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J Biol Chem, Vol. 273, Issue 49, 33073-33081, December 4, 1998
From the McArdle Laboratory for Cancer Research, University of
Wisconsin, Madison, Wisconsin 53706
Epstein-Barr viral nuclear antigen-1 (EBNA-1) is
required for the stable replication of plasmids that contain
oriP, the origin of DNA synthesis used during the latent
phase of the Epstein-Barr virus life cycle. EBNA-1 acts
post-synthetically through unknown mechanisms to facilitate the
continued synthesis of oriP plasmids in ensuing S phases.
In contrast to viral replicons such as that of SV40, DNA synthesis of
oriP is restricted to a single round during each cell
cycle. Large T-antigen of SV40 is a DNA helicase and activates the
synthesis of SV40 DNA by recruiting cellular proteins required for DNA
synthesis to the origin of SV40. Using fusion proteins of EBNA-1 and
large T-antigen, we tested whether tethering large T-antigen to
oriP is sufficient to initiate multiple rounds of DNA
synthesis from oriP during each cell cycle. We report here
that, although these fusion proteins retain the biological activities
of both EBNA-1 and large T-antigen, their constituent proteins do not
confer the properties of one on the other. Thus, it is not possible to
subvert the cellular controls that restrict DNA synthesis from
oriP to a single round per cell cycle. These results also
provide insights into architectural constraints at oriP and
at the SV40 ori.
The Epstein-Barr virus
(EBV)1 genome is maintained
as a plasmid within latently infected cells. Synthesis and maintenance
of this plasmid requires a viral cis-acting sequence,
oriP, and a single viral protein, the Epstein-Barr viral
nuclear antigen 1 (EBNA-1) (1-3). In latently infected cells, the EBV
genome is synthesized only once every cell cycle (4), as are
oriP plasmids in cells that express EBNA-1 (5).
There are 24 binding sites for EBNA-1 within oriP (6). These
sites are organized into two clusters, referred to as the family of
repeats (FR) and the dyad symmetry element (7, 8). FR contains 20 binding sites for EBNA-1, while the dyad symmetry element has 4 binding
sites for EBNA-1, 2 of which are present within an extended palindrome
(1, 8). Unlike the origin-binding proteins of some viruses, EBNA-1 does
not possess enzymatic activities such as a DNA helicase activity that
could contribute directly to synthesis of oriP DNA (9, 10).
Results from a deletion analysis indicate that EBNA-1 is unlikely to
function as an enzyme, as small overlapping deletions in regions other
than the domain required for DNA binding do not disrupt functions of
EBNA-1 (11). We have recently demonstrated that EBNA-1 is not required
for the synthesis of oriP plasmids (12). However, in the
absence of EBNA-1, newly synthesized oriP plasmids are lost
rapidly from proliferating cells. Thus, EBNA-1 bound to oriP
functions post-synthetically to ensure plasmid maintenance and
segregation in dividing cells. EBNA-1 bound at FR has another function
of significant biological consequence to EBV. When bound at FR, EBNA-1
activates two EBV promoters, the BamHI-C promoter and the
LMP1 promoter, the activation of which is critical to the establishment
of viral latency (13-15). In addition to sequence-specific DNA
binding, EBNA-1 molecules bound to cognate binding sites interact with
each other, thereby "linking" the two binding sites (10, 16, 17).
Deleted derivatives of EBNA-1 that lack domains required for DNA
linking are unable to support the stable replication of oriP
plasmids, and fail to activate the EBNA-1-responsive BamHI-C
promoter (18).2 These results
indicate that EBNA-1's ability to link DNA is likely to be important
to EBV.
Replication of the oriP replicon differs substantially from
the replication of other viral replicons such as those of simian virus
40 (SV40) or bovine papilloma virus (BPV) (20, 21). The biochemical
contributions toward DNA synthesis by the origin binding proteins of
these viruses, large T-antigen and E1, are largely understood (20,
22-24). In contrast to oriP, the replicons of SV40 and BPV
are not restricted by cellular controls to a single round of DNA
synthesis during each cell cycle (20, 21). Large T-antigen of SV40 and
E1 of BPV possess an ATP-hydrolysis-dependent DNA helicase
activity in addition to binding specifically to their cognate origins
(22, 25, 26). These proteins also interact directly with cellular
proteins involved in initiation of DNA synthesis such as DNA polymerase
We made fusions between EBNA-1 and the large T-antigen of SV40 to
examine whether localizing a protein competent to assemble an
unregulated DNA synthetic complex to oriP is sufficient to trigger multiple rounds of DNA synthesis from oriP.
Biological characterization of these fusion proteins reveals that they
retain the ability to recognize and replicate their cognate origins but do not confer the DNA synthetic phenotype of one on the other. Fusions
that contain intact EBNA-1 activate transcription and replication
similarly to unfused EBNA-1, indicating that EBNA-1's post-synthetic
contribution to oriP replication is not hampered by the
extraneous domain fused to EBNA-1. A fusion protein, which contains
intact large T-antigen and only the DNA binding and dimerization domain
of EBNA-1, replicates the SV40 ori in a manner
indistinguishable from large T-antigen. While this protein can bind
oriP, it cannot activate DNA synthesis from oriP,
indicating that is not possible to subvert the cellular control of DNA
synthesis imposed at oriP.
Bacterial Strains and Plasmid Preparation--
Escherichia
coli DH5 Reagents--
Taq DNA polymerase (5 units/µl),
proteinase K (18.6 mg/ml), and glycogen (20 mg/ml) were purchased from
Boehringer Mannheim. T4 polynucleotide kinase, T4 DNA ligase, and
restriction endonucleases were purchased from New England Biolabs
(Beverly, MA). All enzymes, except DpnI, were used as
recommended by the manufacturer. DpnI-digestions were
performed in 1× KGB (31). Protein A-Sepharose beads were purchased
from Sigma. Oligodeoxynucleotides were purchased from the Midland
Certified Reagent Co. (Midland, TX). [ DNA Constructions--
Plasmids 1553 and 1606 were used to
express EBNA-1 and large T-antigen, respectively. Plasmid 1553 expresses a fully functional derivative of EBNA-1 that contains only
five copies of the Gly-Gly-Ala repeat (3), under the control of the
human cytomegalovirus immediate early (CMV IE) promoter. The EBNA-1
open reading frame (ORF) in 1553 is flanked by a BssHII
recognition site at its amino terminus and a XhoI
recognition site at its carboxyl terminus, each of which was introduced
by site-directed mutagenesis (32). Plasmid 1606 was created by
replacing EBNA-1 in 1553 with a cDNA copy of the large T-antigen
open reading frame from pTM.TAg as a BssHII-XhoI
fragment (33). SfiI recognition sites, which encode the
peptide linker GQSGPGG, were added independently to the amino and
carboxyl termini of the EBNA-1 and large T-antigen ORFs by site-directed mutagenesis (32). SfiI-modified versions of
EBNA-1 and large T-antigen ORFs were used to create expression plasmids for the EBNA-1-TAg and TAg-EBNA-1 fusion proteins. These plasmids are
1607 and 1609, respectively. The N Tissue Culture and Transfections--
The human cell lines 143B
(37) and 293 (38) and the mouse cell line (10)1 (39) were used in this
study. 143B cells were grown in Dulbecco's modified Eagle's
medium/high glucose supplemented with 10% calf serum and antibiotics.
293 and (10)1 cells were grown in Dulbecco's modified Eagle's
medium/high glucose supplemented with 10% fetal bovine serum and
antibiotics. DNA was introduced into 143B cells by electroporation
(40). 293 and (10)1 cells were transfected by the calcium phosphate
method (31).
Protein Expression--
Protein expression was confirmed on
immunoblots using rabbit anti-EBNA-1 polyclonal antibodies (41) or
hamster anti-large T-antigen polyclonal sera (a gift from Dr. K. Rundell, kindly provided by Dr. J. E. Mertz). Rabbit anti-EBNA-1
antiserum was used at 1:5000 for immunoblots, and 1:500 for
immunoprecipitations. Hamster anti-large T-antigen antiserum was used
at 1:10,000 for immunoblots, and 1:2000 for immunoprecipitations.
Protein expression was quantified using 35S-labeled donkey
anti-rabbit IgG (Amersham Pharmacia Biotech) as described previously
(18). When hamster anti-large T-antigen was used as the primary
antibody, purified rabbit anti-hamster IgG (Cortex) was used as
secondary antibody prior to detection with 35S-labeled
donkey anti-rabbit IgG.
Preparation of Nuclear Extracts--
293 cells were transfected
with vectors expressing EBNA-1, large T-antigen, or a fusion protein
48 h prior to preparation of nuclear extracts. Between 5 × 107 and 108 cells were harvested in 50 ml of
cold phosphate-buffered saline, pelleted, and washed twice with 5 ml of
cold phosphate-buffered saline. The washed pellets were resuspended in
a buffer containing 20 mM HEPES pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM aminoethylbenzoylsulfonyl fluoride (Calbiochem), and
0.1% Triton X-100. After incubation for 10 min on ice, the nuclei were
pelleted by centrifugation at 4 °C for 10 min at 2500 rpm. Nuclei
were resuspended at 108 cell eq/ml in a buffer of the same
composition as the wash buffer, except the NaCl concentration was
raised to 0.42 M. The resuspended nuclei were rocked gently
at 4 °C for 30 min. After transfer to cold Oakridge tubes, nuclei
were centrifuged for 20 min at 20,000 rpm in a Beckman Ty50 rotor. The
supernatant was frozen as aliquots at Protein-DNA Co-immunoprecipitations--
Plasmid 304 (10), which
contains 10 binding sites from the FR, was digested with
ApaLI and EcoRI, and used as probe in
co-immunoprecipitation assays to detect site-specific DNA binding by
EBNA-1. Plasmid 1602, digested with ApaLI and
Bsu36I, was used as probe in co-immunoprecipitation assays
to detect site-specific DNA binding by large T-antigen. A probe was
treated with shrimp alkaline phosphatase (Amersham Pharmacia Biotech),
before 5' end-labeling with [ Measurement of Transcriptional Activation or Repression--
The
plasmids oriP-BamHI-C luciferase, and pSVL were used as
reporters to test the properties of the expressed proteins in the
activation or repression of transcription. Transfections that used the
oriP-BamHI-C-luciferase reporter were performed in 293 cells
or 143B cells, while those that utilized pSVL were performed in (10)1
cells. 4 × 106 293 cells, 2 × 106
143B cells, or 1.6 × 106 (10)1 cells were transfected
with 2 µg of the reporter plasmid and 20 µg of the effector plasmid
under test. Cells were harvested 48 h after transfection, counted,
and lysed at a concentration of 2 × 104 cells/µl in
lysis buffer from the Promega luciferase assay system. Luciferase
assays were performed using extracts from 4 × 105
cells as per the instructions of the manufacturer. Luminescence was
measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory) as described previously (18).
Measurement of DNA Replication--
A total of 10 µg of the
oriP reporter, 994, or the SV40 ori reporter,
1602, was electroporated together with 10 µg of the replication
control plasmid 1381, and 10 µg of the effector plasmid into
107 143B cells. OriP replication was assayed
96 h after electroporation, while SV40 ori replication
was assayed 48 h after electroporation. Plasmid DNAs were
recovered by Hirt extraction (42). The Hirt extracts were digested with
RNase A (100 µg/ml) and proteinase K (200 µg/ml). Samples were then
sequentially extracted with phenol and phenol:chloroform:isoamyl
alcohol (25:24:1). Precipitated samples were resuspended at 5 × 104 cell eq/µl in TE. Samples were incubated with
DpnI for a minimum of 48 h to digest selectively
unreplicated, dam-methylated input DNAs (43).
DpnI-digested samples were digested with BamHI to linearize the plasmids prior to analysis by quantitative, competitive PCR as described (18). Primers used for quantitative, competitive PCR
were 5' end-labeled by T4 polynucleotide kinase using
[ Expression of Fusions between EBNA-1 and SV40 Large
T-antigen--
DNA synthesis from oriP is known to be
restricted to a single round during the S phase of the cell cycle in
human cells (4, 5). Replication of the SV40 replicon has been
biochemically characterized, and the contributions of large T-antigen
to the replication of SV40 DNA have been studied in depth (24, 25, 44,
45). Large T-antigen interacts site-specifically with the origin of
SV40 (20, 22). Large T-antigen possesses a DNA helicase activity, and
interacts with cellular proteins that participate directly in DNA
synthesis (28, 44, 46). In contrast to oriP, the SV40
replicon is not restricted to a single round of DNA replication during
each cell cycle (5, 20). We made fusions between large T-antigen and
EBNA-1 to test whether tethering large T-antigen to oriP is
sufficient to trigger multiple rounds of DNA synthesis during each cell
cycle from oriP.
Schematic representations of EBNA-1, large T-antigen, and the fusion
proteins used in this study are shown in Fig.
1. The derivative of EBNA-1 used in this
study lacks all but 15 residues of the internal Gly-Gly-Ala repeats,
and is referred to as "intact EBNA-1" in this report. Intact EBNA-1
and wild-type EBNA-1 support replication of oriP plasmids,
and activate transcription from EBNA-1-responsive promoters similarly
(3, 47). In two of the fusion proteins described here, intact EBNA-1
was fused to either the amino terminus or the carboxyl terminus of
large T-antigen. These two proteins are referred to as EBNA-1-TAg and
TAg-EBNA-1, respectively. A third fusion protein was constructed by
fusing the domain of EBNA-1 required for site-specific DNA binding
(EBNA-1 amino acids 451-641) to the amino terminus of large T-antigen (N DNA Binding by Fusion Proteins--
Both EBNA-1 and large
T-antigen are site-specific DNA-binding proteins (6, 22, 26). We tested
the fusion proteins for their ability to bind cognate binding sites for
EBNA-1 and large T-antigen using DNA co-immunoprecipitation assays. We
used this assay to avoid the confusion of EBNA-1's linking DNAs to
which it binds such that they do not enter polyacrylamide gels
(17).
Whether EBNA-1 binds DNA site-specifically was tested using the plasmid
304, which contains 10 binding sites from FR. 304 was digested with
ApaLI and EcoRI, and the restriction fragments were 5' end-labeled. Co-immunoprecipitations were performed by the
addition of rabbit anti-EBNA-1 antisera along with protein A-Sepharose
beads, using nuclear extracts from 2.7 × 106 293 cells. The results of this experiment are shown in Fig.
2A, with controls shown in
Fig. 2B. Specific immunoprecipitation of an 896-bp fragment
that contains the EBNA-1-binding sites was observed, demonstrating that
all three of the fusion proteins specifically bind EBNA-1-binding
sites. In contrast, this fragment was not co-immunoprecipitated when
nuclear extracts from 293 cells expressing large T-antigen were used in
conjunction with rabbit anti-EBNA-1 antisera. In three independent
experiments, the FR-containing fragment was preferentially retained in
co-immunoprecipitations from 10-fold to 60-fold better than other
restriction fragments from the same plasmid.
The ability of the fusion proteins to bind binding sites for large
T-antigen was tested using plasmid 1602, which contains the origin of
SV40. 1602 was digested with ApaLI and Bsu36I,
and the restriction fragments were 5' end-labeled.
Co-immunoprecipitations were performed by the addition of hamster
anti-large T-antigen antisera, along with rabbit anti-hamster IgG and
protein A-Sepharose beads using nuclear extracts from 2.7 × 106 293 cells. The result of this assay, which demonstrates
that all three of the fusion proteins specifically co-precipitated a
1695-bp fragment containing the three large T-antigen-binding sites
within the origin of SV40, is shown in Fig. 2C, which was not co-immunoprecipitated when extracts from EBNA-1 expressing cells
were used (Fig. 2D). The SV40 ori-containing
fragment was preferentially retained from 3-fold to 40-fold over other
restriction digest fragments from the same plasmid.
As the fusion proteins synthesized in vivo bind DNA in a
site-specific manner in vitro, we conclude that both the
EBNA-1 and large T-antigen domains of the fusion proteins are likely to
be folded correctly.
Transcriptional Activation of the EBNA-1-responsive EBV BamHI-C
Promoter--
EBNA-1 is known to activate transcriptionally the
BamHI-C promoter of EBV (15, 18). We examined the ability of
the fusion proteins to activate transcription from a luciferase
reporter under the control of the oriP-BamHI-C promoter in
293 cells. The results of this analysis are in Table
I. EBNA-1, EBNA-1-TAg, and TAg-EBNA-1
transactivated the oriP-BamHI-C luciferase
reporter to similar extents (15.6-, 8.2-, and 7.5-fold, respectively). The differences in activation by these proteins reflects their order of
expression, EBNA-1 being expressed at 2-4-fold higher levels than the
fusion proteins. The third fusion protein, N N Transcriptional Repression of the SV40 Early
Promoter--
Repression of the SV40 early promoter by the fusion
proteins is of interest as it requires the large T-antigen moiety of
the fusions to bind cognate binding sites for large T-antigen in a biological context (26). Repression of the early promoter was first
demonstrated in vivo by using temperature-sensitive (tsA) mutants of large T-antigen (49), and later by inhibiting DNA synthesis
in cells infected with wild-type SV40 (50). Transcriptional repression
dependent upon large T-antigen binding the SV40 early promoter has also
been demonstrated in vitro, using extracts from adenovirus-infected cells that express the D2-large T-antigen fusion
(51) or that overexpress large T-antigen (52). We tested the ability of
the three fusion proteins to repress transcription from the early
promoter of SV40 in the luciferase reporter pSVL. This plasmid contains
the SV40 ori in its natural context relative to the early
promoter of SV40. These experiments were performed in murine cells
because SV40 does not replicate in murine cells (20), rendering it
possible to examine transcriptional repression mediated in
vivo by large T-antigen and the fusion proteins without using tsA
mutants or inhibitors of DNA synthesis (50). The results from these
experiments are documented in Table III.
Large T-antigen and N Replication Activities of Fusion Proteins--
Large T-antigen and
the fusion proteins were tested for their ability to support
replication of a plasmid containing the SV40 origin in 143B cells. The
SV40 ori reporter, together with an origin-minus control
plasmid and the appropriate effector plasmid, were electroporated into
143B cells. Hirt extractions were performed on transfected cells
48 h after electroporation (42). This time period was found to be
optimal to detect replication of the SV40 ori reporter in
these cells. Hirt extracts were digested exhaustively with the
restriction endonuclease DpnI to digest selectively the transfected DNA, which is dam-methylated (43). Replication
of the SV40 ori reporter was assessed by measuring the level
of DpnI-resistant, replicated plasmid DNA using
quantitative, competitive PCRs as described under "Experimental
Procedures." Representative results of quantitative, competitive PCR
analysis are shown in Fig. 3A, and summarized in Table IV. Forty-eight
hours after transfection, approximately 370 molecules of replicated
reporter were detected per transfected cell when large T-antigen was
provided as effector. Neither the EBNA-1-TAg or the TAg-EBNA-1 fusion
proteins supported the replication of the SV40 ori reporter
either 48 h after transfection (Fig. 3A, Table IV) or
96 h after transfection (data not shown). The N
The fusion proteins were next tested for their ability to support
replication of an oriP plasmids 96 h after their
introduction into 143B cells. By this time, cellularly synthesized
oriP plasmids are lost from proliferating 143B cells in the
absence of EBNA-1 (12, 18). 143B cells were electroporated with the
oriP replication reporter, origin-minus control plasmid, and
effector plasmid. Hirt extracts were prepared 96 h later and were
digested with DpnI to digest the input bacterially
methylated DNA (42, 43). DpnI-digested Hirt extracts were
used in quantitative, competitive PCR analyses as described under
"Experimental Procedures," to determine the number of replicated
oriP reporter molecules present per transfected cell.
Representative results are shown in Fig. 3B, and summarized
in Table IV. Ninety-six hours after electroporation, 14 molecules of
replicated oriP reporter were detected per transfected cell
when EBNA-1 was provided as effector. Similar levels of replicated oriP reporter were detected when EBNA-1-TAg and TAg-EBNA-1
fusion proteins were provided as effectors (12 and 9 molecules/transfected cell, respectively). No replication was detected
when the N
Our results with the EBNA-1-TAg and TAg-EBNA-1 fusion proteins
demonstrate that the contributions of EBNA-1 to the post-synthetic stabilization of oriP plasmids, and to transcription from
these plasmids, are not impaired by large extraneous protein domains. The results also demonstrate that tethering a helicase competent to
assemble an active DNA synthetic complex to oriP is not
sufficient to activate DNA synthesis from oriP.
EBNA-1 is required for the stable replication of oriP
plasmids in human cells (3, 36). Deletion derivatives of EBNA-1 have
been extensively characterized, and the molecular details of the
interactions between EBNA-1 and oriP DNA have been
elucidated through biochemical and structural studies (54-56). While
the DNA-binding domain of EBNA-1 has been characterized recently to
interact with human RPA in vitro (57), we have failed to
detect this interaction in vivo (12). Additionally, the
DNA-binding domain of EBNA-1, the region of EBNA-1 to which RPA binds,
inhibits the replication of oriP plasmids (18), and RPA also
interacts with proteins that do not participate in DNA synthesis such
as GAL4 and EBNA-2 (58, 59). Thus, the mechanisms by which EBNA-1
facilitates stable replication of oriP plasmids remain
enigmatic. Unlike other viral origin-binding proteins, such as E1 of
BPV, or the large T-antigen of SV40, EBNA-1 has not been found to have
enzymatic activities, such as a DNA unwinding activity, that can
contribute directly to the synthesis of DNA (9, 10), consistent with our recent appreciation that EBNA-1 is not required for DNA synthesis from oriP (12). There are several features of
oriP replication that distinguish it from the replication of
other viral plasmid replicons such as those of SV40 and BPV (1, 20,
21). One central difference between the replication of oriP
and the replication of these other replicons is that
oriP-containing plasmids are synthesized only once every S
phase (5). It is a striking parallel that viral replicons that encode
their own helicases all lack this control exhibited by oriP
and EBNA-1.
Calos and co-workers conducted studies using chimeric plasmids that
contain both oriP and the origin of SV40. Their results indicate that transient expression of large T-antigen within cells that
stably maintain the oriP/SV40 ori chimeric
plasmid increased the copy number of the chimeric plasmid from 10-fold
to 50-fold (60). This result indicates that, while DNA synthesis from
oriP is restricted to a single round per S phase,
oriP present in cis does not impose cellular
regulation on runaway DNA synthesis from an SV40 ori present
on the same plasmid.
We wished to examine whether the cellular control of DNA synthesis from
oriP could be bypassed by localizing to oriP a
protein competent to assemble a DNA synthetic complex that is not
restricted by the cell, and therefore created fusions between EBNA-1
and the large T-antigen of SV40. We chose to fuse the large T-antigen of SV40 to EBNA-1, as it facilitates unfettered DNA synthesis from the
SV40 ori through well studied biochemical mechanisms (44,
45).
We made three fusion proteins, two of which contain intact EBNA-1 and
large T-antigen moieties, while the third contains the domain of EBNA-1
required for site-specific DNA binding and dimerization fused to large
T-antigen. All three of the fusion proteins are competent to bind
binding sites for EBNA-1 and large T-antigen. This has been
demonstrated by DNA co-immunoprecipitation assays, and by examining the
effects of these fusion proteins on transcription from promoters
responsive to EBNA-1 and large T-antigen. That the three proteins bind
both sets of binding sites at levels that correlate with the levels of
expression of the intact fusions indicates that the full-length fusions
function in vivo. While the EBNA-1-TAg and TAg-EBNA-1 fusion
proteins can bind large T-antigen-binding sites in the SV40
ori, they fail to activate replication from the SV40
ori. We speculate that both these proteins either are blocked in their ability to form an active helicase or fail to recruit
cellular proteins required for runaway DNA synthesis from the SV40
ori.
The results we obtained with the N The activities retained by these fusions when bound site-specifically
to DNA, illuminate structural constraints in EBNA-1 and T-antigen.
N Our results also demonstrate that it is possible to fuse large proteins
to EBNA-1 without affecting the activity of EBNA-1 in the activation of
transcription or in the post-synthetic stabilization of oriP
plasmids. Similar fusions of EBNA-1 with the ligand-binding domain of
steroid receptors or GFP may prove useful to study the mechanisms by
which EBNA-1 facilitates the stable replication oriP plasmids.
We thank Steve Johnston and Janet Mertz for
providing a cDNA clone for large T-antigen. We thank Paul Lambert
and Dan Loeb for critiquing this manuscript. The expert technical
assistance of Todd Hopkins in DNA sequencing is gratefully acknowledged.
*
This work was supported by Public Health Service Grants
CA-22442, CA-07175, and T32-CA-09075.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.
The abbreviations used are:
EBV, Epstein-Barr
virus; EBNA-1, Epstein-Barr viral nuclear antigen 1; SV40, simian virus
40; FR, family of repeats; IE, immediate early; CMV, cytomegalovirus; aa, amino acid(s); ORF, open reading frame; bp, base pair(s); PCR, polymerase chain reaction; BPV, bovine papilloma virus; RPA, replication protein A.
2
D. Mackey and B. Sugden, submitted for publication.
Fusions between Epstein-Barr Viral Nuclear Antigen-1 of
Epstein-Barr Virus and the Large T-antigen of Simian Virus 40 Replicate Their Cognate Origins*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/primase and recruit them to the viral origin (27-30), suggesting
one possible mechanism by which the SV40 and BPV replicons bypass the
cellular mechanisms that restrict chromosomal and oriP DNA
synthesis to a single round per cell cycle.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(Life Technologies, Inc.) was used to propagate all
the plasmids used in this study. Plasmid-transformed DH5 cells were
grown in LB supplemented with 100 µg/ml ampicillin (Sigma). Plasmids
were purified by alkaline lysis, followed by isopycnic centrifugation
on cesium chloride density gradients (31).
-32P]ATP (6000 Ci/mmol, 150 mCi/ml) was purchased from NEN Life Science Products.
450 derivative of EBNA-1 was
expressed from plasmid 1160, wherein it is under the control of the CMV
IE promoter (34). 1610, an expression plasmid for the N450-TAg
fusion protein, was constructed by replacing EBNA-1 in plasmid 1607 with the N450 ORF from plasmid 1160. All proteins were expressed
under the control of the CMV IE promoter. The plasmid pcDNA3
(Invitrogen, Carlsbad, CA) was used as a vector control. The ability of
the expressed proteins to activate transcription from the
EBNA-1-responsive BamHI-C promoter of EBV was tested using
the plasmid 1033 (oriP-BamHI-C-luciferase) (18). pSVL, a
plasmid that expresses luciferase under the control of the SV40 ori and
early promoter, was used to monitor the ability of the proteins to
repress the early promoter of SV40 (35). Replication was measured using
994 (36), an oriP plasmid, or 1602, a SV40 ori
plasmid. 1602 was constructed by replacing oriP in 994 with the SV40
ori. 1381, a plasmid that contains neither oriP nor the SV40 ori, and
which can be distinguished by a PCR product-length polymorphism from
994 and 1602, was used as a control in replication assays (18). 1380, a
plasmid distinguished from 994, 1602, and 1381 by a second PCR
product-length polymorphism, was used as competitor DNA in
quantitative, competitive PCR assays (18).
70 °C.
-32P]ATP using T4
polynucleotide kinase. Unincorporated label was removed by
gel-filtration chromatography on Sephadex G-25 spin columns (Boehringer
Mannheim). The DNA concentration of the probe was adjusted to 2.5 ng/µl (304) or 5 ng/µl (1602). 2.5 ng of 304 probe or 5 ng of 1602 probe, 15 µg of sheared salmon sperm DNA, and 10 µg of poly(dI/dC)
were added to 80 µl of a solution containing 13.75 mM
HEPES, pH 7.9, 8.75 mM MgCl2, 2.5 mM dithiothreitol, and 0.05% Triton X-100. To this, 20 µl of nuclear extract were added along with the appropriate
antibodies. For immunoprecipitations to detect site-specific binding by
EBNA-1, 1 µl of a 1:5 dilution of affinity-purified rabbit
anti-EBNA-1 IgG was added. For immunoprecipitations to detect
site-specific binding by large T-antigen, 1 µl of a 1:20 dilution of
hamster anti-large T-antigen antiserum was added, along with 1 µl of
a 1:2 dilution of the affinity-purified rabbit anti-hamster IgG. The
immunoprecipitations were incubated at room temperature for 20 min
before 15 µl of a 20% solution of protein A-Sepharose beads were
added, followed by an incubation for 30 min at room temperature.
Pellets were recovered by centrifugation at 6000 rpm for 5 min. Pellets
were washed five times with 300 µl of a buffer containing 50 mM HEPES, pH 7.9, 150 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100, 0.1 µg/µl
sheared salmon sperm DNA, and 0.05 µg/µl poly(dI/dC). After the
washes, the pellets were resuspended in 100 µl of TE (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0) containing
1% SDS and incubated at 65 °C for 5 min. They were extracted with
an equal volume of phenol. The aqueous phase was extracted sequentially
with equal volumes of phenol:chloroform:isoamyl alcohol (25:24:1), and
chloroform:isoamyl alcohol (24:1). Twenty µg of glycogen were added
to the aqueous phase, and the final salt concentration was adjusted to
0.3 M NaCl. DNA was recovered by precipitation with two
volumes of 100% ethanol, and separated on a 1% agarose gel
electrophoresed in 0.5× Tris-borate-EDTA (31). The gel was fixed in
7.5% trichloroacetic acid, and dried on DE-81 paper using a Bio-Rad
gel dryer for 1 h at 70 °C. Samples were quantified using a
PhosphorImager (Molecular Dynamics).
-32P]ATP to enable detection and quantitation of
products. PCRs in a final volume of 50 µl were performed in 200-µl
microcentrifuge tubes using a thermal cycler with the following
conditions for 20 cycles: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Amplified products were separated on 1.1%
agarose gels electrophoresed in 0.5× Tris-borate-EDTA (31). Gels were
fixed by soaking for 20 min in 7.5% trichloroacetic acid, and dried on
DE-81 paper (Whatman) using a Bio-Rad gel dryer for 1 h at
70 °C. Detected signals were quantified by PhosphorImager analysis
(Molecular Dynamics).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
450-TAg). The EBNA-1 and large T-antigen portions of all three fusion proteins were separated by a short peptide linker, GQSGPGG, which is predicted to be flexible and exposed to an aqueous environment (48). Both these characteristics have been attributed to peptide linkers present between domains of naturally occurring proteins (48).
Immunoblots of extracts from 293 cells expressing EBNA-1, large
T-antigen, or the fusion proteins probed with anti-EBNA-1 and
anti-large T-antigen antisera are shown in Fig. 1 (D and
E). The electrophoretic mobilities of the fusion proteins
are consistent with their predicted molecular weights. The EBNA-1-TAg
and TAg-EBNA-1 fusion proteins appear to be largely intact on the basis
of immunoblot analysis with anti-EBNA-1 and anti-large T-antigen
antisera. There are two bands of significant proportions detected when
extracts from cells expressing N450-TAg are probed on immunoblots
with anti-EBNA-1 antiserum. Because the lower of these two bands is not
detected on immunoblots performed using the anti-large T-antigen antiserum, it is likely to reflect a cleavage event within large T-antigen that removes or disrupts major epitopes in that protein. Quantitative immunoblot analysis performed on extracts from 293 and
murine (10)1 cells indicates that the five effector proteins used in
this study are expressed similarly (within a factor of 10; data not
shown). Their different levels of expression correlate with different
levels of their activities. Next, we examined the ability of these
proteins to bind DNA in a site-specific manner and characterized their
biological activities.
View larger version (23K):
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Fig. 1.
A, schematic representation of EBNA-1.
Wild-type EBNA-1 from the prototypic B95-8 strain of EBV is 641 amino
acids (aa) long. EBNA-1's DNA-binding and dimerization domain lies
between aa 461 and 608 (50, 51). The three DNA-linking domains (17) and
a nuclear localization sequence identified between aa 379 and 386 of
EBNA-1 (50) are indicated. The EBNA-1 protein used in this study
contains only 15 residues of the Gly-Gly-Ala repeat present between aa
90 and 328 of wild-type EBNA-1 protein. This protein, which is 417 aa
long, activates transcription from EBNA-1-dependent
reporters and supports long term replication of oriP
plasmids, similarly to wild-type EBNA-1 (46). B, schematic
representation of SV40 large T-antigen. Large T-antigen is 708 aa long.
The carboxyl-terminal half of large T-antigen contains the domain of
T-antigen responsible for enzymatic ATP hydrolysis. The DNA-binding
domain lies between aa 131 and 259. The ATP hydrolysis-dependent DNA
helicase domain encompasses the domains required for site-specific DNA
binding and ATP hydrolysis. DNA polymerase /primase interacts with large T-antigen within both bracketed regions
marked as POL . C, schematic representation of
the fusions characterized in this study. The EBNA-1-TAg and TAg-EBNA-1
fusions contain intact EBNA-1 and large T-antigen domains fused by a
short linker. They are each predicted to be 1132 aa in length. The
N450-TAg fusion contains aa 379-386 and 451-641 of EBNA-1 fused to
large T-antigen by a short linker. This protein, predicted to be 917 aa
long, contains all of the large T-antigen, and only the DNA-binding
domain and nuclear localization sequence of EBNA-1 (50, 51).
D, immunoblot of extracts from 293 cells transfected with
vectors expressing EBNA-1 or one of the fusion proteins. The blot was
probed with rabbit anti-EBNA-1 antiserum, and developed using alkaline
phosphatase-conjugated goat anti-rabbit IgG. Lane EBNA-1,
extract from 2 × 105 cells; lane
EBNA-1-TAg, extract from 2 × 105 cells;
lane N450-TAg, extract from 7.5 × 105
cells; lane TAg-EBNA-1, extract from 5 × 105 cells. The positions of molecular weight markers are
indicated. E, immunoblot of extracts from 293 cells
transfected with vectors expressing large T-antigen or one of the
fusion proteins. The blot was probed with hamster anti-large T-antigen
antiserum, and developed using alkaline phosphatase-conjugated goat
anti-hamster IgG. Lane Large T-antigen, extract from 5 × 104 cells; lane N450-TAg, extract from
1 × 105 cells; lane EBNA-1-TAg, extract
from 1 × 105 cells; lane TAg-EBNA-1,
extract from 2 × 105 cells. The positions of
molecular weight markers are indicated.
View larger version (56K):
[in a new window]
Fig. 2.
A, results of DNA
co-immunoprecipitation using nuclear extracts from 293 cells
transfected with vectors expressing EBNA-1 or one of the fusion
proteins. 5'-32P-Labeled ApaLI/EcoRI
restriction fragments of plasmid 304 were incubated with nuclear
extracts for 20 min at room temperature. Affinity-purified rabbit
anti-EBNA-1 antiserum was added to the incubations, and protein
A-Sepharose beads were used for immunoprecipitation as described under
"Experimental Procedures." The source of the nuclear extract is
indicated above each lane. The 896-bp restriction fragment from plasmid
304 that contains the FR is indicated by the arrowhead.
B, control DNA co-immunoprecipitations performed using
nuclear extracts from 293 cells transfected with a vector expressing
EBNA-1 or a vector expressing large T-antigen, and 5'-32P
labeled restriction fragments from plasmid 304. The assay was performed
as described in A. C, results of DNA
co-immunoprecipitation using nuclear extracts from 293 cells
transfected with vectors expressing large T-antigen or one of the
fusion proteins. 5'-32P-Labeled
ApaLI/Bsu36I restriction fragments of plasmid
1602 were incubated with nuclear extracts for 20 min at room
temperature. Hamster anti-large T-antigen and rabbit anti-hamster IgG
were added to the incubations, and protein A-Sepharose beads were used
for immunoprecipitation as described under "Experimental
Procedures." The source of the nuclear extract is indicated
above each lane. The 1695-bp restriction fragment from plasmid
1602 that contains the SV40 ori is indicated by the
arrowhead. D, control DNA co-immunoprecipitations
performed using nuclear extracts from 293 cells transfected with a
vector expressing large T-antigen or a vector expressing EBNA-1, and
5'-32P-labeled restriction fragments from plasmid 1602. The
assay was performed as described in C.
450-TAg, did not
activate transcription from this reporter. This result was expected
because the N450 derivative of EBNA-1 has been demonstrated to be
severely impaired in its ability to trans-activate
the oriP-BamHI-C promoter (18). We also observed
transactivation of the oriP-BamHI-C luciferase reporter by
large T-antigen. We think that this low level of transactivation
(3-fold over background) is likely to be nonspecific, as large
T-antigen has been shown to activate promiscuously several promoters
that do not contain binding sites for large T-antigen, perhaps by
interacting with transcriptional repressors (32). These results
indicate that the EBNA-1-TAg and TAg-EBNA-1 fusion proteins retain the
ability to bind cognate EBNA-1-binding sites in vivo, and
activate transcription while bound at these sites, similarly to these
functions of unfused EBNA-1.
EBNA-1 and the fusion proteins activate transcription from an
oriP-BamHl-C luciferase in 293 cells
450-TAg Inhibits Transcriptional Activation by Wild-type
EBNA-1--
The N450 derivative of EBNA-1 is a potent
dominant-negative inhibitor of wild-type EBNA-1's capacity to activate
transcription from the oriP-BamHI-C promoter. The
ability of N450 to bind cognate EBNA-1-binding sites is critical for
this protein to function as a dominant-negative inhibitor of
transcription (18). We examined whether the N450-TAg fusion protein
also functions as a dominant-negative inhibitor of the activation of
transcription by wild-type EBNA-1 in 143B cells (Table
II). Co-expression of N450-TAg with
EBNA-1 reduced transcriptional activation of the
oriP-BamHI-C luciferase reporter by EBNA-1 to approximately
10% of the uninhibited level. We observed a similar inhibitory effect
in experiments in which EBNA-1 and N450 were co-expressed (Table
II). These experimental observations are consistent with the
interpretation that N450-TAg inhibits transcriptional activation by
EBNA-1 by binding cognate EBNA-1-binding sites in the
oriP-BamHI-C luciferase reporter. The observed inhibition
indicates that N450-TAg can bind these sites in vivo.
N450-TAg is a dominant-negative inhibitor of the ability of EBNA-1
to transactivate oriP-BamHI-C luciferase in 143B cells
450-TAg repressed transcription from this
promoter relative to the control containing the vector alone,
indicating that they each bind cognate binding sites in
vivo. Relative to the effect of EBNA-1 on pSVL, the TAg-EBNA-1 and
EBNA-1-TAg fusion proteins significantly inhibit transcription from
this reporter, with a p value of 0.032 as measured by the
Wilcoxon rank-sum test (53). Thus, these two fusions also bind cognate
binding sites for large T-antigen in a biologically relevant context
in vivo.
Large T-antigen and the fusion proteins repress transcription from a
SV40 early promoter-luciferase reporter in murine (10)1 cells
450-TAg fusion
protein supported replication of the SV40 ori reporter to an
extent similar to that for large T-antigen (approximately 270 molecules/transfected cell). This result indicates that N450-TAg,
which has the ability to bind EBNA-1 DNA-binding sites (Fig.
2A, Table II), is competent to assemble a replication complex at the SV40 ori in a manner indistinguishable from
large T-antigen.
View larger version (33K):
[in a new window]
Fig. 3.
A, replication of plasmids containing
SV40 ori reporter measured using quantitative, competitive PCR 48 h after electroporation. 10 µg each of the SV40 ori
reporter, the origin-minus control, and the effector plasmid indicated
above each set of PCRs were electroporated into 107 143B
cells. The positions of the amplified fragments from the competitor
DNA, the SV40 ori replication reporter, and the origin-minus
control are indicated. The amount of competitor DNA used in each PCR is
indicated below each lane. PCRs performed with cells transfected with
the EBNA-1, EBNA-1-TAg and TAg-EBNA-1 effector plasmids contained
DpnI-digested Hirt extract equivalent to 105
cells. PCRs performed with cells transfected with the large T-antigen
and N 450-TAg effector plasmids contained DpnI-digested
Hirt extract equivalent to 104 cells. B,
replication of plasmids containing oriP reporter measured
using quantitative, competitive PCR 96 h after electroporation. 10 µg each of the oriP reporter, the origin-minus control,
and the effector plasmid indicated above each set of PCRs were
electroporated into 107 143B cells. The positions of the
amplified fragments from the competitor DNA, the oriP
replication reporter, and the origin-minus control are indicated. The
amount of competitor DNA used in each PCR is indicated below each lane.
Each PCR contained DpnI-digested Hirt extract equivalent to
105 cells.
Replication of oriP and SV40 ori reporters in 143B cells
450-TAg effector was used, although this protein is
capable of binding oriP as shown by the results in Fig.
2A and Table III, and supports DNA synthesis from the SV40
ori (Fig. 3A, Table IV). N450-TAg behaves like
the N450 derivative of EBNA-1, which has been shown to support
oriP replication to less than 1% the levels of wild-type EBNA-1 at 96 h after electroporation (18).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
450-TAg fusion protein are
informative because this protein facilitates multiple rounds of DNA
synthesis from SV40 ori in a manner indistinguishable from unfused large T-antigen. N450-TAg can also bind oriP, as
demonstrated by DNA co-immunoprecipitations, and by its ability to
function as dominant-negative inhibitor of EBNA-1. While N450-TAg
has the ability to assemble an unregulated DNA synthetic complex at the
SV40 ori, it does not activate synthesis from
oriP. Thus, DNA synthesis from oriP is is
regulated by the cell in a manner that the SV40 replicon is not, and
cannot be subverted by localizing to oriP a helicase
competent to assemble an unregulated DNA synthetic complex. This result
is consistent both with our recent appreciation that EBNA-1 acts
post-synthetically to maintain replication of oriP plasmids
in proliferating cells (12), and that multiple elements within the SV40
ori are necessary for its function (22).
450-TAg supports synthesis of the SV40 ori, indicating that this fusion forms the hexameric structure that T-antigen requires
for its helicase activity (61). N450-TAg is reminiscent of the
naturally occurring, fully functional D2-TAg fusion in which
approximately 130 additional residues are placed close to the amino
terminus of large T-antigen (19). The structure of wild-type dimeric
T-antigen bound to its cognate DNA has not been determined, nor has
that of its hexamer. What is apparent from these studies is that
T-antigen can accommodate at least 209 additional residues at its amino
terminus and still function. That both EBNA-1-TAg and TAg-EBNA-1
function to maintain replication of oriP plasmids indicates
that these molecules which act as dimers can do so with 715 additional
residues at either terminus. EBNA-1 does not bind FR cooperatively, and
its 20 30-bp binding sites would lead to a closely packed array of
EBNA-1, all on one face of the bound DNA. That the fusions function to
maintain replication indicates that either adjacent binding sites
need not be occupied for EBNA-1's functional contributions, or that
the large tethered T-antigen moieties do not hinder those contributions.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 608-262-6697;
Fax: 608-262-2834; E-mail: sugden{at}oncology.wisc.edu.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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