From the Department of Pathology, Stanford University
School of Medicine, Stanford, California 94305-5324 and the
¶ Department of Biochemistry and Molecular Genetics, University of
Alabama at Birmingham, School of Medicine and Dentistry,
Birmingham, Alabama 35294-0005
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ABSTRACT |
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Papovaviruses are valuable models for the study
of DNA replication in higher eukaryotic organisms, as they depend on
host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase Papillomaviruses are members of the small DNA tumor virus family.
They cause papillomas in a wide variety of hosts and certain high risk
human papillomavirus types are strongly linked to the development of
cervical or penile cancer in humans (1). The mode of viral replication
is closely coupled to the differentiation status of the infected
squamous epithelium (for review see Ref. 2). In the basal and parabasal
cells of the squamous epithelium, the virus is maintained as a low copy
number extra-chromosomal episome and undergoes regulated DNA
replication modulated by both viral and host proteins. As cells undergo
progressive differentiation, vegetative viral replication is triggered,
"late" viral genes are expressed, and progeny virions are produced
in a fraction of the terminally differentiated cells in papillomas or
condylomas. The latent stage of papillomaviral replication provides an
ideal system for the study of regulated eukaryotic DNA replication.
We and others (3, 4) have previously reported a cell-free replication
system for bovine papillomavirus type 1 (BPV-1)1 (3) and human
papillomavirus type 11 (HPV-11) (4). Papillomaviral replication
requires the viral proteins E1 and E2 (5, 6), as well as the full
complement of host replication proteins that have previously been
identified in SV40 in vitro replication, including DNA
polymerases The papillomaviral E1 protein is a functional homolog of SV40 large T
antigen, with origin binding activity as well as ATPase and helicase
activity (9-15). It associates as a trimer or a hexamer on its cognate
E1-binding site in the viral origin (ori) with relatively low affinity
and low sequence specificity (3, 4, 16-23). As a result, high
concentrations of E1 can bind DNA nonspecifically and initiate
ori-independent replication at low efficiency in vitro (3,
4). It has been proposed that the replication competent form of BPV-1
E1 is a multimeric complex of 10-12 E1 molecules (20). Recently, the
HPV-11 E1 protein has been shown to bind to the human chaperone protein
Hsp40, and in its presence, a dihexameric E1 complex forms on the ori
(23), mirroring the structure of SV40 T antigen on the SV40
ori (24) as well as other known Escherichia coli helicases
(25). In addition to its role in initiation, HPV-11 E1 is also required
during elongation in vitro, suggesting its helicase activity
may be critical at the replicating forks (26).
E2 is a viral transcriptional transactivator that is essential for
viral DNA replication in vivo (27, 28). It binds as a dimer
with high affinity to its conserved binding sites (E2-binding site) in
the viral genome, including several sites in the viral origin of
replication (29-34). It forms a complex with E1 (35), and this E1·E2
complex has increased sequence specificity for binding to the E1 and E2
cognate sites in the viral origin (36, 37). Thus one of the critical
functions of E2 in viral DNA replication is to interact with and
recruit E1 to the viral origin of replication by virtue of the stronger
DNA binding affinity and specificity for E2 (20, 35-41). Therefore,
the addition of E2 protein to a cell-free replication assay enhances
ori-dependent and suppresses ori-independent replication.
Based on these data the following model of E2/E1 interaction during
initiation of bovine papillomaviral DNA replication has been proposed
(20). Once the first molecule of E1 is loaded onto the origin by E2, E2
is then released from the origin, allowing E1 to multimerize into a
replication-competent form. However, more recent studies in HPV
replication have suggested that the role played by E2 extends beyond
the recruitment of E1; E2 is necessary for the formation of the entire
pre-initiation complex, although it is dispensable during elongation
(26). This interaction between E2 and E1 is extremely important for HPV
ori replication, as only the E2-binding site is absolutely essential
for origin-specific viral replication, whereas the E1-binding site is
dispensable for in vivo or in vitro viral DNA
replication (4, 31, 42, 43). In addition, certain heterologous
combinations of E1 and E2 either do not support replication, or do so
poorly, whereas the homologous pairs of viral proteins always support replication effectively (28, 40, 41), suggesting type-specific interactions between E1 and E2.
DNA pol In this report, we describe the physical and functional interactions
between HPV-11 E1 and the catalytic subunit, p180, as well as the p70
subunit of human DNA pol Plasmids and Proteins--
The HPV-11 ori plasmid pUC7874-99 has
been described (4, 31). Native HPV-11 E2 protein and HPV-11 E1 protein
tagged at the amino terminus with the Glu-rich (EE) epitope from the
polyomavirus middle T antigen (57) were purified from recombinant
baculovirus-infected Sf9 cells as described (4). p180 was
purified from baculovirus-infected Sf9 cells using the
monoclonal antibody SJK237-71 as described previously (58), except for
the following modifications to accommodate the cell-free replication
assay. After elution from the monoclonal antibody column with 3.2 M MgCl2, the protein was dialyzed for two
changes against 50 mM Tris-HCl (pH 8.0), 10 mM
EDTA, 20% glycerol, and 1 mM Sucrose Gradients--
Five µg of purified proteins were
layered onto a 4-ml 10-30% (v/v) sucrose gradient in 200 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.1% Tween
and centrifuged for 6 h at 48,000 rpm at 15 °C in a Beckman
SW60 rotor. Fractions (15 drops) were collected from the bottom of the
tube using a 21-gauge needle. Ten µl of each fraction were combined
with an equal amount of 2× SDS loading buffer and run on a 9%
SDS-polyacrylamide gel. The sucrose density of each fraction was
measured. When indicated, equimolar amounts of E1 protein were
incubated together on ice with p70 for 20 min prior to loading on the
gradient. Western analysis was performed using either anti-EE
monoclonal antibody against E1, or anti-p70 polyclonal antibody to
determine the elution profile for each protein and compared with both
internal standards (aldolase, 158 kDa) as well as to external standards
run in a parallel gradient.
Gel Filtration--
When indicated, equimolar amounts of
proteins were incubated together on ice for 20 min prior to loading
onto the column. For analysis of the interaction between E1 and p70, 10 µg of each protein was applied to a Bio-Rad SE1000/17 column at a
flow rate of 0.25 ml/min in 10% glycerol, 200 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 0.1% Tween. Two hundred-µl
fractions were collected, and 15 µl of each fraction was analyzed by
10% SDS-polyacrylamide gel electrophoresis followed by Western
blotting with either anti-EE monoclonal antibody for detection of E1 or
anti-p70 polyclonal antibody. Gel filtration of p70 and E2 was
performed using the Amersham Pharmacia Biotech SMART® system. Two µg
of each protein were preincubated for 20 min on ice prior to injection
on a Superose 6 PC 3.2/30 column at a flow rate of 0.05 ml/min. Column
was eluted at room temperature in the buffer system described above.
Fifty-µl fractions were collected, and protein profiles were analyzed
via Western blot using anti-p70 polyclonal antibody and anti-E2
polyclonal antibody.
Enzyme-linked Immunosorbent Assay (ELISA)--
ELISA assays were
performed as described (49), with the following modifications.
One-half-µg of the primary protein was used to coat the 96-well
plate. After blocking with 3% bovine serum albumin, increasing amounts
of the second protein were added, and incubation proceeded for 1 h. The plate was washed thoroughly 5 times with phosphate-buffered
saline and incubated with a monoclonal antibody specific for the second
protein for 1 h at room temperature. Incubation with a horseradish
peroxidase-conjugated goat anti-mouse antibody and development
proceeded as described previously (50). Five overlapping p180 fragments
named 1 Construction of Myc-tagged p70 and in Vitro Transcription and
Translation of HPV-11 E1 and E2, and p70--
Oligonucleotides were
constructed to allow polymerase chain reaction amplification of the Myc
epitope from a previously existing construct. The primer for
amplification of the 5' side of the tag was constructed to contain an
NheI site (5' CGC CGC TAG CCA TGC TGA GGA GCA A 3'), and the
primer for amplification of the 3' side contains a BamHI
site (5' GCC GGC TAG CGG ATC CCA TAT GTA AGT CCT C 3'). The 113-base
pair fragment resulting from this amplification contains two sequential
Myc epitopes (EQKLISEED). This fragment was then cloned into the
NheI-BamHI sites of pET11a (Novagen). Subsequent
digestion with BamHI allowed the insertion of the coding
sequence of the human p70 gene after liberation from pQE9-hup70 with
BamHI digestion. Orientation and appropriate coding frame
were confirmed through direct sequence analysis. Construction of
pET-EE-E1 and pET-BB-E2 will be described
elsewhere.2 BB epitope was
derived from CMVpp65 (a phosphoprotein encoded by UL83 kindly suggested
to us by Dr. William Britt).
In vitro transcription and translation of pET-EE-E1 and
pET-BB-E2 and pET-Myc-p70 were carried out using the
Transcription and Translation-coupled Reticulocyte Lysate System from
Promega. Due to extreme inefficiency of labeling human p70 with either [35S]methionine or [35S]cysteine, this
protein was labeled using L-[4,5-3H]leucine
(156 Ci/mmol, 1 mCi/ml, Amersham Pharmacia Biotech). All other proteins
were labeled using L-[35S]methionine (1000 Ci/mmol, 15 mCi/ml, Amersham Pharmacia Biotech).
Co-immunoprecipitation--
Sf9 insect cells were grown
to 80% confluence in a T-150 flask. They were then infected at 10 multiplicity of infection with baculovirus expressing either p180
(AcHDP
Co-immunoprecipitation of in vitro translated proteins was
performed as follows. Defined amounts of reticulocyte lysate containing the labeled protein of interest were mixed together in 0.5 ml of 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 10%
glycerol, 2% bovine serum albumin and allowed to rock gently at
4 °C for 30 min, after which 5 µg of antibody (either monoclonal
anti-EE or polyclonal anti-p70) that had been pre-cleared by
centrifugation in a TLS-55 at 100,000 × g for 30 min
was added to the mixture. Incubation continued with rocking for 1 h, after which 20 µl of protein A-agarose (Sigma) was added and
rocking continued overnight at 4 °C. Beads were washed 4 times with
20 mM Tris-HCl (pH 7.5), 200 mM KCl, 5%
glycerol, 0.2% Triton X-100, resuspended in 2× SDS loading buffer,
and run on a 10% denaturing polyacrylamide gel. Gels were fixed,
soaked in "Amplify" (Amersham Pharmacia Biotech), and dried before
being exposed to film.
Cell Extracts and Cell-free Replication Assays--
Suspension
293 cells were maintained in Joklik's medium with 5% calf serum. The
preparation of whole cell extracts was modified from previously
published protocols (61, 62) as described (4). The conditions for
HPV-11 cell-free replication assays have been described (4, 26) with
minor modifications. Briefly, 40 ng of ori-plasmid (pUC7874-99), 100 µg of protein from 293 cell extracts, viral proteins (12 ng of E1 and
8 ng of E2, unless otherwise specified), and all four ribonucleoside
and four deoxyribonucleoside triphosphates in the replication buffer
were incubated for 1 h at 37 °C before 2.5 µCi of
[ HPV-11 E1 Interacts with p180 Subunit of Human DNA Polymerase
Since it is difficult to quantitatively control the relative levels of
protein expression by these recombinant baculovirus in insect cells, we
further tested the interaction by enzyme-linked immunosorbent assay
(ELISA) with immunopurified p180 from AcHDP
The ELISA results led us to investigate further the possible
interaction of HPV-11 E1 and p180 by fast protein liquid chromatography over a Superdex 200 column. The DNA polymerase activity of p180 alone
eluted at a molecular mass of approximately 180 kDa, with a
reproducible second peak at approximately 210 kDa. Upon preincubation of p180 with HPV-11 E1, which migrates as an 82-kDa protein in SDS-polyacrylamide gel electrophoresis (28), a portion of the polymerase activity shifted to an elution position corresponding to a
molecular mass of 340-480 kDa with the peak at 340 kDa (data not
shown). The shift of the polymerase activity from 180 kDa to a higher
molecular mass after preincubation with E1 again suggests that there is
an interaction between HPV-11 E1 and the p180 subunit of human pol
The elution profile of the p180·E1 protein complex suggests that the
interaction between E1 and p180 is not stoichiometric and perhaps more
than one molecule of E1 interacts with one molecule of p180, in
agreement with previous observations that E1 can exist as a multimer in
solution (14, 20, 21, 23). Finding that only a portion of the p180
associated with E1 during gel filtration and a small percentage of the
total polymerase Purified p180 Inhibits the HPV-11 Cell-free Replication
Reaction--
We have previously shown that when increasing amounts of
purified p180 were added to the cell-free SV40 replication reaction, a
proportional inhibition of the replication was observed (50). Therefore
we hypothesized that when exogenously added p180 was included in the
HPV-11 cell-free replication reaction, inhibition of replication might
also be observed, through the sequestration of either E1 or other
replication proteins in the cellular extracts. To test these
possibilities, we conducted an HPV-11 cell-free ori-dependent replication reaction in the presence of
excess p180. Inhibition was observed when 250 ng of purified p180 was
added to a standard reaction containing 12 ng of E1 and 8 ng of E2
(data not shown). To determine whether inhibition is due to a
competition for cellular replication factor(s) or to interference in
the interaction between E1 and E2, replication assays were performed in
the presence or absence of 8 ng of E2 with 20 or 40 ng of E1, as
E2-independent replication only takes place at elevated E1 protein
levels (4, 10). Replication produces slow-migrating replication
intermediates and fast-migrating form I and form II circular molecules.
As shown in Fig. 2, there were more
replication products in the presence of the E2 protein than in its
absence (compare lane 1 with lane 5 and
lane 3 with lane 7). Upon addition of excess
p180, a reduction of both the E2-dependent and
E2-independent replication levels was observed relative to the control
reactions without exogenously added p180 (lanes 2, 4, and
8). This inhibition was not completely rescued by the
addition of excess cellular extracts (data not shown), suggesting the
effect is mediated at least in part through the viral proteins. These
data indicate that p180 may be binding to and sequestering HPV-11 E1
away from the viral origin of replication, in an interaction similar to
one that occurs between p180 and SV40 T antigen.
The 70-kDa Subunit of pol
To verify the ELISA results, we further analyzed the interaction
between E1 and p70 by chromatography over the SE1000/17 Bio-Rad column.
Under the conditions used, E1 alone eluted at a molecular mass >669
kDa, suggesting that E1 exists as a large homocomplex (data not shown).
Upon preincubation of equimolar amounts of E1 and p70, most of the p70
eluted as a 140-kDa complex indicative of a dimer of p70,
similar to that seen with p70 alone and
consistent with our previous observations3 (Fig.
3, A and B).
However, there was a reproducible and distinct fraction of p70 which
co-eluted with the multimeric E1 (Fig. 3B). To verify the
presence of E1 in the p70-containing fractions, the immunoblot in Fig.
3B was stripped and reprobed with anti-EE antibody (Fig.
3C). This profile confirms the interaction between these two
proteins.
The interaction was further substantiated by sedimentation analysis in
10-30% sucrose density gradient. E1 alone sedimented at a range
between 300 kDa to a very large complex of >1000 kDa, indicating again
that under these conditions HPV-11 E1 exists as multimers in solution
(Fig. 4A). In the absence of
E1, p70 sedimented as a dimer and was not detectable in the higher
sucrose density fractions even upon overexposure of the Western blot
(Fig. 4B and data not shown). Upon preincubation of
equimolar amount of E1 with p70 prior to sedimentation in the sucrose
gradient, it was again possible to detect a large E1·p70 complex,
although the majority of p70 remained unassociated and sedimented as a dimer (Fig. 4C). Since the proteins were incubated in
equimolar amounts but most of the p70 remains unassociated, the
relative stoichiometry of E1 to p70 seems as before to be much greater than 1:1. One possible explanation for this result is that a single molecule of p70 interacts with a multimer of E1. Similar
results were seen when the two proteins were analyzed in 20-40%
glycerol gradients (data not shown). These results indicate that the
70-kDa subunit of pol Purified p70 Inhibits Cell-free HPV-11 Ori-dependent
Replication--
To evaluate whether the physical interaction between
HPV-11 E1 and p70 is functionally significant in HPV-11
ori-dependent replication, we tested the effect of adding
purified p70 to a standard cell-free replication reaction. Increasing
amounts of purified p70 inhibited replication in a
dose-dependent manner (Fig.
5, lanes 5-7). In contrast,
addition of identical amounts of the recombinant p58·p49 primase
subunit complex had little or no effect on replication, although it was
expressed and purified from E. coli in an identical fashion
as p70 (Fig. 5, lanes 2-4). This suggests that the primase
subunits of DNA polymerase
To investigate possible mechanisms of this inhibition, we conducted
replication assays with a constant amount of E2 (8 ng) and increasing
amounts of E1 (Fig. 6A) or,
conversely, a constant amount of E1 (12 ng) and increasing amounts of
E2 (Fig. 6B). In the absence of p70, increasing amounts of
replication products were observed when the E1 concentration in the
reaction was increased (Fig. 6A, lanes 1, 3, 5, 7, and 9), in agreement with our previous results
(4). However, with the addition of p70, a similar reduction in
replication products was observed (Fig. 6A, lanes 2, 4, 6, 8, and 10). In reactions with a constant 12 ng of
E1, it appears that increasing, saturating, levels of E2 were able to reduce slightly the inhibition by p70 (Fig. 6B, lanes 2, 4,
and 6).
p70 Does Not Interact with HPV-11 E2--
One possible explanation
of the above result is that p70 competes with E2 for binding to E1. If
E2 is unable to bind E1 and bring it to the origin, replication would
be inhibited. Increasing amounts of E1 should increase both the amount
of E1 available for p70 binding as well as for binding to E2, resulting
perhaps in a minimal rescue of replication. An alternative explanation for the slight rescue by E2 is that p70 may also bind to E2. Thus, at
higher concentrations of E2, more E2 is available to bind to E1 and
recruit it to the origin, resulting in a decrease in inhibition.
To distinguish between these two possibilities, we analyzed the ability
of p70 to bind E2 by gel filtration on a Superose 6 column. As shown in
Fig. 7, both the 43-kDa E2 protein and
the p70 subunit eluted as homodimers (Fig. 7, A and
C). Upon preincubation of equimolar amounts of E2 and p70,
neither E2 nor p70 shifted to an elution volume of a larger complex
(Fig. 7, B and D). This result rules out the
possibility that p70 is sequestering E2 away from the productive
reaction. Together with the data in Fig. 6B, it suggests
that p70 may be competing with E2 for binding to E1 and that the
inhibition by p70 may be due to this competition.
p70 Stimulates the E2-independent Replication Activity of
E1--
If the above hypothesis is correct, no inhibition by p70
should be detected under conditions allowing E2-independent
replication. However, if the inhibitory effect of p70 is due to
interference by p70 in an interaction between E1 and the pol
Elevated amounts of E1 (required in the absence of E2) were used in
reactions with either an ori-containing plasmid (pUC7874-99) (Fig.
8A, lanes 1-8) or pUC-19
(lanes 9-12). In the control reaction with 8 ng of E2,
replication was inhibited by the addition of 250 ng of purified p70
(Fig. 8A, compare lanes 1 and 2, and lanes 3 and 4), consistent with the above results.
Interestingly, in the absence of E2, the addition of p70 stimulated the
replication of both the ori-containing template and pUC-19 in an
identical fashion (Fig. 8A, compare lanes 5-12).
Thus, in the absence of E2 but in the presence of high concentrations
of E1, p70 stimulates replication in both ori-dependent and
ori-independent replication.
To gain further insight into the opposing effects of p70 on HPV-11
replication under different conditions, we compared the relative
efficiency of E2 and p70 to stimulate ori-specific replication at
levels of E1 that replication by E1 alone is very inefficient. In a
reaction containing 10 ng of E1, no replication was observed in the
absence of E2 (Fig. 8B, lane 1), but extensive replication took place upon the addition of 8, 16, and 32 ng of E2 (Fig. 8B, lanes 9-11). In contrast, the addition of up to 250 ng of p70 failed to stimulate replication at 10 ng of E1 (Fig. 8B,
compare lanes 2-4 to lane 1). When 20 ng of E1
protein were used in the reactions, a low level of E2-independent
replication was observed (Fig. 8B, lane 5). At this
concentration of E1, the addition of p70 also stimulated the
replication activity (Fig. 8B, lanes 6-8) but not nearly as
efficiently as the E2 protein in the presence of 10 ng of E1 (Fig. 8,
lane 9). Interestingly, the level of stimulation decreases
at the highest amount of p70 added (Fig. 8B, lane 8), suggesting that the stoichiometry between the two proteins may be
important. This point will be addressed in greater detail below. These
results demonstrate that the stimulation by p70 is rather weak relative
to that by E2.
p70 Competes with E2 to Bind E1--
In order to evaluate better
the relative interactions between p70, E1, and E2, we utilized
radioactively labeled, in vitro transcribed and translated
proteins. p70 was constructed with a Myc tag to avoid the
cross-reactivity between the anti-EE antibody and the histidine tag on
the recombinant p70. Due to internal initiation sites within the
Myc tag, p70 appears as a triplet, whereas E1 is translated
as a doublet (Fig. 9, A and
B). Immunoprecipitation with the anti-EE antibody
precipitates E1 and the associated p70 protein (Fig. 9B, lanes
3-5). Paradoxically, more E1 is immunoprecipitated by the anti-EE
antibody in the presence of p70 than in its absence (Fig.
9B, compare lanes 2, 3, and 4).
Furthermore, this effect is dependent on a particular stoichiometry of
the two proteins, as the higher amounts of p70 caused a reproducible
decrease in the amount of E1 immunoprecipitated (Fig. 9B,
compare lanes 4 and 5). Perhaps the multimeric
conformation of E1 is stabilized by the inclusion of p70 in the
reaction, allowing each antibody molecule to precipitate multiple
molecules of E1 protein. Alternatively, p70 may induce a conformational
change in E1 that makes the EE tag more accessible. This effect may
help in part to explain the stimulation of E2-independent replication
by p70 seen in Fig. 8. The reduction in precipitable E1 at a high ratio
of p70 to E1 seen in Fig. 9B (lane 5) appears to parallel
the reduced stimulatory effect by p70 on E2-independent replication
described in Fig. 8B (lane 8) and may emphasize
the importance of the dual role played by p70 in viral replication
initiation in our assays. However, the nature of the in
vitro transcription and translation reactions and the fact that
p70 and E1 are radiolabeled with different isotopes preclude reliable
quantification of the relative amounts of E1 and p70 in this assay.
To assess the potential competition between p70 and E2 for E1 binding,
we performed co-immunoprecipitation of E2 in the presence of E1 using
the anti-EE antibody. Increasing amounts of unlabeled His-tagged p70
were preincubated with in vitro translated labeled E1
protein prior to the addition of E2. Interaction between E1 and E2 is
decreased at 20 ng of p70 (Fig. 9C, compare lane
1 to lane 2). This reduction suggests that p70 competes
with E2 for binding to E1. Again, at high amounts of p70 (100 and 400 ng), lower levels of E1 were precipitated, similar to that observed in
Fig. 9B, lane 5.
Results of these experiments indicate the following: (i) p70 inhibits
an HPV-11 cell-free replication reaction that contains optimal levels
of E1 and E2 and the ori-containing template; (ii) in the absence of
E2, p70 stimulates, rather than inhibits, the replication activity of
E1, and this stimulation is not dependent on the presence of an HPV-11
origin; (iii) stimulation by p70 of the replication reaction requires
higher levels of E1 than stimulation by E2, but the level of
stimulation achieved is much lower than that by E2; (iv) p70 does not
physically interact with E2; and (v) excess p70 can interfere with the
interaction between E1 and E2. Collectively, these results strongly
suggest that the apparent inhibitory effect of p70 in replication
reactions containing both E1 and E2 proteins may be due to competition
between p70 and E2 for binding to E1.
In this study, we have shown by ELISA, gel filtration, sucrose
density gradient sedimentation, and co-immunoprecipitation that HPV-11
E1 can exist as a multimeric complex in solution and can physically
interact with two DNA pol E1 as well as other viral helicases has been postulated to function in
large oligomeric complexes (for review see Ref. 64). Electron
microscopy revealed that HPV-11 E1 exists in solution as particles of
different sizes. But when bound to ori, only particles of hexameric
sizes were observed. However, in the presence of Hsp40, the majority of
the DNA-bound E1 is a dihexamer (23). In this study, we observed that
HPV-11 E1 exists as a large multimer in solution (Figs. 3 and 4), in
excellent agreement with the previous observations. Thus, in the
absence of E2 and DNA, E1 may exist as a mixture of monomeric and
oligomeric complexes. This equilibrium could be affected by many
factors, including protein concentration, the presence of DNA, or the
interacting viral or host proteins. Perhaps the high concentrations of
E1 protein, trace amounts of DNA in our E1 preparations, or buffer
conditions used in the studies favor the formation of oligomeric
complexes. Collectively, our results suggest that a dynamic interplay
occurs between E1, E2, DNA, and the DNA polymerase The interactions between E1 and the DNA pol p180 and p70 Exhibit Differential Effects in HPV-11 Cell-free
Replication--
We have shown in this study that exogenously added
p180 inhibits cell-free HPV-11 viral replication. This inhibition is
similar to that seen in the SV40 in vitro replication
reaction in the presence of excess p180 (50) and is independent of E2
function (Fig. 2). Increasing amounts of cellular extracts were not
able to completely rescue replication, indicating that E1 is limiting. The simplest explanation for the inhibition is that p180 sequesters the
E1 oligomers, shifting the equilibrium between monomeric E1 and
oligomeric E1, so that there is less E1 available for the formation of
a productive pre-replication complex on the ori.
We observed two opposing effects when exogenously added p70 was
included in the HPV-11 cell-free replication reaction. In the presence
of both E1 and E2, p70 inhibited replication in a manner similar to the
inhibition by p180 (compare Fig. 2 with Fig. 5). Increasing amounts of
E1 could not alleviate this inhibition (Fig. 6A). In
contrast, increasing levels of E2 in the replication reaction may
partially restore replication activity (Fig. 6B). One
explanation for this effect supported by our experimental data is that
p70 and E2 may compete for binding to the E1 protein (Fig.
9C), either through an overlapping recognition site on E1 or
through a conformational change that precludes simultaneous binding by
E2 and p70. During the preparation of this manuscript, Masterson et al. (66) reported an
interaction of HPV-16 E1 with only the p70 subunit of DNA polymerase
Stimulation of the cell-free replication by p70 in the absence of E2 at
elevated levels of E1 (Fig. 8) suggests that the mechanism of p70
stimulation of E2- and ori-independent replication is different from
that employed by E2. First, E2 stimulates ori-specific replication and
represses ori-independent replication (4). Second, E2 stimulation is
much stronger than that achieved by p70 (Fig. 8B). Third,
the stimulatory effect of p70 is observed only at a concentration of E1
where E2-independent replication occurs (Fig. 8). In the presence of
E2, the two proteins compete for binding to E1, and the presence of p70
at these low levels of E1 inhibits replication activity. However, the
exact cause of the stimulation of replication by p70 remains to be investigated.
A Proposed Model for Replication Initiation--
The possibilities
presented above for stimulation and inhibition of replication activity
by p70 are not mutually exclusive. Together with previous studies of
both BPV-1 and HPV E1 and E2 interactions we suggest a model of
sequential associations between viral and host proteins during normal
viral origin replication.
We propose that E1 exists in solution in a dynamic equilibrium of
monomers and multimeric complexes. The high affinity of E2 for the
E2-binding site allows it to recruit E1 to the ori at relatively low E1
concentrations. Interactions between E1 and the pol
In the absence of E2, elevated levels of E1 can multimerize on DNA
nonspecifically and initiate replication, albeit in a very inefficient
manner. We propose that the exogenous p70 forms nonproductive complexes
with E1 multimers in solution. But the small amounts of E1 that
successfully assemble on the DNA are stabilized by the exogenous p70,
allowing subsequent productive interactions with the DNA pol
Overall, the elucidation of the multiple interactions among viral and
host proteins may aid in the understanding of the initiation of
chromosomal replication as well. Given the important role played by the
p70 subunit of human DNA polymerase /primase (pol
/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol
/primase interact with HPV-11 E1. The interactions of E1 with p180
and p70 are functionally different in cell-free replication of an
HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both
E2-dependent and E2-independent cell-free replication of
HPV-11, whereas p70 inhibits E2-dependent but stimulates
E2-independent replication. Our experiments indicate that p70 does not
physically interact with E2 and suggest that it may compete with E2 for
binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.
INTRODUCTION
Top
Abstract
Introduction
References
and
(7, 8). It is therefore probable that physical
interactions between the host initiation enzymes and the papillomaviral
initiation proteins E1 or E2 occur during initiation of viral DNA replication.
/primase is the principal enzyme for the initiation of DNA
synthesis and is required for both leading and lagging strand DNA
replication (44-46). Pol
/primase is essential for the initiation
of cell-free replication of SV40 (50), BPV-1 (3, 51), and HPV-11 (4).
DNA pol
/primase has also been shown to be the major host factor
responsible for species-specific replication of SV40 and polyomaviral
DNA in vitro (54-56). These findings not only indicate the
critical function of DNA pol
/primase in the initiation of viral DNA
replication but also reveal the important role played by this enzyme
complex in the timing and control of DNA replication in eukaryotic
cells. pol
/primase consists of four subunits as follows: a
polymerase catalytic subunit of 180 kDa (p180), two smaller subunits of
58 and 49 kDa (p58 and p49) containing the primase activity, and a
70-kDa subunit (p70 or B-subunit) which is a cell
cycle-dependent phosphoprotein (47, 48) with no known
catalytic function (44, 45). The catalytic subunit p180 has been shown
to interact physically with both the SV40 T antigen (49, 50) and with
the BPV-1 E1 protein (51, 52), and the interaction between p180 and T
antigen is required for SV40 viral DNA replication in vitro
(50). The p70 subunit has also been shown to interact physically with
SV40 T antigen in vitro, although the functional
significance of this interaction is not understood (53). DNA polymerase
activity is essential for the initiation of cell-free replication
of SV40 (50), BPV-1 (3, 51), and HPV-11 (4). It is probable that
analogous interactions between DNA pol
/primase and the host
counterparts of the viral initiators may also play a critical role in
the control of chromosomal DNA replication.
/primase. HPV-11 is the causative agent of
genital condylomata and laryngeal papillomas and shares extensive
sequence homology with the high risk HPV types. Thus, investigation of
the homo-species interactions between the human pol
/primase
subunits and the HPV E1 and E2 proteins will both advance our knowledge
of the molecular mechanisms of the initiation process of DNA
replication in mammalian cells and also address potential therapeutic
approaches to this family of prevalent human pathogens. By using
biochemical and functional assays, we found that interactions between
the E1 protein with both p180 and p70 are critical for the initiation
of viral DNA replication. In addition, we showed that p70 may compete
with E2 for binding to E1, suggesting a model of sequential
interaction between the proteins during origin-dependent
initiation of HPV-11 replication.
EXPERIMENTAL PROCEDURES
-mercaptoethanol, followed
by dialysis for two changes against 50 mM Tris-HCl (pH
8.0), 1 mM EDTA, 20% glycerol, and 1 mM
-mercaptoethanol. A final concentration step was performed by
coating the dialysis tubing with dry G-50 Sephadex. The cloning of the
70-kDa subunit of DNA pol
/primase and the production in rabbit of
anti-p70 polyclonal antibody will be described elsewhere. Five
hundred-ml cultures of M15[pREP4] (Qiagen Inc.) transformed with an
amino-terminal histidine-tagged p70 cDNA cloned into the pQE9
plasmid (Qiagen Inc.) were grown in 2× YT broth to
A595 = 0.7-0.9, induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside and grown for
2.5 h at 20 °C. Purification of overexpressed protein was
carried out as described previously (59). One-ml fractions were
collected, and protein-containing fractions were immediately dialyzed
against 10% glycerol, 50 mM Tris-HCl (pH 8.0), 20 mM KCl, 1 mM EDTA, and 1 mM
-mercaptoethanol for several changes. Protein was then concentrated
via dialysis against 50% glycerol, 50 mM Tris-HCl (pH
8.0), 20 mM KCl, 1 mM EDTA, and 1 mM
-mercaptoethanol.
, 2
, 3
, 4
, and 5
constructed as GST fusion
proteins were expressed and purified as described (50).
) (58) or HPV-11 EE-E1 (4) or co-infected with both viruses.
After 48 h, the cells were harvested, washed once in
phosphate-buffered saline, resuspended in 20% ethylene glycol, 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM of EDTA,
-mercaptoethanol, phenylmethylsulfonyl
fluoride, and sodium bisulfite, and lysed by sonication with a Branson
Sonifier 450 with a 10-s pulse at 50% power three times. Lysates were
cleared by centrifugation at 18,000 × g and incubated
with increasing amounts of CNBr-Sepharose coupled to the anti-EE
monoclonal antibody for 1 h at 4 °C with gentle rocking. Beads
were washed three times with 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5% glycerol, 0.04% Triton X-100 and then assayed
for polymerase activity (60) with the following modifications. To the
washed beads, 80 µl of assay mix was added, and the beads were gently
rocked at 37 °C for 30 min. The reactions were then terminated,
precipitated by trichloroacetic acid, and the acid-insoluble products
were quantified as described previously (60).
-32P]dCTP was added. The delay in
[
-32P]dCTP addition reduces the amount of noise from
repair synthesis while having little effect on the signal of
replication (4). As indicated, the p180, p70, or primase subunits were
added prior to the preincubation. After another 1 h incubation,
reactions were stopped, and replication products were purified and
analyzed in 0.8% agarose gels. The gel was exposed to Hyper-Film
(Amersham Pharmacia Biotech) and quantified by PhosphorImager
(Molecular Dynamics).
RESULTS
--
We have previously shown that the catalytic subunit of human
DNA pol
, p180, physically interacts with BPV-1 E1 and that pol
/primase activity is required for the cell-free replication of BPV-1
and HPV-11 (3, 4, 51, 52). To test whether HPV-11 E1 interacts with
p180, we initially infected Sf9 cells with either the
recombinant baculovirus AcHDP
expressing the human DNA pol
p180
subunit (58) or a recombinant baculovirus expressing the HPV-11 E1
protein tagged with a Glu-rich epitope at the amino terminus called
EE-E1 (4, 57), or we performed a co-infection with both viruses. The
lysates were then subjected to immunoprecipitation with anti-EE
antibody-coupled Sepharose. Polymerase assays of the immunoprecipitates
showed that a small but increasing proportion of the total polymerase
activity in the lysate precipitated with increasing amounts of
antibody-coupled Sepharose from cell lysates co-infected with AcHDP
and HPV-11 E1 expressing virus but not from either of the
singly-infected cell lysates (Fig.
1A). The anti-EE
immunoprecipitates from the singly- and co-infected insect cells were
also analyzed on an SDS gel and stained with Coomassie Blue (Fig.
1B). An approximately 200-kDa insect cell protein was
precipitated from all lysates, including uninfected control (lane
1). E1 protein was immunoprecipitated by anti-EE antibody in E1
recombinant baculovirus singly-infected cell lysates (lane
2). A 180-kDa protein was observed in the E1-recombinant virus and
AcHDP
co-infected cell lysates (lane 4) and was not detected in the AcHDP
singly-infected lysates (lane 3).
To ensure that the 180-kDa protein is indeed human pol
p180, the
AcHDP
singly-infected cell lysate was immunoprecipitated with the
monoclonal anti-pol
antibody SJK237. A protein of 180 kDa was
immunoprecipitated (lane 5). In addition, the identities of
the p180 and HPV-11 E1 proteins in the immunoprecipitates were
confirmed through Western blotting (data not shown). Together these
results suggest that there is an interaction between the p180 subunit
of human pol
/primase and E1 under physiological conditions in
insect cells.
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Fig. 1.
p180 subunit of the human DNA polymerase
physically interacts with HPV-11 E1 protein.
A, co-immunoprecipitation with anti-EE antibody of p180 and
EE-tagged HPV-11 E1 from insect cell lysates that were singly or
co-infected with recombinant baculovirus AcHDP
expressing p180 or
recombinant baculovirus expressing EE-tagged HPV-11 E1 protein. DNA
polymerase
activity in the immunoprecipitates was measured as
described under "Experimental Procedures." B, SDS gel
analysis of immunoprecipitates from A. Shown are the
Coomassie stains of immunoprecipitates of insect cell lysates by
anti-EE antibody. Lane 1, from uninfected cells; lane
2, from cells infected by HPV-11 EE-tagged E1 HPV-11 recombinant
virus; lane 3, from AcHDP
-infected cells; lane
4, from insect cells co-infected with AcHDP
and HPV-11
EE-tagged E1 virus; lane 5, immunoprecipitate by monoclonal
anti-polymerase
SJK237-71 antibody from AcHDP
-infected insect
cells. p180 and E1 are marked with a dot. C,
confirmation of E1 and p180 complex formation by ELISA. Half of a µg
of the p180 subunit, five p180 GST fusion peptides, or p70 subunit were
immobilized in the wells of an ELISA plate and incubated with the
indicated amounts of purified EE-tagged E1. After washing, the bound
EE-E1 proteins were detected with monoclonal anti-EE antibody with a
horseradish peroxidase-conjugated goat anti-mouse secondary antibody as
described under "Experimental Procedures." Shown are the increasing
chromogenic signals from which background levels of each amount of E1
incubated in the absence of respective proteins have been subtracted.
The chromogenic signals of each protein were normalized as percentage
of the maximum signal (A405 = 0.18).
-infected insect cells
(63) and immunopurified EE-tagged HPV-11 E1 (4) as described under
"Experimental Procedures." A fixed amount (0.5 µg) of the highly
purified p180 subunit was immobilized in the wells of an ELISA plate
and incubated with increasing amounts of the purified E1 protein.
Complex formation was detected using the monoclonal anti-EE antibody
and a horseradish peroxidase-coupled goat anti-mouse secondary
antibody. An increasing chromogenic signal was detected with increasing
amounts of E1 (Fig. 1C). It has been previously shown by
ELISA that the amino-terminal region of human DNA polymerase
p180
subunit from residues 195 to 313 interacts with SV40 large T antigen
(50). Therefore, five overlapping GST fusion peptides of p180 spanning
the entire open reading frame of p180 that had been previously used for
the T antigen interaction study were tested for their ability to
interact with HPV-11 E1 in this assay. Contrary to the findings with
SV40 T antigen-interacting amino-terminal fusion, none of the GST-human
pol
fusion proteins, including the T antigen interacting
amino-terminal fusion fragment (1
), yielded an equivalent signal to
that of the full-length intact p180 (Fig. 1C). These results
indicate that none of the GST-human pol
fusion proteins alone
contains an intact interacting domain and the interaction requires a
correctly folded full-length p180. Nonetheless, these GST-human pol
fusion proteins provide a negative control for the ELISA experiments.
The positive signals seen between p180 and E1 by ELISA thus confirm
that there is a physical interaction between these two proteins and
that the interaction is specific. The region(s) of p180 necessary and
sufficient for the interaction, however, is different from that
required for interaction with SV40 T antigen.
.
activity co-immunoprecipitated from lysates of
co-infected insect cells (data not shown) leads us to the hypothesis
that E1 may exist as a multimer in solution. This hypothesis will be
further addressed below. Together, these results indicate that HPV-11
E1, similar to SV40 T antigen and BPV-1 E1, is able to physically
interact with the human DNA pol
catalytic subunit p180.
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Fig. 2.
p180 subunit inhibits both
E2-dependent and E2-independent HPV-11 ori replication
in vitro. Cell-free replication reactions were
performed as described under "Experimental Procedures" with 40 ng
of HPV-11 ori-plasmid pUC7874-20 and 120 µg of total protein from 293 cell extracts. The amounts of E1 protein, E2 protein, and p180 subunit
of DNA pol used in the reactions are indicated. Presence or
omission of a protein is indicated by a plus or minus sign,
respectively. Replication products were separated in a 0.8% agarose
gel and processed as described under "Experimental Procedures." The
slow migrating replication intermediates (R.I.) and fast
migrating form I and II DNA are marked. Labeled form I topoisomers and
form II DNA in lanes 5 and 6 were due to repair
synthesis as there were no detectable replication intermediates.
Interacts with HPV-11 E1--
The
p70 subunit of human pol
has also been previously shown to interact
with SV40 T antigen (53). Given the similarity in function between T
antigen and E1 as well as the observed interaction between E1 and p180,
we tested whether HPV-11 E1 also interacts with p70. An affinity of the
EE epitope tag of E1 to Ni2+-nitrilotriacetic acid resin
and a moderate affinity of anti-EE antibody to the His-tag on E. coli-expressed recombinant p70 precluded the possibility of
testing for an interaction between E1 and His-tagged p70 by
co-immunoprecipitation. Therefore the interaction between HPV-11 E1 and
p70 was first analyzed by ELISA. A fixed amount of recombinant
His-tagged p70 protein purified from E. coli as described
under "Experimental Procedures" was immobilized in the wells of an
ELISA plate. As with p180, incubation with increasing amounts of E1
resulted in increasing chromogenic signals above the background values,
indicative of complex formation between E1 and p70 (see Fig.
1C).
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Fig. 3.
p70 subunit co-elutes with EE-tagged HPV11 E1
during gel filtration. Aliquots of each fraction from an SE1000/17
gel filtration column (Bio-Rad) were immunoblotted to reveal the
protein of interest. Molecular mass standards used were as follows:
proliferating cell nuclear antigen trimer (90 kDa) eluted around 10.3 ml, catalase (232 kDa) about 8.6 ml, ferritin (440 kDa) around 7.6 ml,
and thyroglobulin (669 kDa) around 6.3 ml. A, elution
profile of p70 subunit alone. B, elution profile of p70
subunit after preincubation with HPV11 EE-E1. C, to confirm
the presence of HPV-11 EE-E1 in the high molecular mass fractions of
B, the immunoblot of B was stripped and re-probed
with anti-EE antibody.
/primase physically interacts with HPV-11 E1. Furthermore, these interactions may occur when E1 is in a multimeric conformation in solution.
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Fig. 4.
p70 subunit associates with HPV-11 E1 during
sucrose density gradient sedimentation. Sucrose gradient
sedimentation analysis was performed as described under "Experimental
Procedures." Shown are aliquots of each fraction immunoblotted by
their respective antibodies. Sucrose density of fractions are indicated
under the elution profiles. A, sedimentation profile of
EE-tagged E1 alone. B, sedimentation profile of p70 alone.
C, sedimentation profile of p70 after preincubation with
EE-tagged E1. Molecular mass standards: bovine serum albumin (68 kDa)
sediments around sucrose density of 15.4% (w/w), catalase (232 kDa)
around 19.0%, and thyroglobulin (669 kDa) around 23.8%.
do not interact with E1, and the
inhibitory effect of p70 on HPV-11 cell-free replication is
specific.
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Fig. 5.
The p70 subunit but not the p58·p49 primase
complex inhibits HPV-11 ori-dependent in vitro
replication. HPV-11 cell-free replication reactions were
performed as described above in Fig. 2. Lane 1, a standard
HPV-11 ori-dependent replication reaction with 12-ng E1 and
8-ng E2 proteins in the absence of exogenously added host primase or
p70. Increasing amounts of primase to 250 ng (lanes 2-4)
and increasing amounts of p70 subunit to 250 ng (lanes 5-7)
as indicated were added to the standard HPV-11 in vitro
replication reactions. Inhibition is seen only in the presence of p70
subunit. R.I., replication intermediates.
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Fig. 6.
Inhibition of the cell-free replication
reactions by the p70 subunit is independent of E1 concentration but
partially alleviated by elevated E2 levels. HPV-11
ori-dependent cell-free replication reactions were
performed as described under "Experimental Procedures."
A, increasing amounts of E1 protein as indicated were mixed
with 8 ng of E2 proteins in the absence (odd-numbered lanes)
or the presence (even-numbered lanes) of 250 ng of p70
protein. B, increasing amounts of E2 protein as indicated
were added in reactions with 12 ng of E1 in the presence
(even-numbered lanes) or in the absence (odd-numbered
lanes) of 250 ng of the p70 subunit. R.I., replication
intermediates.
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Fig. 7.
p70 was unable to associate with HPV-11 E2
protein during gel filtration. Two µg of p70 and E2 each were
preincubated together and fractionated on a Superose 6 PC 3.2/30 column
as described under "Experimental Procedures." Proteins in each
fraction were analyzed by immunoblot using anti-p70 polyclonal antibody
and anti-E2 polyclonal antibody.
/primase holoenzyme or other host factors, p70 inhibition should
persist in E2-independent replication reactions. To distinguish between
these two possibilities, we tested the effect of excess p70 in
E2-independent replication reactions.
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Fig. 8.
The p70 subunit has a weak stimulatory effect
on E2-independent replication in the presence of elevated E1 protein
concentrations. A, p70 activated E2-independent
replication in the presence or in the absence of an ori. Forty ng of
HPV-11 ori-plasmid pUC7874-99 (lanes 1-8) or the cloning
vector pUC-19 (lanes 9-12) were used in cell-free
replication reactions. In the presence of 8 ng of E2, addition of p70
inhibited replication in reactions containing either 40 ng (lanes
1 and 2) or 60 ng of E1 (lanes 3 and
4). In the absence of E2 with 40 and 60 ng of E1, p70
stimulated replication of both the ori-containing plasmid pUC7874-99
(lanes 5-8) and the vector plasmid pUC-19, which lacks an
origin of replication (lanes 9-12). B, the
stimulatory effect of p70 was weak relative to E2. Forty ng of the
ori-plasmid pUC7874-99 was used in each reaction. In E2-independent
reactions, no replication products were observed in reactions with 10 ng of E1 at any level of p70 (lanes 1-4). In reactions
containing 20 ng of E1, increasing amounts of p70 stimulated
replication (lanes 5-8). The stimulatory effect of E2 at
low levels of E1 (10 ng) was much higher than that of the p70 (compare
lanes 1-4 with lanes 9-11). Omission of a
protein is indicated by a minus sign. R.I., replication
intermediates.
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Fig. 9.
p70 competes with E2 for E1 binding. p70
and HPV-11 E1 and E2 proteins were in vitro transcribed and
translated as described under "Experimental Procedures."
A, shown are 2 µl of the translation reactions of
35S-labeled EE-E1 (lane 1),
3H-labeled Myc-p70 (lane 2), and
35S-labeled BB-E2 (lane 3). B,
co-immunoprecipitation of the in vitro transcribed and
translated EE-tagged E1 and p70 by anti-EE antibody. Lane 1 is 2 µl of 3H-labeled p70 as a reference. Lanes
2-5 are anti-EE immunoprecipitates of 2 µl of EE-E1 with 0, 2, 4, and 6 µl of 3H-labeled p70; lane 6 is the
anti-EE immunoprecipitate of 6 µl of 3H-labeled p70 alone
(C). p70 competes with E2 for E1 binding. Shown are the
co-immunoprecipitations of 2 µl each of 35S-labeled E1
and 35S-labeled E2 by anti-EE antibody in the absence of
added p70, (lane 1) and after preincubation with 20, 100, and 400 ng of unlabeled His-p70 protein (lanes 2-4,
respectively).
DISCUSSION
/primase subunits, p180 and p70.
Furthermore, the HPV-11 cell-free replication assay demonstrates that
both of these interactions are functionally significant. Here we
discuss these interactions and propose a model of how these proteins
participate in initiation of viral DNA replication.
holoenzyme
during the initiation of viral DNA replication.
/primase subunits,
p180 and p70, appear to be of a different nature. Gel filtration and
sucrose density gradient experiments suggest that many more molecules
of E1 are present in the complex than either p180 or p70, indicating
the two cellular DNA polymerase subunits are able to associate with E1
in its multimeric conformation. It has been reported that the hexameric
T antigen must be assembled on the origin-containing DNA for productive
interactions to occur, whereas pre-formed T antigen hexamer in solution
is replication-incompetent (65). Similarly, the E1 multimers alone or
the E1 multimers complexed with p180 or p70 formed in solution may not
be replication-competent.
/primase but not the p180 subunit. Their results also suggest that
HPV-16 E2 competes with p70 for binding to HPV-16 E1.
Increasing E1 concentration would increase both the highly
replication-efficient E1·E2 complex and a less replication-efficient
E1·p70 complex, resulting in no apparent increase in replication. The
fact that the exogenously added p70 is in excess in the reaction and
the unique stoichiometry-dependent interactions between the
two molecules observed in Figs. 8B and 9B may
also explain why increasing amounts of E1 are unable to overcome
replication inhibition. In contrast, an increase in E2 levels would tip
the balance toward the formation of replication-competent E1·E2
complexes, leading to a slight restoration of activity. An alternative
explanation is that although p70 and E2 do not physically interact
in vitro (Fig. 7), it is possible that E2 may recruit other
host proteins to the ori-bound E1 (26). The exogenous p70 may somehow
interfere with these interactions between E2 and host proteins,
inhibiting the assembly of the preinitiation complex.
holoenzyme
subunits may then displace E2 and replication is initiated.
/primase to occur. Thus, p70 was able to stimulate replication
weakly but only at higher E1 concentrations (Fig. 8). However, the
stimulatory effect is much lower than that seen with E2 (Fig. 8),
perhaps due to the relatively inefficient binding of E1 to the DNA in
the absence of E2 or to the important role of E2 involvement in
pre-initiation complex formation (26). In the presence of E2, the
exogenous p70 competes with E2 for binding to E1. The formation of a
non-productive p70-multimeric E1 complex in solution then results in a
decrease in the amount of highly productive E1·E2 complex, inhibiting replication.
, it will be interesting to
investigate the protein partners of p70 in an uninfected cell.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. I. R. Lehman and members of the Wang laboratory for helpful discussions and critical reading of the manuscript and Chuen-Sheue Chiang for cloning of the human p70 subunit.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA14835 from the National Cancer Institute (to Wang laboratory) and by National Institutes of Health Grant CA36200 from the National Cancer Institute (to Chow laboratory).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Cancer Biology Predoctoral Training Grant CA09302 from Stanford University.
To whom correspondence should be addressed.
The abbreviations used are: BPV, bovine papillomavirus; HPV, human papillomavirus; pol, polymerase; ELISA, enzyme-linked immunosorbent assay; ori, origin.
2 L. T. Chow, unpublished results.
3 K. L. Conger and T. S. F. Wang, unpublished observation.
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REFERENCES |
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