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J Virol, April 1998, p. 3436-3441, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Carboxyl-Terminal Region of the Human
Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity
during DNA Replication
Nianxiang
Zou,
Jen-Sing
Liu,
Shu-Ru
Kuo,
Thomas R.
Broker, and
Louise T.
Chow*
Department of Biochemistry and Molecular
Genetics, University of Alabama at Birmingham, Birmingham, Alabama
35294-0005
Received 7 August 1997/Accepted 17 December 1997
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ABSTRACT |
The mechanism of DNA replication is conserved among
papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit
with different efficiencies, indicating a certain degree of specificity
in E1-E2 interactions. We report that, in the assays of our study, the
human papillomavirus type 11 (HPV-11) E1 protein functioned with the
HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no
detectable activity with the HPV-11 E2 protein. Taking advantage of
this distinction, we used chimeric E1 proteins to delineate the E1
protein domains responsible for this specificity. Hybrids containing
HPV-16 E1 amino-terminal residues up to residue 365 efficiently
replicated either viral origin in the presence of either E2 protein.
The reciprocal hybrids containing amino-terminal HPV-11 sequences
exhibited a high activity with HPV-16 E2 but no activity with HPV-11
E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues
from either E1 had an intermediate property, but both collaborated more
efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras
with a junction in the putative ATPase domain showed little or no
activity with either E2 protein. We conclude that the E1 protein
consists of distinct structural and functional domains, with the
carboxyl-terminal 284 residues of the HPV-16 E1 protein being the
primary determinant for E2 specificity during replication, and that
chimeric exchanges in or bordering the ATPase domain inactivate the
protein.
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TEXT |
Human and animal papillomaviruses
contain a double-stranded circular DNA genome. Replication of plasmids
containing a papillomaviral origin sequence (ori) in cell-free systems
is dependent on virus-encoded E1 and E2 proteins and the host DNA
replication machinery (17, 43). Transfection of E1 and E2
expression plasmids into mammalian cells can support transient
replication of an ori-containing plasmid (7, 42). The ori
sequences are highly conserved among all papillomavirus types and
consist of a known or putative E1 protein binding site and multiple
copies of E2 protein binding sites in close proximity (for reviews, see
references 8 and 37). The E1
protein binding sites are similar but not identical in sequence among
different virus types, whereas all E2 proteins bind to the consensus
sequence ACCN6GGT. Of all papillomavirus proteins, E1 is
the most conserved. It is an ATPase and helicase (5, 15, 16, 25,
36, 44) and has sequence homology to the ATPase domain of the
simian virus 40 (SV40) T antigen (9, 26), the initiator for
SV40 ori replication (see Fig. 2A). As does the T antigen, the E1
protein binds to the ori and unwinds DNA in the presence of the host
single-stranded DNA binding protein RPA and topoisomerase I. The human
papillomavirus (HPV) and bovine papillomavirus type 1 (BPV-1) E1
proteins are thought to function as a helicase at the replication fork,
since each is required during elongation (20). The BPV-1 E1
protein is known to interact with the 180-kDa catalytic subunit of the
host DNA polymerase 
, thereby bringing host replication proteins to
the unwound ori (3, 30). The E2 protein is a transcription
factor but also serves multiple functions in ori replication. It
interacts with E1, stabilizing its binding to the ori (12, 24, 28,
34, 38, 43), and it also helps recruit host replication proteins into the initiation complex (20). In addition, E2 may
prevent nucleosome formation around the ori in vivo (19). By
virtue of its strong and specific affinity to the E2 protein binding site and its interaction with the E1 protein, the E2 protein is critical to the initiation of replication from ori (4, 6, 17, 22,
31, 33, 40), but it appears to be dispensable during elongation
(20).
Because of the homologous nature of the ori and of E1 and E2 proteins,
proteins from one virus type can efficiently replicate either a
homologous or heterologous viral ori. In contrast, mixed pairs of
proteins support ori replication with various levels of efficiency
(2, 7, 10, 13, 39). These observations indicate that a
degree of specificity in E1-E2 protein interactions must exist. Binding
studies in vitro and the yeast two-hybrid system have yielded
conflicting results with regard to the E1 domain which interacts with
the E2 protein. Some investigators have reported that the
amino-terminal portion of the E1 protein of BPV-1 and HPV type 31 (HPV-31) contains the major DNA binding and E2 interaction domains
(1, 11, 18, 41). However, others have demonstrated that the
C-terminal portion of the BPV-1 E1 protein (residues 162 to 605), the
HPV-16 E1 protein (residues 144 to 649), or the HPV-33 E1 protein
(residues 312 to 644) was necessary for E2 binding (23, 29, 32,
45) (see Fig. 2A). A study to examine the functional interactions
between E1 and E2 proteins during replication would be of significant
interest. Here we report such an investigation.
To examine the interactions between the E1 and E2 proteins, we tested
combinations of HPV-11 and HPV-16 E1 and E2 proteins for their ability
to support ori replication in transfected human 293 cells. The HPV-11
proteins were each expressed from the eukaryotic vector pMT2 as
described previously (7). The interrupted HPV-16 E1 open
reading frame (ORF) in the prototype genomic HPV-16 clone (35) was repaired by inserting a G residue between
nucleotide positions (nt) 1138 and 1139 (27) by
site-directed mutagenesis. The PvuII-HincII and
AvaII-MstII restriction fragments spanning the
HPV-16 E1 ORF (nt 685 to 3211) and the E2 ORF (nt 2714 to 4337),
respectively, were cloned into pMTX, a derivative of the pMT2 vector
containing a multiple cloning site. The ori plasmid contained either
the HPV-11 ori (nt 7730 to 7933/1 to 99) or the HPV-16 ori (nt 7455 to
7906/1 to 111) in pUC19.
ori replication by matched or mixed pairs of HPV-11 and HPV-16 E1
and E2 proteins.
Transient replication assays were conducted as
previously described (7). Briefly, 48 h after
electroporation of 5 µg each of the expression vectors and 0.5 µg
of the ori plasmid, low-molecular-weight DNA was harvested by alkaline
Hirt lysis and digested with HindIII alone, which
linearized all three plasmids, or with both HindIII and
DpnI, which eliminated all unreplicated input DNA and
linearized the replicated DNA. The digestion products were separated
electrophoretically in a 0.8% agarose gel and then revealed by
Southern blot hybridization with [-32P]dCTP-labeled
ori plasmid probes generated by random-priming reactions. These results
showed that the matched protein pairs were able to replicate both
HPV-11 and HPV-16 ori plasmids efficiently (Fig. 1, lanes 1 and 2, 7 and 8, and 11 and 12; see also Fig. 4B, lane
5). Cotransfection of the HPV-11 E1
expression vector with the HPV-16 E2 expression plasmid supported ori
replication with a slightly reduced efficiency relative to that
achieved with either homologous protein pair (Fig. 1, compare lanes 5 and 6 to 1 and 2 and to 7 and 8). In contrast, the HPV-16 E1 protein consistently failed to replicate either HPV ori plasmid in the presence
of HPV-11 E2 (Fig. 1, lanes 3 and 4 and lanes 9 and 10; see also Fig.
3, lane 7, and Fig. 4A, lane 1). Furthermore, these results were
reproducible when the cells were transfected with E1 and E2 expression
plasmids in a wide range of relative quantities (data not shown). The
stringent discrimination between the two E2 proteins exhibited by
HPV-16 E1 and the lack of specificity by the HPV-11 E1 provided us with
an opportunity to investigate the domains involved in functional
interactions during replication by using chimeric E1 proteins.
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FIG. 1.
Transient replication by combinations of HPV-11 and
HPV-16 E1 and E2 proteins. Replication assays of an HPV-11 ori plasmid
(nt 7730 to 7933/1 to 99) (lanes 1 to 8) or an HPV-16 ori plasmid (nt
7455 to 7906/1 to 111) (lanes 9 to 12) were conducted with human 293 cells as described in the text. The E1 and E2 expression vector
plasmids used are indicated above each pair of lanes.
Low-molecular-weight DNA was harvested 48 h posttransfection. Half
of the recovered DNA from one 100-mm plate was subjected to
HindIII and DpnI double digestion (DpnI +).
The other half was subjected to HindIII digestion, which
linearized the three plasmids (DpnI  ). The products were separated
electrophoretically in a 0.8% agarose gel and transferred to a
nitrocellulose membrane. The membrane was probed with the
[-32P]dCTP-labeled, origin-containing plasmid and
exposed to X-ray film overnight. Only replicated DNA was resistant to
DpnI digestion (marked Ori), whereas the unreplicated DNA
was digested to small fragments and migrated ahead of the linearized,
newly replicated ori plasmid (see Fig. 3 and 4).
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Reciprocal HPV-16/11 and HPV-11*/16 hybrid proteins.
Nine
hybrid E1 protein genes were constructed (Fig.
2). Each hybrid E1 gene encodes a protein
identical in length (649 amino acids) to that encoded by the wild-type
HPV-11 or HPV-16 gene or a modified HPV-11 E1 gene (designated 11*;
described below). The junctions of the hybrid proteins were selected
based on one of two criteria or both. Either they were located near a
boundary of functional domains inferred from the homologous BPV-1 E1
protein or they were within a highly conserved region to minimize the probability of generating a misfolded, nonfunctional protein (Fig. 2B).
Five HPV-16/11 (16/11) hybrid E1 genes contained HPV-16 E1 sequences at
the 5' portion and the balance from HPV-11 E1 sequences at the 3'
portion. We prepared four HPV-11/16 (11/16) hybrid E1 protein genes in
which the sequences encoding the amino-terminal portion were derived
from HPV-11, with the balance from HPV-16. To facilitate a quantitative
comparison among various wild-type and hybrid E1 genes, we modified the
5' untranslated sequence and the first 5 amino acids of the 11/16
hybrid gene to those of HPV-16 E1 (Fig. 2A, designated HPV-11*/16 E1H1,
E1H2, E1H4.5, and E1H5). This modification resulted in a net change of
2 amino acids from MADDS of HPV-11 to MADPA of HPV-16. These two
altered residues are not conserved among different HPVs. The reason for making the HPV-11*/16 hybrid genes is the following. We note that HPV-16 proteins always supported a higher level of replication than
HPV-11 proteins (Fig. 1, compare lanes 7 and 8 to lanes 1 and 2). This
distinction could in part be due to differences intrinsic to the viral
proteins, to differences in levels of protein expression, or both. By
constructing clones that had identical sequences in the 5' untranslated
region and around the translation initiation codon, we could at least
reduce differences in replication that may have originated from
variances in protein translation efficiency among the different E1
genes. To provide a reference protein, we also prepared an HPV-11 E1
gene with the same modification and designated it HPV-11*E1. In
transient replication assays, HPV-11*E1 supported the replication of
either the HPV-11 or HPV-16 ori plasmid in the presence of either E2
protein as efficiently as that promoted by HPV-16 E1 and E2 proteins
(Fig. 3, compare lanes 1 and 8 to lane
14; Fig. 4A, compare lanes 6 and 12 to
lane 7; and data not shown). These results demonstrate that HPV-11*E1 is fully functional and that the difference previously observed between
the wild-type HPV-11 and HPV-16 E1 proteins in the presence of their
respective homologous E2 proteins is due at least in part to the levels
of protein expression. This modification minimized the difference and
made it feasible to quantify the levels of replication by the panel of
E1 proteins.
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FIG. 2.
Structures, functional domains, and relative replication
activities of wild-type and hybrid HPV E1 proteins. (A) Schematic
representations of wild-type and hybrid E1 proteins and relative
replication activities. The shaded boxes represent regions derived from
HPV-16 E1, and the open boxes represent those derived from HPV-11 E1.
To minimize replication differences that might be attributable to mRNA
translation efficiencies, the 5' untranslated sequence and first 5 codons of HPV-11 and of 11/16 hybrid proteins were all replaced with
those from HPV-16 E1 and are denoted with an asterisk in the
designation. This modification resulted in a net change of two amino
acids. The BPV-1 E1 protein is shown as a thick line. Numbers refer to
amino acid residues. Thin solid lines represent the BPV-1 E1 protein
domains defined to be involved in E2 interactions (lines c and d) or
DNA binding (line a), based on protein interaction assays in vitro or
in the yeast two-hybrid system (1, 18, 23, 32, 41), or the
ATPase domain (line b) based on the homology to the SV40 T antigen and
mutagenic analysis (8, 26, 28). The dotted line (e)
approximates the E1 domain of HPV-16 and HPV-33 involved in interaction
with the E2 protein (29, 45). The dashed line (f) signifies
the domain of HPV-16 E1 responsible for E2 specificity during
replication (this study). After subtraction of background signals, the
relative replication activities (Rel Activity) of each E1 protein in
the presence of HPV-11 or HPV-16 E2 protein were determined by
PhosphorImager quantification of the results shown in Fig. 3 and 4A.
The results are shown to the right of each clone. The activities
achieved with HPV-16 E1 and E2 were taken as 100% and are assigned a
score of ++++. Scores: , relative activity of less than 5%,
including signals detectable only after a long exposure; +, activity
between 5 and 10%; ++, activity between 10 and 50%; +++, activity
between 50 and 90%; ++++, activity over 90%. (B) Peptide sequences
spanning the junctions of hybrid E1 proteins. The numbers flanking the
arrows are amino acid residues where protein coding switches from one
virus type to the other.
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FIG. 3.
Transient replication of an HPV-11 ori plasmid by
HPV-16/11 E1 hybrid proteins in combination with either HPV-16 or
HPV-11 E2 protein. Replication assays were conducted as described in
the legend to Fig. 1. The low-molecular-weight DNA was digested
overnight with both DpnI and HindIII.
Fragments that migrated faster than the linearized ori plasmid were
derived from DpnI-sensitive input DNA. Similar results were
obtained when an HPV-16 ori plasmid was used (data not shown).
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FIG. 4.
Transient replication by HPV-11*/16 E1 hybrid proteins
in combination with either HPV-16 or HPV-11 E2 protein. (A) Replication
of an HPV-11 ori plasmid; (B) replication of an HPV-16 ori plasmid.
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Hybrid genes 16/11 E1H1 through E1H4 were generated by PCR
amplification followed by exchanging a restriction fragment with
the
wild-type HPV-16 E1 gene in the expression vector. Hybrid
gene 16/11
E1H5 was prepared similarly except that a
HindIII site
was introduced downstream of the hybrid E1 gene during PCR
amplification.
Using a similar strategy, we constructed hybrid genes
containing
the amino terminus of HPV-11 E1 and the balance from HPV-16
E1
except for the extra steps to replace their 5' ends with that
of the
HPV-16 E1 gene as follows. We took advantage of a
PstI
site
located in the fifth codon of the HPV-16 E1 gene. A
PstI
site was generated by PCR amplification at the comparable site
in
HPV-11 E1 or 11/16 hybrid E1, and an
EcoRI site was
introduced
at the 3' flanking sequence. After digestion with
PstI and
EcoRI,
the fragment containing the bulk
of the E1 gene was ligated to
the vector fragment of similarly digested
pMTX-16E1. Hence, all
of the E1 sequences were located at the same site
in the pMTX
vector and had identical sequences at the 5' ends of the
mRNAs.
All junction sequences or the entire sequences generated by
PCR
were confirmed by sequencing.
The domain of HPV-16 E1 protein which confers E2 protein
specificity during replication.
The 16/11 E1H1 and 16/11 E1H2
hybrid proteins contain amino acids 1 to 237 and 1 to 365, respectively, from HPV-16 E1 and the balance from HPV-11 E1. The
junctions are located in the middle or at the carboxyl-terminal
boundary of the putative DNA and E2 binding domains by analogy to BPV-1
(Fig. 2A). Figure 3 presents the results of transient replications of
an HPV-11 ori with the expression vectors of 16/11 hybrid E1 proteins
in combination with the HPV-11 or HPV-16 E2 expression vector. Unlike
the wild-type HPV-16 E1 protein, chimeric proteins 16/11 E1H1 and E1H2
supported efficient replication in the presence of either E2 protein, a property shared with HPV-11 E1 (Fig. 3, compare lanes 1 to 3 and 7 to
lanes 9 to 11 and 14). Similar results were observed when the HPV-16
ori plasmid was used in the test (data not shown). These results
demonstrate that the amino-terminal region up to residue 365 of HPV-16
E1 is not responsible for functional discrimination of the two E2
proteins during replication. By inference, the carboxyl-terminal domain
of the HPV-16 E1 protein is expected to constitute the determinant. We
then tested the reciprocal 11*/16 hybrid E1 genes. Transient
replication assays of an HPV-11 or HPV-16 ori showed that neither
11*/16 E1H1 nor 11*/16 E1H2 had detectable replication activity with
HPV-11 E2 while both supported efficient replication of either viral
ori in the presence of HPV-16 E2 (Fig. 4A, compare lanes 1, 2, and 3 to
lanes 7, 8, and 9; Fig. 4B, compare lanes 1, 2, and 5 to lanes 6, 7, and 10). These properties are similar to those of the HPV-16 E1 protein
and support the previous conclusion that the carboxyl-terminal region
of the HPV-16 E1 is responsible for E2 specificity. This conclusion is
supported by the replication properties of reciprocal H5 hybrid
proteins described below.
Inactivation of the E1 protein by a chimeric junction in the ATPase
domain.
For further delineation of functional domains, we tested
16/11 E1H3, E1H4, and E1H5, which contain amino acid residues 1 to 443, 1 to 526, and 1 to 605 from HPV-16 E1, respectively, and the balance
from HPV-11 E1. The junctions reside within or border on the putative
ATPase domain (Fig. 2A). 16/11 E1H3 showed a very low replication
activity with HPV-11 E2, and the activity with HPV-16 E2 was detectable
only after prolonged exposure (Fig. 3, lanes 4 and 11). 16/11 E1H4 had
no detectable activity with either E2 protein (Fig. 3, lanes 5 and 12).
In contrast, 16/11 E1H5 had an activity comparable to that of HPV-16 E1
or HPV-11* E1 in the presence of HPV-16 E2 (Fig. 3, compare lane 13 to
lanes 8 and 14). Interestingly, this hybrid now gained a moderate
activity in collaboration with HPV-11 E2 (Fig. 3, lane 6).
Two 11*/16 hybrid genes with a junction near the carboxyl terminus were
tested. The exchange in 11*/16 E1H4.5 occurs between
residues 559 and
560, whereas the 11*/16 E1H5 is the reciprocal
hybrid of 16/11 E1H5
(Fig.
2). Transient replication assays of
the HPV-11 or HPV-16 ori by
11*/16 E1H4.5 revealed no detectable
activity with either HPV-11 E2 or
HPV-16 E2 (Fig.
4A, lanes 4
and 10; Fig.
4B, lanes 3 and 8). Only after
extended exposure
was a very weak replication signal observed, and only
with HPV-16
E2 (data not shown). Transient replication assays of 11*/16
E1H5
showed a low activity with HPV-11 E2 and a higher activity with
HPV-16 E2 (Fig.
4A, compare lanes 5 and 11; Fig.
4B, compare lanes
4 and 9). Therefore, our results showed that 16/11 E1H3, 16/11
E1H4, and
11*/16 E1H4.5 were severely crippled in their function
in transient
replication assays. Since the reciprocal H5 hybrids
have a property
intermediate between the HPV-11 E1, HPV-11*E1
(or 16/11 E1H1 or E1H2),
and HPV-16 E1 (or 11*/16 E1H1 or E1H2),
we suggest that the
carboxyl-terminal 44 amino acids of HPV-16
contribute to but do not
comprise the entire determinant for E2
specificity.
The 16/11 E1H3 and E1H4 hybrid proteins had little or no detectable
replication activity, whereas most other hybrid proteins
had a moderate
to high activity in the presence of at least one
of the E2 proteins. To
ascertain that these hybrid genes indeed
express full-length proteins,
we tested for the presence of E1
proteins in transfected COS-7 cells.
To eliminate variations in
antibody reactivities with hybrid proteins,
HPV-16 E1 and each
of the 16/11 hybrid E1 genes were recloned into pMT2
with a glutamic
acid-rich (EE) epitope tagged at the amino terminus.
EE-epitope-tagged
HPV-11 and HPV-16 E1 proteins function in transient
and in cell-free
replication systems (
17,
21). After
transfection of individual
expression vectors into COS-7 cells, the E1
proteins were detected
by immunoblot with anti-EE monoclonal antibody
(
14). Figure
5 shows that all
had mobilities identical to that of the EE-epitope
tagged HPV-16
protein purified from insect Sf9 cells infected
with a recombinant
baculovirus (rightmost lane) and that there
were no significant
differences in expression levels under these
conditions. These results
indicate that the inactivity of the
H3 and H4 (and possibly that of
H4.5) chimeras is likely mainly
due to a loss of function, with at most
a minor effect from variations
in steady-state protein levels in the
transfected cells.
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FIG. 5.
Expression of wild-type and HPV-16/11 hybrid E1 proteins
in COS-7 cells. The EE-epitope-tagged wild-type and hybrid E1 proteins
were each expressed from the pMT2 vector. After 24 h of
incubation, the transfected cells were disrupted in 200 µl of lysis
buffer. Forty microliters of each lysate was subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. The E1 proteins were detected by monoclonal
antibody to the EE epitope (14). The rightmost lane contains
EE-epitope-tagged HPV-16 E1 protein purified from insect Sf9 cells
infected with a recombinant baculovirus (21).
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Multiple structural and functional domains of the E1 protein.
The extent of replication shown in Fig. 3 and 4A was quantified by
PhosphorImager and compared to the activities achieved by HPV-16*E1 and
HPV-11 E2. The results are shown in Fig. 2A. Several conclusions can be
drawn. (i) The HPV-11 E1 protein is more flexible in its collaboration
with a heterologous E2 protein than is the HPV-16 E1 protein, which
does not function in the presence of HPV-11 E2 (Fig. 1, 3, and 4). (ii)
The E1 protein can be divided into at least three structural and
functional domains on the basis of the ability of hybrid proteins to
function in transient replication assays. The amino-terminal residues 1 to 237 or 1 to 365 (E1H1 and E1H2) and the carboxyl-terminal 44 amino acids (E1H5) can be exchanged between the two viral proteins and result
in hybrid proteins that exhibit low (5 to 10%), moderate (10 to 50%),
or high (50 to 90%) replication activity in the presence of one or
both E2 proteins. However, residues 366 to 605, which comprise the
putative ATPase domain (H3, H4, and H4.5), are very sensitive to
changes within the domain or in the flanking regions, since the hybrid
proteins are largely or entirely nonfunctional in the presence of
either E2 protein (Fig. 3 and 4). Whether this loss of function is due
to the inactivation of the ATPase and helicase activities or to other
defects is not known. (iii) The amino-terminal 56% of the HPV-16 E1
protein (residues 1 to 365) does not play a role in E2 protein
specificity, as 16/11 E1H1 and E1H2 exhibited high replication
activities with both E2 proteins (Fig. 3). That the carboxyl-terminal
44% of the HPV-16 protein (residues 366 to 649) is responsible for
this discrimination is substantiated by the reciprocal 11*/16 E1H1 and
E1H2 proteins. These latter hybrid proteins exhibited functional
properties similar to those of the wild-type HPV-16 E1 and replicated
either HPV-16 or HPV-11 ori only in the presence of HPV-16 E2 (Fig. 4).
(iv) The reciprocal E1H5 proteins functioned with either E2 protein. Since the discrimination between the two E2 proteins by the hybrid protein containing residues 606 to 649 of HPV-16 is not as stringent as
that by the HPV-16 or 11*/16 E1H2 protein that contains HPV-16 E1
residues 366 to 649 (Fig. 3 and 4), we suggest that the domains spanning residues 366 to 605 and residues 606 to 649 of HPV-16 E1 both
contribute to the selectivity for the HPV-16 E2 protein. Conversely, in
16/11 H5 and H2, the corresponding domains of HPV-11 E1 confer
increasing activities with the HPV-11 E2 protein. Considered together,
these two domains spanning residues 366 to 649 of HPV-16 E1 comprise
the primary determinant for E2 protein specificity during replication.
This conclusion, however, does not necessarily rule out the possibility
that the amino-terminal domain of the E1 protein also interacts with
E2. But such an interaction does not lead to an E2 selectivity. Taking
into account previous reports that the carboxyl domain of HPV E1 binds
to the E2 protein, a mechanistic interpretation of our observations is
the following. The noncooperativity of the HPV-16 E1 protein with the
HPV-11 E2 protein is due to steric interference involving subregions within the carboxyl-terminal 44% of the protein, thereby negating or
preventing positive interactions between other subregions within the
same domain. (v) The E2 specificity is not related to the ori plasmid
used, since similar results were also obtained with an HPV-16 ori
plasmid (Fig. 1 and 4B and data not shown). In summary, we have
constructed reciprocal chimeric E1 proteins between HPV-11 and HPV-16.
The results of transient replication assays demonstrate that the E1
protein consists of at least three structural and functional domains
and that the region of HPV-16 E1 responsible for discriminating E2
proteins resides within the carboxyl-terminal 44% of the protein.
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ACKNOWLEDGMENTS |
This work was supported by USPHS grant CA36200.
We thank Harald zur Hausen and Lutz Gissmann for providing cloned
HPV-11 and HPV-16 DNAs and Biing-Yuan Lin for technical assistance.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Genetics, University of Alabama at
Birmingham, 1918 University Blvd., Birmingham, AL 35294-0005. Phone:
(205) 975-8300. Fax: (205) 975-6075. E-mail: ltchow{at}uab.edu.
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J Virol, April 1998, p. 3436-3441, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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