ABSTRACT
OriLyt, the
cis
-acting element of Epstein-Barr virus lytic origin of replication, consists of upstream and downstream
components. The upstream component plays a dual role in transcription and replication. The downstream component contains a homopurine-homopyrimidine sequence which forms an H palindrome. We show that the
downstream component can adopt a triple helix structure
in vitro
, that the 5
'
border of the homopyrimidine sequence is sensitive to P1 nuclease when carried by a supercoiled plasmid and that an oligonucleotide complementary
to the homopyrimidine strand is taken up by a plasmid carrying the OriLyt H palindrome. We also show
that all mutations which alter the H palindrome impair both oligonucleotide
uptake and OriLyt-dependent replication. Interestingly, compensatory mutations which restore an H palindrome also restore oligonucleotide uptake by the mutated plasmids and their OriLyt-dependent replication. Thus, there is a strong correlation between the inability of the OriLyt H palindrome to form a non-B-DNA structure
in vitro
and impairment of OriLyt-dependent replication. This suggests that the presence of a non-B-DNA structure in the OriLyt downstream component is required for OriLyt-dependent replication.
Epstein-Barr virus (EBV) is a human gammaherpesvirus which is associated with different malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma (for a review see
1
).
In vivo
it is supposed that EBV infection is latent in B lymphocytes but productive in epithelial cells within the oropharynx (
2
).
In vitro
EBV infects and immortalizes human B lymphocytes. In such immortalized B cells
the viral genome is maintained as an extrachromosomal, nuclear multiple copy
episome (
3
), from which 11 genes are expressed, defined as type III latency. The products of these viral genes are implicated in B cell immortalization and in EBV genome persistance. The
cis
-acting elements which mediate DNA replication during type III latency have
been identified and called Ori-P (for plasmid origin of replication). Viral DNA replication occurs once
per cell cycle (
4
), proceeds bidirectionally from Ori-P (
5
) and is dependent both on cellular proteins and on the viral protein EBNA1 (
6
,
7
).
In vitro
EBV production occurs spontaneously in a few infected B cells and can be
induced in many cells by treatment with agents such as 12-
O
-tetradecanoylphorbol-13-acetate associated with butyrate or by cross-linking of surface immunoglobulin (
8
,
9
). The productive cycle is characterized by the sequential activation of viral
gene expression, which begins with expression of the viral transcription
factors EB1 (also called Z, ZEBRA or Zta) and R (also called Rta) (
10
-
14
). Once made, R and EB1 induce expression of the EBV early genes, whose products are implicated in amplification of the viral genome via an origin of replication different from Ori-P, called OriLyt (Fig.
1
) (
15
). A co-transfection-replication assay has demonstrated that seven EBV proteins are required for OriLyt-dependent replication: BALF5 (DNA polymerase), BMRF1 (polymerase processivity factor), BALF2 (single-stranded DNA binding protein), BSLF1 (primase), BBLF4
(helicase), BBLF2/3 (helicase/primase-associated protein) and EB1 (
16
,
17
). Several non-essential proteins with enzymatic activities involved in the biochemical
pathways of nucleotide synthesis and phosphorylation are also encoded by the
virus. These replication proteins, with the exception of EB1, have homology with known herpes simplex virus (HSV) and cytomegalovirus (CMV) replication proteins (
18
,
19
). One factor that has not been identified in EBV is the origin binding protein,
the equivalent of HSV UL9 protein. Although the viral
trans
-acting factors and the
cis
-acting elements have been identified, the mechanisms which underlie
herpesvirus replication are not yet understood.
The EBV OriLyt overlaps with the divergent promoters of the BHLF1 and BHRF1
early genes (Fig.
1
). OriLyt consists of two essential and several auxiliary elements which are
required for full activity. Auxiliary elements which are only poorly defined
are non-essential but influence the efficiency by which OriLyt- containing plasmids replicate in transient replication assays (
15
). The two essential or core elements, called respectively the upstream and the downstream components, constitute the minimal origin of DNA replication (Fig.
1
) (
20
).
The upstream OriLyt component is localized within the promoter region of the
BHLF1 gene, which contains four Z-responsive elements (ZREs). Mutation of these sites completely abolishes OriLyt-dependent replication, indicating that EB1 binding to these sites is
required to mediate OriLyt-dependent replication (
21
). In effect, exchange of the OriLyt ZREs with other activator binding sites
does not rescue replication, even if transcription of the BHLF1 gene is
unaffected. The EB1 domain implicated in activation of replication seems to co-localize with the transcriptional activation domain (
22
).
The downstream OriLyt component is located 440 bp from the upstream component
and is 90 bp long. In contrast to the upstream component, the downstream
component is only dedicated to activation of OriLyt-mediated DNA replication and does not appear to be involved in
transcriptional activation of the BHLF1 and BHRF1 genes. Several cellular
proteins, including SP1, bind specifically to this element
in vitro
, but they are probably not implicated in OriLyt-dependent replication (
23
).
The contribution of the downstream component in OriLyt- dependent replication is as yet unknown. It contains a pyrimidine-rich sequence which can be divided into two parts (Fig.
2
A). The first part represents a homopurine-homopyrimidine sequence, made up of a mirror repeat, also called an H
palindrome, in which any introduced mutation impairs OriLyt-dependent replication in a transient replication assay (
20
,
23
). The second part, located 3' of the homopurine-homopyrimidine sequence is C rich. We have focused our attention
on the homopurine-homopyrimidine sequence element and we show that
in vitro
it can form a triple helix. We also present experimental evidence demonstrating
that every mutation in the H palindrome that impairs formation of the triple
helix
in vitro
also impairs OriLyt-dependent replication in a transient replication assay and that mutations
which do not dramatically alter folding of the H palindrome
in vitro
allow some replication of plasmids carrying these mutations. Thus, OriLyt-dependent replication of the EBV genome appears to require that the homopurine-homopyrimidine sequence of the OriLyt downstream component adopts a non-B-DNA structure. It also appears probable that a
specific, as yet unknown,
trans
-acting factor contributes to activation of this element.
D98HR1 cells were derived from a somatic cell hybrid made between the EBV genome-positive Burkitt lymphoma cell line P3HR1 and the human epithelial cell
line D98 (
24
). This adherent cell line was maintained in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum.
Plasmid DNA was prepared by alkali lysis and supercoiled DNA molecules were
purified by ethidium bromide/cesium chloride centrifugation. Plasmid p968.22,
which carries the complete OriLyt element (EBV B95-8 nucleotide coordinates 48848- 56084;
25
) has been described in detail elsewhere (
20
). Oligonucleotide-directed mutations were introduced into this plasmid as described
elsewhere (
20
). The mutations relevant to this study are shown in the figures, which include
sequence information on specific mutations. Plasmid p1562 contains the OriLyt downstream element (EBV B95-8 nucleotide coordinates 53337-53428;
25
) cloned in the pBluescript SK- (Stratagene) vector.
The different single-stranded oligonucleotides were radiolabeled with
32
P using T4 polynucleotide kinase and then purified on a 10% polyacylamide gel
according to standard procedures. Cstr refers to the pyrimidine-rich DNA strand of the OriLyt downstream element. Gstr refers to the other
purine-rich DNA strand. The different oligonucleotides used in the
oligonucleotide uptake assays have been previously described, as have the
mutant sequences and wild-type plasmids containing the OriLyt downstream element (
23
). The M13 sequencing primers -40 and reverse sequencing primer were purchased from Pharmacia LKB.
Supercoiled plasmid pBSK or p1562 was incubated with different amounts (between
1 * 10
-4
mg/ml and 1 * 10
-2
mg/ml) of P1 nuclease (Boehringer) in 50 [mu]l P buffer (10 mM Tris-HCl, pH 7.6, 10 mM MgCl
2
, 50 mM NaCl) for 10 min at 37oC. The reaction was stopped by addition of EDTA (37 mM final
concentration). The DNA was purified by phenol/chloroform extraction and
digested with
Pvu
II. The resulting DNA fragments were separated on 1% agarose gels. Gels were stained with ethidium bromide and the DNA fragments visualized with UV light.
The P1-treated DNAs were incubated in 17 [mu]l buffer H (25 mM Tris-HCl, pH 7.2, 5 mM MgCl
2
) with 2 * 10
5
c.p.m. of a 5'-end-labeled primer for 4 min at 90oC and then for 20 min at 45oC. The primer extension reaction was performed on
the C-rich strand using the reverse sequencing primer and on the G-rich strand using the -40 primer. After addition of 5 mM dithiothreitol and 4 mM
dNTP, 5 U Klenow enzyme were added and the DNA was allowed to polymerize for 10
min at 37oC. The reaction was stopped by addition of 10 [mu]l of a solution containing 20 mM EDTA and 0.5 M NH
4
Ac. The primer-extended products were resolved on a 6% polyacrylamide-urea denaturing gel.
Aliquots of 200 ng supercoiled (S) or linearized (L) plasmid DNA were incubated
for 2 h at 25oC in 10 [mu]l Hyb. buffer (10 mM Tris-HCl, pH 7.5, 50 mM sodium acetate, 2.5 mM MgCl
2
) containing 1 * 10
4
c.p.m. radiolabeled oligonucleotide. The reaction mixtures were then analyzed
on a 0.8% agarose gel in TAE buffer containing 20 mM sodium acetate, pH 7.5.
After ethidium bromide staining, the positions of the supercoiled and
linearized plasmids were visualized with UV light. The gel was then dried under
vacuum onto DE81 paper (Whattman) and autoradiographed to detect labeled
oligonucleotides.
Transient replication assays were performed in D98HR1 cells by co-transfections of plasmids carrying either the wild-type (p968.22) or the mutated OriLyts together with plasmid pCMV-BZLF1, which efficiently expresses EB1 protein (
15
). Two days after co-transfection, DNA was prepared by the Hirt technique, digested with
Dpn
I and
Bam
HI, subjected to electrophoresis through a 0.7% agarose gel, transferred to a
nylon N+ membrane (Amersham) and probed with random primer
32
P-labeled pUC19. The replication efficiency of plasmids carrying OriLyt was
quantified by scanning the Southern blot autoradiogram.
The OriLyt downstream component contains a homopurine- homopyrimidine sequence which forms a mirror repeat element, also called
the H palindrome (Fig.
2
A). Analysis of the nucleotide sequence of the H palindrome suggested that it
might form a DNA triplex structure. Formation of an intramolecular triplex
structure
in vitro
occurs on supercoiled DNA and induces a single-stranded DNA loop sensitive to specific nucleases (
26
). We thus tested whether the homopurine-homopyrimidine sequence of the OriLyt downstream component present in
supercoiled DNA (p1562) is sensitive to the single-strand-specific nuclease P1. The supercoiled plasmid p1562 was treated with
increasing amounts of P1 nuclease and then digested with
Pvu
II (Fig.
2
B). As shown in Figure
2
C, two fragments of 2.5 and 0.5 kb were generated by digestion of plasmid p1562
with
Pvu
II (lane 5). In the presence of P1 nuclease two additional fragments of ~0.2 kb were also generated, as expected if the OriLyt sequence carried by
the supercoiled plasmid contains a single-stranded DNA region (lanes 6 and 7). With the pBSK supercoiled vector,
devoid of the OriLyt downstream component, only the two fragments of 2.5 and
0.45 kb were detected (lanes 1-3), indicating that the OriLyt downstream component cloned in the pBSK vector was necessary for P1 nuclease sensitivity. When the p1562 or pBSK plasmids were digested with
Pvu
II before treatment with P1 nuclease, the two small 0.2 kb fragments were not
observed (lanes 4 and 8), demonstrating that sensitivity of the plasmid to the
action of P1 nuclease was dependent on supercoiling. These results suggest that
single-stranded DNA is present in the OriLyt downstream component carried by a
supercoiled plasmid.
If the 5'-part of the C strand in the H palindrome is single stranded, then it should hybridize with a radiolabeled complementary single-stranded oligonucleotide. To test this hypothesis, several
radiolabeled single-stranded oligonucleotides complementary to the OriLyt downstream component
(Fig.
4
A) were incubated with supercoiled or linearized plasmid p1562. In an agarose
gel the hybridized radiolabeled oligonucleotide should co-migrate with the supercoiled p1562 plasmid but not with the linearized
plasmid. As shown in Figure
4
B, only two single-stranded oligonucleotides, T1.2.G (lane 2) and T2.G (lane 3), detectably
hybridized with the supercoiled plasmid p1562. However, T2.G and T2.C did not hybridize with linearized plasmid p1562 (Fig.
4
B, lanes 11 and 12) nor with supercoiled plasmid pBSK (Fig.
4
B, lanes 13 and 14). Moreover, uptake of oligonucleotide T1.2.G by the
supercoiled plasmid p1562 was less efficient than that of oligonucleotide T2.G
(Fig.
4
B, compare lanes 2 and 3). It should be noted that the sequences of the T1.2.G
and T2.G oligonucleotides are complementary to a region in the OriLyt
downstream component that was strongly sensitive to P1 nuclease (Fig.
3
C). These results strongly indicate that the 5'-border of the C-rich sequence of the OriLyt downstream component is single
stranded when carried by a supercoiled plasmid. Taken together, these results
suggest that DNA in the OriLyt downstream component has the potential to form a
triplex structure
in vitro
when carried by a supercoiled plasmid and that the 5'-part of the C strand is likely to be single stranded (Fig.
4
C).
We next evaluated whether mutations in the H palindrome of the OriLyt downstream
component that impaired replication in a transient replication assay (Fig.
5
A;
23
) also impaired uptake of oligonucleotide T1.2.G
in vitro
. As shown in Figure
5
B, oligonucleotide T1.2.G hybridized very efficiently with supercoiled plasmid p1562 (lane 1) but not with H palindrome mutants that were inactive in
the replication assay (lanes 2-4 and 7). Mutant p1568, whose replication efficiency was only 5% of that
of plasmid p1562, also hybridized weakly with oligonucleotide T1.2.G (lane 6).
In the replication assay, mutant p1567 was 80% less efficient than the wild-type OriLyt plasmid p1562 and interestingly uptake of radiolabeled
oligonucleotide T1.2.G by this mutated plasmid was ~20% of that observed with the wild-type OriLyt plasmid p1562 (lane 5). Thus there seems to be a good
correlation between efficiency of replication of the H palindrome mutants and
efficiency of uptake of oligonucleotide T1.2.G by these mutants.
If our conclusion is valid, any mutation introduced into the 5'-part or into the 3'-part of the mirror repeat of the OriLyt downstream
component should impair formation of a triple helix
in vitro
and the function of OriLyt
in vivo
. However, a compensatory mutation which recreates a mirror repeat but does not
modify the homopurine-homopyrimidine nature of the sequence should restore the structure and
possibly the function of OriLyt. We generated the mutants depicted in Figure
6
A and first analyzed the efficiency of oligonucleotide T1.2.G uptake by
supercoiled plasmids carrying the mutations. As already shown, an OriLyt
downstream component containing the wild-type mirror repeat cloned in replication test plasmid p968 took up
oligonucleotide T1.2.G (Fig.
6
B, lane 1). Mutants with a C -> T transition in either the left (p1648 and p1658) or right (p1637 and
p1662) half of the mirror repeat (Fig.
6
A) did not hybridize with oligonucleotide T1.2.G (Fig.
6
B, lanes 2, 3, 5 and 6). In agreement with our prediction, mutants in which the
mirror symmetry was restored (p1649 and p1659; Fig.
6
A) took up oligonucleotide T1.2.G (Fig.
6
B, lanes 4 and 7). However, uptake of T1.2.G by mutants p1649 and p1659 was less
efficient than that observed with the wild-type mirror repeat. Nevertheless, our results demonstrate that homopurine-homopyrimidine mirror sequences similar to that found in the OriLyt
downstream component could form a triplex
in vitro
.
Chromosomal or viral origins of replication are composed of different
cis
-acting elements consisting of a putative DNA unwinding element aligned
with clusters of scaffold-associated region, autonomously replicating sequence and pyrimidine tracts
(
27
,
28
). These
cis
-acting elements are sites of interaction with origin-specific DNA binding proteins, which initiate DNA replication in
this region (
29
). DNA replication origins are often associated with transcriptional units (
30
). The origin of replication OriLyt of EBV seems to be structurally slightly different from other eukaryotic
origins of replication. It is composed of two essential core elements which lie
>400 bp apart and are unique in their DNA sequence. However, their function in OriLyt-dependent replication is still unknown. The upstream component co-localizes with a basal promoter element which is concomitantly activated with
OriLyt. Induction of this component for both transcription and replication is
transactivated by EB1 (
20
,
21
). The downstream component has been shown previously to contain binding sites
for several cellular proteins, including Sp1 (
23
). However, these factors do not appear to be directly implicated in OriLyt-dependent replication. In an attempt to understand the function of the downstream
component, we show here that the downstream component contains an H palindrome
capable of forming a non-B-DNA structure
in vitro
which is most likely a triple helix. In addition, we found that all mutations
which disturb the structure of the OriLyt downstream component in a supercoiled
plasmid
in vitro
also impair the replication efficiency
in vivo
of a plasmid carrying an OriLyt that contains these mutations. Thus it seems
that the OriLyt downstream component has to adopt a particular structure in
order to be functional.
DNA triplexes are formed by polypyrimidine/polypurine sequences. In this
structure a DNA strand (donor strand) from one half of the sequence folds into
the major groove of the other half duplex, forming Hoogsteen base pairs and
leaving the other strand in a single-stranded state. Some triplex structures are formed only at acidic pH,
while others are stabilized by the presence of magnesium in the buffer and can
form at neutral pH. Triplex DNA formation
in vitro
always requires DNA supercoiling to compensate for the high nucleation energy required (reviewed in
26
,
31
). Our results indicate that the OriLyt downstream component can adopt a non-B-DNA structure
in vitro
in a supercoiled plasmid. The structure formed at pH 7.6, which is an
indication that triplex formation does not require protonation of the C-rich strand to become the donor strand. The DNA triplex is of a Pu-Py/Pu type. Confirmation of this hypothesis was given by fine
mapping of the P1 nuclease-hypersensitive sites and by oligonucleotide uptake by supercoiled
plasmids. Indeed, the pyrimidine-rich strand was more sensitive to P1 nuclease digestion than the purine-rich strand and might therefore be single stranded. This was
confirmed by the observation that only oligonucleotides complementary to the
pyrimidine strand were taken up by a supercoiled plasmid carrying the H
palindrome. One interpretation of these results is that the DNA triplex
structure formed utilizes the homopurine strand as the donor strand and leaves
the homopyrimidine strand single stranded, as shown in Figure
4
C. Surprisingly, oligonucleotide T3.G, which shares some sequences with
oligonucleotide T2.G, was not, or at least only very weakly, taken up by
supercoiled plasmid p1562 (Fig.
4
B, lane 4). This oligonucleotide is composed of a guanine-rich DNA sequence which
in vitro
has been shown to be capable of associating to form a stable, parallel four-stranded structure termed G4 DNA under our assay conditions (
32
). Folding of oligonucleotide T3.G could explain why it could not interact with
supercoiled plasmid p1562. Formation of the triplex DNA structure required only
the presence of the mirror repeat sequence and did not appear to be influenced
by the C-rich region which is located near the H palindrome. In effect, a plasmid
carrying only the H palindrome sequence took up oligonucleotide T1.2.G as
efficiently as a plasmid carrying the H palindrome sequence together with the C-rich sequence (not shown). However, The C-rich sequence is also important for function of the downstream
component and it is possible that these two elements contribute differently to
OriLyt-dependent replication. We estimate that <10% of the supercoiled plasmids used in the oligonucleotide uptake assay
were in the triple-stranded form. This low proportion can be explained by the short length of
the homopurine-homopyrimidine sequence implicated in the triplex structure and by the
fact that the triplex structures are in rapid equilibrium with normal B-DNA structures. This rapid equilibrium could be detrimental to interaction between the plasmid and the
oligonucleotide.
We do not, however, have strong evidence that the DNA triplex exists
in vivo
. In cells latently infected with EBV, no S1-hypersensitive sites were detected in the OriLyt downstream component (not shown). This does not mean that this region is not folded like H-DNA. It could be that the triplex is present but protected from
nuclease attack by a cellular(s) factor(s). It is also conceivable that the DNA
triplex structure is only transient. The fact that the OriLyt
cis
-acting elements co-localize with two divergent promoters could suggest that the
transcription process can influence and/or stabilize formation of a non-B-DNA structure. It is well established that negative supercoiling is
an energetic prerequisite for triplex formation under physiological conditions.
Moreover, it has been shown that actively transcribing RNA polymerase complexes
generate positive supercoiling in front of and negative supercoiling behind the
elongation complex: this has been observed
in vitro
and
in vivo
in both prokaryotes and eukaryotes (
33
-
38
). Thus, transcription induced at the BHLF1 or BHRF1 promoter could facilitate
DNA replication from OriLyt by introducing negative supercoiling favoring DNA
triplex formation in the OriLyt downstream component.
DNA secondary structure has been shown to be important for replication origin
activity of some bacterial plasmids, phages and eukaryotic viruses (
39
-
42
) and may be involved in initiation of DNA synthesis in mammalian cells (
43
). In this study we have observed that all mutations which disturb the DNA
sequence of the H palindrome and impair formation of the DNA triplex in the
OriLyt downstream component
in vitro
also alter the replication efficiency
in vivo
of plasmids carrying the mutated OriLyt. These results suggest that the
structure of this component is probably important for its function in
replication. A direct implication of a non-B-DNA structure in EBV OriLyt replication is strongly suggested by
comparing the effects of mutations that change both the sequence and the
structure of the H palindrome (p1637, p1648, p1662 and p1658) with those that
alter only the sequence of the H palindrome (p1649 and p1659). Replication of
plasmids which contain a 1 bp change in the left or right side of the mirror
repeat and modify both the sequence and structure is impaired. Compensatory
double mutants in the left and the right side of the mirror repeat retain part
of the DNA triplex structure and replicate. However, the replication efficiency
of these mutants is greatly reduced, suggesting that the sequence of the H palindrome, in addition to its structure, is important for efficient replication. One
possibility is that the mutations reduce the stability of base pairing in the
triplex compared with the wild-type sequence. In effect, in the mutants we have substituted two cytosines
by two thymines, which is known to reduce folding stability.
In vitro
these compensatory mutations are also detrimental to the structure.
Alternatively, the structured sequence may contain a recognition motif involved
in origin function. This is consistent with the observation that exchange of
the OriLyt H palindrome for a different homopurine/homopyrimidine sequence is detrimental to replication (not shown).
Taken together, our results suggest that both the sequence and structure of the
OriLyt downstream component contribute to EBV replication during the lytic
cycle. A similar component to the OriLyt downstream component, called the Y
block, is present in the OriLyt of human cytomegalovirus, which, like EBV, does
not have an origin binding protein. Interestingly, Huang
et al
. have shown that some transcripts, called SRT, overlap the Y block and are
complementary to the C-rich strand (
44
). These RNAs might cooperate with the Y block to initiate or stabilize strand
separation, as observed in mitochondrial heavy strand replicator, in which a
similar C-rich component, called the CSBII box, promotes formation of a stable RNA-DNA hybrid forming a locally opened region, a D loop, coincident
with the origin of DNA synthesis (
45
,
46
). This type of system could explain, at least in part, the relative complexity
of OriLyt and the lack of a requirement for a specific origin binding protein.
We thank Dr W.Hammerschmidt for continuous interest and helpful suggestions. We
thank Conrad B.Bluink for reading the manuscript and our colleagues for
discussions. S.P.-S. was supported by an MRT fellowship. Research in the laboratory is
financially supported by INSERM and the Association pour la Recherche contre le
Cancer (contract ARC 2049 to H.G).
*To whom correspondence should be addressed. Tel: +33 472 72 81 76; Fax: +33 472
72 86 86; Email: hgruffat@popserver.ens-lyon.fr
REFERENCES
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