Epstein-Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments.
ABSTRACT Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.
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ABSTRACT: The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs.PLoS Pathogens 08/2013; 9(8):e1003506. · 8.14 Impact Factor
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ABSTRACT: Nuclear domains 10 (ND10), alternatively termed PML nuclear bodies (PML-NBs) or PML oncogenic domains (PODs), have been discovered approximately 15 years ago as a nuclear substructure that is targeted by a variety of viruses belonging to different viral families. This review will summarize the most important structural and functional characteristics of ND10 and its major protein constituents followed by a discussion of the current view regarding the role of this subnuclear structure for various DNA and RNA viruses with an emphasis on herpesviruses. It is concluded that accumulating evidence argues for an involvement of ND10 in host antiviral defenses either via mediating an intrinsic immune response against specific viruses or via acting as a component of the cellular interferon pathway.Biochimica et Biophysica Acta 11/2008; · 4.66 Impact Factor
Epstein–Barr virus origin of lytic replication
mediates association of replicating episomes with
promyelocytic leukaemia protein nuclear bodies
and replication compartments
Wolfgang Amon,3 Robert E. White and Paul J. Farrell
Paul J. Farrell
Department of Virology, Imperial College Faculty of Medicine, St Mary’s Campus, Norfolk Place,
London W2 1PG, UK
Received 6 October 2005
Accepted 6 January 2006
Epstein–Barr virus (EBV) establishes a latent persistence from which it can be reactivated to
undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it
is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the
the viral genome forms replication compartments, which are usually adjacent to promyelocytic
leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain–enhanced
green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline
operator sequence array. ori lyt mediated the production of plasmid replication compartments
that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were
visualized in the same cells and replicated in similar regions of the nucleus, further supporting the
validity of the plasmids for studying late-gene regulation.
Like all herpesviruses, the life cycle of Epstein–Barr virus
(EBV) is biphasic. After EBV establishes latency in the
human host, it can be reactivated to enter the lytic cycle.
episome. Similar to the host chromosomal DNA, it is pack-
aged in nucleosomal arrays with cellular histones (Dyson &
Farrell, 1985), replicates once during the S phase of the cell
cycle (Adams, 1987) and is divided between the daughter
cells during mitosis (Yates et al., 1984).
In contrast to latent replication, multiple rounds of replica-
tion are initiated within the viral origin of lytic replication
(ori lyt) during the lytic cycle (Hammerschmidt & Sugden,
1988). ori lyt-mediated replication involves two different
meric plasmid progeny (theta mode). In the second phase,
the viral genome is amplified 100- to 1000-fold through a
rolling-circle mechanism, leading to large, head-to-tail con-
EBV genomes (Pfu ¨ller & Hammerschmidt, 1996). Late-gene
to the same inhibitors (phosphonoacetic acid and acyclovir)
that block lytic replication (Summers & Klein, 1976).
We recently showed that the expression of late-lytic genes
depends on the presence of ori lyt in cis on stably transfected
luciferase reporter plasmids (Amon et al., 2004). Efficiency
of lytic replication, late-gene expression and sensitivity to
inhibitors were functions of the size of ori lyt used. With the
so-called big ori lyt (bol), which comprised the BamHI H
fragment of EBV, we properly reconstituted lytic DNA
replication and late-gene expression on small plasmids. Our
results contrasted with those of Serio et al. (1997), who
found no requirement for the ori lyt sequence in cis for late-
gene expression in a transient assay, implicating a trans-
acting factor. Because of this difference, we have further
characterized the importance of the EBV ori lyt, analysing
the nuclear location of small plasmids containing ori lyt
during the lytic cycle.
Lytic DNA replication occurs at discrete sites in the nucleus,
called replication compartments (Daikoku et al., 2005).
Promyelocytic leukaemia protein nuclear bodies (PML-
NBs), speckled subnuclear structures with reported func-
tions in tumour suppression, apoptosis and interferon-
regulated antiviral defence (Ahn & Hayward, 2000; Guo
et al., 2000; Li et al., 2000), are found co-localized with the
associated with PML-NBs during the lytic cycle as replica-
tion compartments develop, and not during latency (Bell
et al., 2000). During replication, the PML-NBs become
disrupted. Although the association of EBV with PML-NBs
during lytic replication is established, which viral sequences
are required for this process was so far unknown. We show
here that ori lyt is required for the appearance of replicating
3Present address: Nuclear Oncogene Team, CRUK Centre for Cell and
Molecular Biology, Institute of Cancer Research, 237 Fulham Road,
London SW3 6JB, UK.
0008-1589 G 2006 SGMPrinted in Great Britain1133
Journal of General Virology (2006), 87, 1133–1137
episomes that are associated with PML-NBs. Furthermore,
plasmids carrying ori lyt frequently occupy the same replica-
tion compartments as EBV in a pattern that suggests that
replication compartments may derive from one or a few
The plasmid pMOVE-bol, carrying ori lyt (Amon et al.,
2004), was visualized under the confocal microscope
through multiple binding of an enhanced green fluorescent
protein (EGFP)-fusion protein (Fig. 1a), using a strategy
similar to that of Sourvinos & Everett (2002). The plasmid
contains multiple arrays (100–150 copies) of the tetracycline
operator sequence (TetO), which is recognized by the
tetracycline repressor DNA-binding domain (TetR). TetR
was expressed as a fusion with EGFP. Expression of the pro-
left). As a control, cells expressing an EGFP–nuclear locali-
zation signal (nls) fusion protein lacking the TetR domain
were also generated (Fig. 1b, middle). The replicating plas-
mids binding EGFP–nls–TetR could be detected through
confocal imaging. For immunofluorescence, cells were trans-
ferred to Cytospin microscope slides and fixed in a 1:1 solu-
tion of ice-cold methanol and acetone for 30 min at 220uC,
which reduced the background level of unbound EGFP.
Fig. 1. (a) Plasmid pMOVE is a bacterial
artificial chromosome (BAC) carrying the big
ori lyt and the multiple Tet operator-binding
array (TetO). pMOVE also contains oriP for
expressed under the control of the cyto-
megalovirus immediate-early promoter from a
separate plasmid. (b) Fluorescence micro-
scopy of cells transfected with the EGFP–
EGFP–nls expression plasmid (middle) and
untransfected cells (right). (c) Visualization
of replicating plasmids. Confocal image of
Akata 2003 cells showing the formation of
plasmid replication compartments appearing
as green dots (arrowed) 48 h post-induction.
The replication compartments only appear
in induced cells carrying pMOVE-bol (big
Replication compartments were not visible in
Akata 2003/pMOVE-bol cells expressing the
EGFP protein without the TetR DNA-binding
domain (right). (d) Immunofluorescence of
cells stained for replicating plasmids (EGFP,
green) and the single-stranded DNA-binding
protein BALF2 (red) 10 h post-induction.
BALF2 was stained with antibody OT13B
(Zeng et al., 1997).
1134 Journal of General Virology 87
W. Amon, R. E. White and P. J. Farrell
By using this system, it was possible to visualize the plasmid
replication compartments, where the concatemeric TetO
plasmids are concentrated in certain areas of the nucleus
during the EBV lytic cycle (Fig. 1c). The left image shows
several cells containing the TetO plasmids with the ori lyt
(pMOVE-bol) and the EGFP–nls–TetR expression plasmid.
To disrupt latency, cells were treated with anti-IgG for 48 h.
In the cells that have entered the lytic cycle, the plasmid
pMOVE-bol underwent lytic DNA replication and, through
binding of the EGFP-fusion protein to the replicating plas-
mids, this was observed as green dots (see arrows). Each dot
represents one replication compartment. The occurrence of
these green dots is not a result of animbalanced distribution
of the EGFP-fusion protein in the cells, but depends on the
binding of EGFP–nls–TetR to the plasmids carrying the
TetO-binding sites. In cells expressing the EGFP–nls protein
without TetR, which cannot bind to the TetO plasmids,
no green dots were detected (Fig. 1c, right image). The
individual plasmid episomes were not detectable during
latency with this system against the background level of
The single-stranded DNA-binding protein BALF2, part of
the replication complex, co-localized with the replicating
plasmids, confirming the observed green areas to be replica-
tion compartments (Fig. 1d). Complete co-localization was
not expected, as EBV genomes are also replicating in the
We previously demonstrated (Amon et al., 2004) that
small plasmids containing bol replicate in parallel with the
endogenous EBV genome. bol supported late-gene expres-
sion and mediated sensitivity to inhibitors of lytic DNA
replication. As these plasmids reflected the behaviour of the
virus closely, it seems likely that the presence of ori lyt plays
akeyrole intheseevents.We therefore testedwhether,when
plasmids carrying bol form their replication sites, PML-NBs
could be found in the vicinity, as was observed for EBV (Bell
et al., 2000). A rabbit polyclonal antibody against human
PML (Santa Cruz) was used as a primary antibody and a
goat anti-rabbit IgG–tetramethylrhodamine isothiocyanate
conjugate served as the fluorescent secondary antibody. To
detect cells that have entered the viral lytic cycle, the EBV
immediate-early protein BZLF1 was also stained (BZ-1
2003 cells transfected with the EGFP–nls–TetR expression
IgG and samples were taken after 6, 12 and 24 h. Fig. 2(a)
cells that have entered the lytic cycle are already expressing
the immediate-early protein BZLF1 (blue). At this early
stage of the lytic cycle, the PML-NBs (red) are still intact.
Plasmid replication has not yet started, as judged by the
absence of green dots (EGFP–nls–TetR) indicating replica-
Twelve hours after induction of the lytic cycle (Fig. 2b),
replication sites have started to form (green). In many cases,
these sites were in the vicinity of PML-NBs (red), indicating
an association between plasmid replication factories and
PML-NBs (see arrows). However, not all sites of plasmid
Fig. 2. (a) Replication compartments (green) are not yet detect-
able 6 h post-induction. The PML-NBs (red) are still intact. Cells
that have entered the lytic cycle (indicated) express BZLF1.
(b) Replication compartments (green) have formed 12 h post-
induction. Cells that have entered the lytic cycle (indicated)
express BZLF1 (blue). Plasmid replication compartments fre-
quently form in association with PML-NBs (red) (arrows).
(c) Disruption of PML-NBs 12 h after induction. Some cells were
found to contain enlarged red spots (arrows). (d) PML-NBs are
not detected after 24 h in those cells that have entered the lytic
cycle. Plasmid replication compartments (EGFP) are shown as
green spots. Cells were stained for PML (red) and BZLF1 (blue).
EBV ori lyt and replication compartments
replication shown in the images have a PML body next to
them. In many cases, the PML-NBs were not in the same
confocal plane, as three-dimensional projections revealed.
In other cases, the PML-NBs might already be disrupted.
Some of the cells in the lytic cycle were found to have
abnormally large PML-NBs (Fig. 2c). This was never
observed in latent cells and thus seems to be connected
with the EBV lytic cycle. Interestingly, these enlarged PML-
NBs were always close to a plasmid replication compart-
ment. The enlarged red areas could therefore represent
disrupting PML-NBs that are dispersing their PML protein.
After 24 h treatment with anti-IgG, all PML-NBs have been
disrupted in those cells that have entered the EBV lytic cycle
The results of the immunofluorescence experiments indi-
cate an association between plasmid replication sites and
PML-NBs, similar to that of the viral genome. The plasmids
also contain the EBV oriP sequence, so we cannot exclude
the possibility that this makes a contribution, but it is likely
that ori lyt is sufficient for the replication and association
The observed association of replicating ori lyt plasmids with
PML-NBs still leaves the question of whether the plasmids
actually replicate at the same sites in the nucleus as the EBV
genome. The system described here allows observation of
cell. For this purpose, the ori lyt plasmids and the viral
episomes were stained with two different colours by using
fluorescence in situ hybridization (Fig. 3a). Confocal imag-
ing revealed the formation of plasmid (green) and virus
(red) replication compartments 12 h after induction of the
lytic cycle (Fig. 3b). Only certain areas of the cell nuclei
contained lytically replicating DNA. With a few exceptions,
these replication areas contain both replicating virus and
plasmid DNA. Replicating plasmids appear more focused,
because each green dot represents a large, concatemeric
molecule of several hundred plasmid copies that cannot be
cleaved due to the lack of terminal repeats. In contrast, the
viral DNA (red) is spread over a larger area, presumably
because the single virus amplicons are free to diffuse in the
nucleus or are transported actively to the sites of virus
assembly. This co-localization of virus and plasmid DNA
supports the conclusion that the ori lyt sequence is sufficient
to mediate the association of small plasmids with the virus
replication compartments. The frequent co-localization of
plasmid and viral genomes in replication compartments,
with a few containing either plasmid or viral DNA uniquely,
suggests that each replication compartment derives from
one or a few initial plasmid or viral episomes.
After finding that the ori lyt sequence is required in cis for
late-gene expression (Amon et al., 2004), a process linked
to lytic DNA replication through inhibitors such as phos-
phonoacetic acid and acyclovir, we have now shown that ori
lyt is also required for the production of virus replication
compartments associated with PML-NBs. Our results do
simplex virus 1 (HSV-1), the viral origin of replication OriS
is sufficient for the association of plasmids with PML-NBs
(Tang et al., 2003). Similarly, in Simian virus 40, the
minimum sequence required for the association with PML-
NBs contained the viral core origin of replication, although
the origin alone was not sufficient (Tang et al., 2000).
Association of the viral episome with PML-NBs during
reactivation might contribute to the link between lytic DNA
Fig. 3. (a) Fluoresence in situ hybidization. Viral DNA was
hybridized with a digoxigenin (DIG)-labelled probe against part
of the BamHI C fragment and detected with an anti-DIG rhod-
amine-conjugated antibody. The ori lyt plasmid pMOVE-bol was
detected with a biotin-labelled probe against the BAC back-
bone and avidin–fluorescein. Cells were fixed in a 1:1 solution
of ice-cold methanol and acetone for 30 min at ”206C and
denatured in 70% formamide in 26 SSC for 15 min at 806C.
(b) EBV (red) and the ori lyt plasmid pMOVE-bol (green) form
their replication compartments in the same areas of the nucleus
(12 h post-induction).
1136Journal of General Virology 87
W. Amon, R. E. White and P. J. Farrell
replication and late-gene expression. All three processes
(association with PML-NBs, lytic replication and late-gene
expression) depend on the presence of ori lyt in cis. Upon
reactivation, the viral episome was located in an ori lyt-
dependent manner adjacent to PML-NBs. These are not
only the sites for lytic DNA replication, but might also be
sites of active transcription (Everett, 2001), which could
promote late-gene expression.
The plasmid system allowed us to study two independently
replicating units during the lytic cycle within the same cell.
Our fixed-time data cannot distinguish whether replicating
EBV plasmids move to become adjacent to PML-NBs, as
previously suggested for EBV reactivation (Bell et al., 2000),
or whether the PML-NBs form adjacent to replication foci,
as was concluded in recent studies of HSV-1 infection
(Everett et al., 2004; Everett & Murray, 2005). However, the
similar behaviour of the small plasmids containing ori lyt
and the EBV genome in nuclear location is consistent with
characteristics very similar to those of the complete viral
We thank Roger Everett for the EGFP–TetR and –TetO plasmids and
advice on plasmid visualization, Jaap Middeldorp for the BALF2
antibody, Richard Greaves for plasmid pRG201 and Kenzo Takada for
the Akata cell line. P.J.F. is a research affiliate of the Ludwig Institute
for Cancer Research, who supported part of this work.
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EBV ori lyt and replication compartments