 proteins were
detected by Western blotting with anti-E2 serum. Negative control
reactions containing EE-E1 with no E2 are shown in lanes 1, 9, and 19. The amounts of input HPV-16 E2 and E39A used in lanes 1 to 16 are shown
in lanes 17 and 18. The input amount of E2 used in the HPV-11 E2
experiment (lanes 19 to 25) is shown in lane 26. (B) In vitro
replication of HPV-11 origin plasmid p11ori(7874-99) using increasing
amounts of purified HPV-11 EE-E1 in the presence (lanes 6 to 9) or
absence (lanes 2 to 5) of 6 ng of HPV-16 E2. The reaction mixture in
lane 1 contained no HPV proteins. (C) The ability of E2N-WP15 to
inhibit in vitro replication of the HPV-11 origin using HPV-11 EE-E1
and HPV-16 E2 was tested (lanes 4 to 8). Effects of equivalent
concentrations of the E2N-MP15 peptide were also determined (lanes 9 to
11). Lanes 1 to 3 contained no HPV protein.
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The ability of the E2N-WP15 peptide to inhibit HPV-11 E1-mediated
replication in vitro was also tested. As shown in Fig.
6B
(lanes 1 to
9), HPV-11 E1 could cooperate with HPV-16 E2 in supporting
dose-responsive in vitro replication of a plasmid containing the
HPV-11
origin. (These proteins also functioned in the replication
of the
HPV-16 origin but less efficiently than in the replication
of the
HPV-11 origin; data not shown.) The E2N-WP15 peptide inhibited
HPV-16
E2-HPV-11 E1-dependent replication of the HPV-11 origin
with
approximately the same effectiveness as it inhibited HPV-16
E1-E2-mediated in vitro replication (Fig.
5B, lanes 11 to 16).
The E39A
HPV-16 E2 mutant had only a slight effect on replication
in these
assays (lanes 17 to 19). Thus, the effects of inhibitory
peptides on in
vitro replication mirrors those observed in E1-E2
interaction assays.
The dynamics of E1-E2 interaction.
Little is known about the
ordered assembly of the papillomavirus E1 and E2 proteins at the origin
of replication and the formation of the replication initiation complex.
Two types of BPV E1-E2-origin complexes have been observed. One complex
contains both BPV E1 and E2 (22, 34); the other complex
contains only E1, which forms an oligomeric ring around the DNA
(33). Although the latter complex can form at high E1
concentrations in the absence of E2 (22, 34), E2 stimulates
assembly of the oligomeric E1 complex.
As a preliminary step in examining the assembly of replication factors
in HPV-16 DNA replication, we performed experiments
to determine the
timing of E1-E2 complex formation. In these experiments,
EE-E1 was
prebound to the EE epitope antibody and protein G beads
prior to the
indicated additions of E2 protein and an inhibitory
peptide. As shown
in Fig.
7, HPV-16 E1-E2 complexes form
rapidly;
the binding reaction was 50% complete after approximately 3 min,
and this binding reached a saturated steady state within 10 min
(lanes 2 to 7). We also sought to utilize the E2N-WP15 peptide
to study
the E1-E2 binding reaction itself by adding the peptide
(50 µM final
concentration) at various times after starting the
E1-E2 binding
reaction. Addition of the peptide at 5 min resulted
in significant
inhibition of the subsequent E1-E2 interaction,
but addition of the
peptide after 10 min had little or no effect
on the E1-E2 interaction.
We interpret this to mean that complex
formation between HPV-16 E1 and
E2 is rapid and that E2N-WP15
inhibits the formation of this complex
but has no effect upon
the E1-E2 complex in the binding reaction after
its formation.
View larger version (39K):
[in this window]
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|
FIG. 7.
Kinetics of interaction between HPV-16 EE-E1 and E2 in
in vitro binding assays. In vitro E1-E2 binding assays were carried out
for the indicated times (lanes 2 to 7), and E1-E2 complexes were
determined by immunoprecipitation with an anti-EE monoclonal antibody,
followed by Western blotting with antiserum to the C terminus of HPV-16
E2. In parallel reactions, the E2N-WP15 peptide (50 µM final
concentration) was added to the E1-E2 binding reaction mixture at the
indicated times (lanes 8 to 12), and the reactions were continued
through termination at 60 min, when binding between EE-E1 and E2 was
determined as described for Fig. 6A.
|
|
| |
DISCUSSION |
The papillomavirus E1 and E2 proteins carry out the critical step
necessary for viral DNA replication: assembly of the replication initiation complex at the origin of replication. This process requires
specific interaction of the E2 amino terminus with the E1 protein.
Papillomavirus replication is otherwise dependent on host cell
polymerases and other components of the host cell replication
machinery. Little is known about the formation and composition of this
replication initiation complex or about the subsequent onset of
elongation and termination. A greater understanding of the regulation
and coordination of papillomavirus DNA replication would enhance our
knowledge of both basic mechanisms of DNA replication in mammalian
cells and the specific manner in which papillomavirus DNA replication
is carried out.
We have mapped a number of viral DNA replication-associated activities
within the HPV-16 E1 protein (45). The E2 interaction domain
of HPV-16 E1 resides within the carboxy-terminal 223 amino acids (amino
acids 421 to 647), a region that also includes the ATP-binding domain.
This region is also capable of interacting with other E1 molecules in
two-hybrid assays (46). Many missense mutations within this
domain of HPV-16 E1 simultaneously disrupt multiple E1 activities,
including transient replication, E1-E1 association, formation of E1-E2
complexes, ATPase activity, and the ability of E1 to interact with
cellular proteins in two-hybrid assays. The pleiotropic effects of many
of these HPV-16 E1 mutations have led us to speculate that such E1
functions are tightly clustered or functionally interdependent
(46).
The initial deletion mapping studies of the BPV type 1 (BPV-1) E2 amino
terminus identified domains necessary for transcriptional activation
and viral DNA replication (24, 42) but failed to discriminate between these two E2 functions. Nonoverlapping deletions within the conserved amino terminus of BPV E2 (e.g., 
1-15,
92-161, and 195-282) disrupted both transcriptional activation
and replication functions. These results implied either that the E2
transcriptional activation and replication functions coexist within an
extended domain of the amino terminus or that such deletion mutations
cause concurrent disruption of multiple functional moieties within the amino terminus.
More recent studies of the HPV-16 E2 and BPV-1 E2 amino-terminal
domains that employed alanine substitution mutagenesis successfully separated the E2 amino-terminal replication and transcriptional activation functions. Mutation of the conserved glutamic acid residue
at position 39 of HPV-16 E2 to alanine specifically abrogates its
ability to stimulate transient papillomavirus replication. This mutant
no longer interacts with E1 but is proficient in transcriptional activation. Alternatively, substitution of alanine for the conserved isoleucine at position 73 of HPV-16 E2 abrogates transcriptional activation but has no effect on E1 interaction and DNA replication (31). Similar directed mutagenesis studies have extended
these results to BPV E2 (1, 6, 7, 10). A genetic screen
based on BPV E2 transcriptional activation in yeast has identified
additional E2 mutants that have lost either or both of these functions
(6, 13).
Such studies have provided convincing evidence that the papillomavirus
E2 amino terminus is composed of at least two separable functional
domains: one domain, defined by isoleucine 73, that is required for
transcriptional activation and a separate domain, defined by the
glutamic acid residue at position 39, that is required for E1
interaction and replication. In this sense, it is notable that the BPV
E2 deletion mutants mentioned above that disrupted both transcriptional
activation and replication overlap neither the E39 nor the I73 domain.
Indeed, even missense mutations scattered throughout the first 200 amino acids of E2 are capable of abrogating either or both functions
(1, 6, 13, 31, 42). Thus, although the transcriptional
activation and replication functions of the E2 amino terminus are
functionally independent, the domains required for these respective
functions do not necessarily retain their functional integrity when
removed from their normal context. Furthermore, in our mapping studies
of HPV-16 E2, we defined a domain encompassing amino acids 1 to 190 as
the smallest domain capable of interaction with HPV-16 E1 in two-hybrid
assays (45). For these reasons, it was uncertain whether a
peptide derived from the E39 region of HPV-16 E2 would be capable of
competing with intact E2 proteins for specific interaction with the
HPV-16 E1 carboxy terminus. In this study, we have demonstrated that a
15-amino-acid peptide derived from the amino terminus of HPV-16 E2 and
centered upon E39 can inhibit both HPV-16 E1-E2 interaction and
papillomavirus replication in vitro. This inhibition is specific, since
an equivalent peptide containing an alanine substitution at position 39 had minimal inhibitory activity in both assays. The ability of this
peptide to act as a competitive inhibitor of in vitro E1-E2 interaction
suggests that a specific E1 recognition activity is contained within a
relatively small part of the E2 amino terminus and that this domain is
able to adopt a conformation suitable for occupancy of the E2 binding
site of E1.
The E1-E2 interaction and peptide inhibition experiments presented here
indicate functional conservation in E1-E2 recognition and interaction
between the E1 and E2 proteins of HPV-11 and HPV-16. The E39 residue of
E2 appears to be a conserved functional component of E1-E2 interaction
among these proteins, since this residue is required for interaction
between HPV-16 E1 and E2, as well as HPV-11 E1 and HPV-16 E2. E39 is
also critical for the replication function of BPV-1 E2 (10).
Our peptide inhibition experiments have reiterated the importance of
this residue in both HPV-16 and HPV-11 E1-E2 interaction and
replication, since E2N-WP15 disrupted interaction between all
combinations of the HPV-16 and HPV-11 E1 and E2 proteins. The lack of
interaction we observed between HPV-16 E1 and HPV-11 E2 in binding
assays is consistent with the inability of these proteins to support
transient replication when transfected into 293 cells (47).
HPV-16 E1 seems to be particularly selective in selection of its E2
partner, compared to HPV-11 E1, which can cooperate functionally in
replication assays with the E2 proteins of HPV-6, HPV-11, HPV-16, or
HPV-18 (9, 38). Experiments using HPV-11 and HPV-16 chimeric
E1 proteins have identified the carboxyl-terminal 284 amino acids of
HPV-16 E1 as the domain that mediates selective binding of HPV-16 E1 to HPV-16 E2 (47). This selectivity has been attributed to
steric interference within this domain of HPV-16 E1 that somehow
prevents HPV-11 E2 binding. Testing the ability of an extended set of
peptides derived from the E39 region of a variety of E2 proteins to
inhibit interaction between other E1 and E2 proteins may provide
greater insight into the exact nature of this selectivity and thus
allow an estimation of the spectrum of HPV subtypes against which an individual peptide or small molecule might be effective.
Demonstration that a peptide could inhibit in vitro E1-E2 interaction
and papillomavirus DNA replication constitutes an essential first step
in assessing the potential efficacy of small compounds as
papillomavirus antiviral agents and validates this approach to the
design of papillomavirus antiviral compounds. We also used the E2N-WP15
peptide to examine early events in the formation of E1-E2 complexes and
initiation of viral DNA replication. Formation of the HPV-16 E1-E2
complex occurred quickly, within 10 min, under the conditions of both
of the in vitro E1-E2 interaction assays. We have not tested the
ability of these peptides to inhibit HPV DNA replication in cell-based
assays; peptides composed of conventional amino acids are not
themselves typically useful as pharmacological reagents, since they are
often unstable or unable to enter cells. We will explore the
incorporation of peptide modifications known to increase peptide
solubility, permeability, and bioavailability (e.g., rhodamine
conjugation or amide bond methylation) to facilitate the use of the
E2N-WP15 peptide or similar peptides to inhibit HPV DNA replication in
vivo. Regardless of whether E2N-WP15 or similar inhibitory peptides are
effective replication inhibitors in vivo, the peptides defined in these
studies may be useful in defining structures that would serve as a
starting point for the design or discovery of clinically useful
antiviral compounds that inhibit papillomavirus replication.
| |
ACKNOWLEDGMENTS |
We thank Louise Chow (University of Alabama, Birmingham) for
providing HPV-11 E1 and E2 reagents. We also thank Susanna Schmid for
critical reading of the manuscript.
This work was sponsored in part by a Sponsored Research Agreement to
Harvard University from the Terumo Corporation of Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-2892. Fax: (617) 432-0727. E-mail:
jbenson{at}warren.med.harvard.edu.
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Abstract
Introduction
