HSV-1 Genome Subnuclear Positioning and Associations
with Host-Cell PML-NBs and Centromeres Regulate LAT
Locus Transcription during Latency in Neurons
Fre ´de ´ric Catez1,2¤a, Christel Picard3, Kathrin Held4, Sylvain Gross1,2,5, Antoine Rousseau3,
Diethilde Theil4¤b, Nancy Sawtell6, Marc Labetoulle3,7, Patrick Lomonte1,2,5*
1Virus and Centromere team, Centre de Ge ´ne ´tique et de Physiologie Mole ´culaire et Cellulaire CNRS, UMR5534, Villeurbanne, France, 2Universite ´ de Lyon 1, Lyon, France,
3Institut de Virologie Mole ´culaire et Structurale, CNRS-UPR-3296, Gif-sur-Yvette, France, 4Department of Clinical Neurosciences, Ludwig Maximilian University, Munich,
Germany, 5Laboratoire d’excellence, LabEX DEVweCAN, Lyon, France, 6Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio,
United States of America, 7Ophtalmology Department, Hopital Bice ˆtre, APHP, Universite ´ Paris-Sud, Kremlin-Bice ˆtre, France
Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host.
During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of
herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant
induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic
program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated.
Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1
genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.).
Latent HSV-1 genomes display two major patterns, called ‘‘Single’’ and ‘‘Multiple’’, which associate with centromeres, and
with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image
reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML
partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG
and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML.
‘‘Single’’ and ‘‘Multiple’’ patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT
expression is significantly favored within the ‘‘Multiple’’ pattern, which underlines a heterogeneity of LAT expression
dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-
knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate
the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are
functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction.
Citation: Catez F, Picard C, Held K, Gross S, Rousseau A, et al. (2012) HSV-1 Genome Subnuclear Positioning and Associations with Host-Cell PML-NBs and
Centromeres Regulate LAT Locus Transcription during Latency in Neurons. PLoS Pathog 8(8): e1002852. doi:10.1371/journal.ppat.1002852
Editor: Roger D. Everett, University of Glasgow, United Kingdom
Received April 16, 2012; Accepted June 26, 2012; Published August 9, 2012
Copyright: ? 2012 Catez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by grants from CNRS (ATIP program, to PL, http://www.cnrs.fr), ANR (ANR-05-MIIM-008-01, CENTROLAT, http://www.agence-
nationale-recherche.fr), the FINOVI Foundation (http://www.finovi.org/:fr:start), LabEX DEVweCAN (ANR-10-LABX-61, http://www.agence-nationale-recherche.fr),
l’Association pour la Recherche contre le Cancer (ARC-7979 and ARC-4910, http://www.arc-cancer.net), la Ligue Nationale Contre le Cancer (LNCC, http://www.
ligue-cancer.net), and INCa (EPIPRO program, http://www.e-cancer.fr). SG was supported by the French Ministry for Education and Research (http://www.
enseignementsup-recherche.gouv.fr) and the Fondation pour la Recherche Me ´dicale (FRM, http://www.frm.org). FC and PL are CNRS researchers. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: INSERM U1052, CNRS UMR5286, Centre de Recherche en Cance ´rologie de Lyon, F-69000, Lyon, France
¤b Current address: Novartis Institute for Biomedical Research, Basel, Switzerland
Herpes simplex virus type 1 (HSV-1), a major human pathogen,
is a persistent human neurotropic virus and a model of long-term
interaction between a host cell and a parasite. HSV-1 establishes a
long-term latent infection in neurons of the trigeminal (or
Gasserian) ganglia (TG) of the peripheral nervous system, from
which it reactivates periodically to replicate and spread . The
establishment of latency is dependent on a sequence of physiolog-
ical and molecular events involving the host immune system, the
cellular antiviral response, and the ability of the virus to initiate a
latent gene expression program.
Latent HSV-1 dsDNA genomes localize in the nucleus of the
host neuron where they remain as multi-copy chromatinized
episomes, which do not integrate into the host-cell genome [2,3].
During latency, HSV-1 lytic gene expression is strongly repressed;
although some lytic transcripts could be detected at low level, by
highly sensitive techniques [4–6]. The latency-associated transcript
(LAT) locus is the only gene to be highly expressed throughout the
persistent stage, from establishing latency to reactivation . LAT
is a noncoding RNA, synthesized as an 8.3-kb polyadenylated,
unstable primary transcript, and is rapidly processed into a stable
2-kb intron lariat and several microRNAs [8–11]. LAT expression
has been linked to several aspects of the latency process, including
PLoS Pathogens | www.plospathogens.org1 August 2012 | Volume 8 | Issue 8 | e1002852
neuron survival, viral genome chromatin status, lytic gene
expression, number of latently infected neurons, and efficiency
of reactivation in animal models [2,3,10,12–17]. Although LAT
appears to regulate latency and reactivation, several studies have
shown that LAT is probably expressed only in a subset of latently
infected neurons, implying that latency is intrinsically a heteroge-
neous event [18–23]. The heterogeneity of HSV-1 latency has also
been observed at the level of the viral genome copy number in
individual neurons, which has been directly correlated with
reactivation probability, suggesting that it is a functionally
significant parameter [19,20,24]. How these variable parameters
impact on the biology of the latent virus and the reactivation
process remains unclear. Moreover, host-cell factors and the
cellular environment can be anticipated to also account for the
variability of latency and for determining the ability of HSV-1 to
reactivate. Therefore, the study of latency requires experimental
approaches in which the heterogeneity can be fully assessed with
regard to viral genome features, viral gene expression, and host-
cell nuclear components. In situ fluorescence-based strategies offer
such a possibility, through a multi-parametric reading of a cell
population at the single-cell level.
The mammalian cell nucleus is a highly organized compartment
containing the chromosomes and several nuclear domains, which
reflect the various molecular activities taking place in the nucleus.
Numerous studies reported that the position of a gene within the
nucleus is correlated with its transcriptional status [25,26]. The
predetermined nuclear positions of genetic loci within the nuclear
architecture are key determinants of gene expression, together
with transcription factors and epigenetic chromatin modifications
[25,27,28]. Nuclear structures known to influence gene expression
include the nuclear envelope, telomeres, centromeres and
pericentromeres, and nuclear domains such as promyelocytic
leukemia (PML) nuclear bodies (NBs, also called ND10),
transcription factories, polycomb group complexes, and the
nucleolus [26,29–34]. Among these nuclear domains, PML-NBs
are proteinaceous structures that reorganize in response to various
cellular stressors [33,35,36]. PML-NBs provide a nuclear envi-
ronment that can be associated with transcription of cellular genes
[32,37,38]. However, PML-NBs contain repressor proteins such as
HP1, ATRX, and hDaxx [39,40], which have an inhibitory effect
on transcription and replication of RNA and DNA viruses,
supporting the silencing activity of PML-NBs [41,42]. In cultured
infected cells, the association of PML-NB with genomes of several
viruses, including HSV-1, has led to the hypothesis that PML-NBs
may operate as a nuclear relay for innate host-cell defense
mechanisms, blocking replicative infection by creating an envi-
ronment unfavorable for viral gene expression [32,42–50].
However, how nuclear domains impact in vivo on the biology of
persistent viruses such as HSV-1 and whether they may intervene
in the latency process, in particular in the acquisition of essential
parameters involved in latency maintenance and reactivation, is
In this study, we took advantage of a physiologically well-
characterized mouse model of HSV-1 infection, to develop an
efficient fluorescent in situ hybridization (FISH) approach for
detecting HSV-1 genomes during latency in neurons from infected
mouse TG. Using a high-resolution visualization technique, we
described the intra-nuclear distribution of the latent HSV-1
genome in neurons, and correlated HSV-1 patterns with LAT
expression. We found that HSV-1 genomes were non-randomly
associated with two nuclear domains, PML-NBs and centromeres.
Using infected PML knockout (KO) mice, we showed that PML/
PML-NBs influence viral genome distribution and negatively
regulate expression of LAT. Finally, we demonstrated that HSV-1
genomes associated with PML-NBs or centromeres were negative
for the expression of LAT.
In situ detection of latent HSV-1 genomes by FISH
The lack of an efficient in situ detection method of viral genomes
has been a major technical limitation to the study of herpes virus
infection and disease both in animal models and human samples.
Detection of HSV-1 genomes by FISH in latently infected mouse
tissues has remained unsuccessful despite attempts of many groups
. To determine the intra-nuclear organization of the multiple
copies of HSV-1 and its influence HSV-1 gene expression, we
developed a DNA-FISH protocol and applied it to an established
lip-inoculation mouse model in which HSV-1 establishes signifi-
cant latency in the TG (Figure 1A; ). Infected and mock-
infected mice were sacrificed at 28 d.p.i., a time point at which
HSV-1 latency is known to be fully established , and the TGs
were cryo-sectioned. Our FISH protocol efficiently detected latent
HSV-1 genomes in mouse neuronal tissues (Figure 1D; see
Materials and Methods for details). The DNA-FISH probes
recognized a 90 kb region of the viral genome, excluding the LAT
locus (named hereafter ‘‘HSV-1 genome probes,’’ Figure 1B).
Importantly, our protocol did not include a signal amplification
procedure and thus is well suited for the study of intra-nuclear
organization by high-resolution microscopy. Signal specificity was
assessed through several control experiments, including FISH
analysis of mock-infected mice, FISH with control probes without
HSV-1 sequences (Figure 1C), and a comparison between our
probe and a commercially available probe (Figure S1).
In TG sections from infected mice sacrificed at 28 d.p.i., the
FISH signal for HSV-1 DNA was observed only in the nuclei of
neurons, where it appeared as a dotted pattern comprising spots of
various numbers, sizes, and intensities (Figure 1D and S1). The
presence of the HSV-1 genome in neurons and not in satellite cells
is consistent with the results of previous in situ PCR and single-cell
PCR studies [20,24]. Two main intra-nuclear patterns were
After an initial lytic infection, many viruses establish a life-
long latent infection that hides them from the host
immune system activity until reactivation. To understand
the resurgence of the associated diseases, it is indispens-
able to acquire a better knowledge of the different
mechanisms involved in the antiviral defense. During
latency, viral genomes of nuclear-replicative viruses, such
as herpes simplex virus type 1 (HSV-1), are stored in the
nucleus of host cells in a non-integrated form. Latency
establishment is associated with a drastic change in HSV-1
gene expression program that is maintained until reacti-
vation occurs. The last two decades of research has
revealed that the functional organization of the cell
nucleus, so-called nuclear architecture, is a major factor
of regulation of cellular genes expression. Nonetheless, the
role of nuclear architecture on HSV-1 gene expression has
been widely overlooked. Here we describe that the
genome of HSV-1 selectively interacts with two major
nuclear structures, the promyelocytic nuclear bodies (PML-
NBs or ND10) and the centromeres. We provide evidence
supporting that these nuclear domains directly influence
the behavior of latent viral genomes and their transcrip-
tional activity. Overall, this study demonstrates that
nuclear architecture is a major parameter driving the
highly complex HSV-1 latency process.
HSV-1 Nuclear Positioning and LAT Expression
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observed: a single, bright, round spot (termed ‘‘single’’) and
numerous spots of non-uniform size and shape (termed ‘‘multiple’’)
(Figure 1D–E). Both patterns were observed in different inocula-
tion models (lip, eye, and whisker pads), animals (mice of different
inbred strains and rabbits), and viral strains (SC16, 17syn+, KOS/
M, and McKrae), indicating that they are characteristic of neurons
latently infected with HSV-1. The occurrence of each pattern was
variable (Figure 1E), suggesting that the strain of virus and or the
route of infection may affect how the viral genome accumulates in
neurons. In addition to these two primary patterns, a single spot
accompanied by one or two smaller spots (termed ‘‘single+’’) and a
multiple pattern that filled the nucleus (termed ‘‘super-multiple’’)
were less frequently observed (Figure S1). The presence of multiple
genome spots in a large proportion of infected neurons is
consistent with the results of earlier single-cell quantitative PCR
(qPCR) analyzes [19,20]. We further confirmed that the sizes of
the spots observed by FISH were consistent with the presence of
several copies per spot (Table 1). The spot in the single pattern was
Figure 1. In situ detection of the HSV-1 genome in mouse TG sections. (A) Models of latent infection. (B) Schematic representation of the
HSV-1 genome and the LAT locus. The RNA- and DNA-FISH probes used in this study are indicated in grey. (C) Control DNA-FISH experiments were
performed on mock-infected mouse tissue by using an HSV-1-specific probe and on HSV-1-infected mouse tissue by using an empty cosmid vector
(Cos64) probe. Stained tissue sections were imaged by wide-field microscopy. Dashed lines indicate the position of the nucleus. Scale bar=5 mm. (D)
Illustration of the main latent HSV-1 genome pattern. DNA-FISH was performed on TG sections obtained from infected mice 28 d.p.i. as in (A), using a
mix of HSV-1 cosmids 14, 28, and 56 as indicated in (B). Stained sections were imaged by wide-field microscopy. Dashed lines indicate position of the
nucleus. Scale bar=5 mm. (E) Quantification of HSV-1 genome patterns during latency. Two groups of mice were infected according to the two
models presented in (A). For SC16/lip-infected mice, n=6 (4671 infected neurons); for 17syn+/eye mice, n=4 (1000 infected neurons). Bars show the
standard error of the mean. (F) Distribution of latently infected neurons along the TG. The data obtained in (E) from five SC16/lip-infected mice were
plotted as total number of HSV-1 genome-positive neurons per section. (G) Distribution of the single/single+ and multiple/super-multiple patterns
along the TG. Data from one mouse shown in (F) is shown as example.
HSV-1 Nuclear Positioning and LAT Expression
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0.8060.14 mm wide (n=48), a size similar to that of in vitro-
induced quiescent genomes , which were estimated by qPCR
to contain four to five copies of the genome. The spots of the
multiple pattern varied from 0.40 to 3 mm in diameter (more in the
case of large aggregates). We measured the sizes of individual
FISH-detected HSV-1 genomes in in vitro-infected cells to define a
reference. Single-copy parental genomes entering the nucleus
appeared as spots that were 0.5160.08 mm wide (n=76), which
was similar to the width of isolated spots within the multiple
pattern (0.4460.07 mm; n=51), indicating that these spots may
represent single genomes. Based on this analysis, it can be
predicted that the single-spot pattern contains more than one copy
of the genome and that in the multiple-spot pattern, the genome
can be either isolated or aggregated.
We next used serial sectioning to explore whether HSV-1
established latency with a topographical preference within the TG.
Neurons shown by FISH to be positive for HSV-1 were distributed
all along the TG (one of the three sections analyzed), without any
enrichment along the antero-posterior axis (Figure 1F). Similarly,
the frequencies of the single and multiple patterns were equivalent
throughout the TG (one mouse is shown as an example in
Figure 1G). The frequency varied from section to section, and no
reproducible pattern could be detected in a group of six mice.
Overall, these results show that the HSV-1 latent genome in
mouse neuronal tissues can be detected by FISH, with sufficient
efficiency and quality for the analysis of intra-nuclear distribution.
During latency in neuron nuclei, the HSV-1 genome is present as
multiple copies, as shown previously using other methods ,
and adopts a non-random intra-nuclear organization.
LAT expression is linked to a specific nuclear HSV-1
Data from several groups suggest that LAT is expressed in a
fraction of infected neurons during latency. We set up a dual
RNA/DNA-FISH assay based on tyramide signal amplification
(TSA) technology to co-detect LAT transcripts and HSV-1
genomes (Figure 1B and 2A). We challenged the sensitivity of
our RNA FISH method by using up to 20 times the amount of
probe (1000 ng/assay instead of 50 ng) and increasing the TSA
time. We failed to detect neurons with weak LAT signals,
indicating that our test efficiently detected LAT-expressing
neurons. In mice at 28 d.p.i., 18 to 31% of the HSV-1 DNA-
containing neurons were positive for the 2-kb LAT RNA, thus
confirming the results previously obtained by different approaches
[18,20]. Notably, fewer than 10% of neurons with a single pattern
were positive for 2-kb LAT (Figure 2B). In contrast, 40.369.5% of
the multiple-pattern neurons expressed LAT, suggesting that the
multiple pattern reflects conditions favorable for LAT transcrip-
tion (Figure 2B). A reciprocal analysis showed that 83.0% of LAT-
positive neurons contained the HSV-1 genome in a multiple
pattern (Figure 2C). This suggests that the organization of the
HSV-1 genome in a multiple pattern is necessary, but not
sufficient, to support LAT transcription. These data demonstrate
Table 1. Estimation of the number of genome copy per FISH spot.
In vitro infected cellsLatent genome
Smaller spots in
the ‘‘Multiple’’ pattern
Size of the spot (mm) 0.51+/20.080.80+/20.140.44+/20.07
Estimated number of HSV-1
genome per spot
1 2 to 51
The diameter of FISH spots was measured using Metamorph software at the equatorial plan of each spot. In vitro HSV-1 infected cells fixed at 2 hours post infection
were used as a reference. At this time of infection, the genome replication has not started and single spots represent single copy of the genome. Note that the
difference of chromatin status of lytic vs latent genomes prevents any precise calibration of the spot size/copy number relationship to be established.
Figure 2. LAT expression correlates with HSV-1 genome
pattern. (A) TG sections obtained from SC16/lip-infected mice at
28 d.p.i. were processed for RNA-FISH using a 2-kb LAT RNA-FISH probe
and an HSV-1 DNA-FISH probe as in Figure 1D. The dotted lines outline
the nucleus. Each labeling is shown as separated channels on the right
panel. Wide-field imaging. Scale bar=5 mm. (B) TG sections obtained
from three mice at 28 d.p.i. were processed as in (A). The 2-kb LAT RNA-
FISH signal and DNA-FISH pattern were quantified and plotted as the
fraction of LAT+ neurons among neurons with the various patterns. (C)
Same data set as in (B), plotted as the fraction of each pattern in LAT+
neurons. Bars show the standard error of the mean.
HSV-1 Nuclear Positioning and LAT Expression
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that transcription of the LAT locus is linked to the intra-nuclear
pattern of the viral genome.
Latent HSV-1 genomes co-localize with centromeres in
The distribution of latent HSV-1 in neuron nuclei did not bear
any resemblance to the patterns of known nuclear domains, and it
remained unclear whether the viral genome associated with
particular structures. As HSV-1 gene expression has been shown
to involve viral chromatin [2,3], we first focused on nuclear
structures that are known to control cellular gene transcription
through heterochromatin domains: the nuclear envelope, telo-
meres, centromeres, and pericentromeres. HSV-1 latent genomes
were rarely found at the periphery of the nucleus, thus excluding a
preferential association with the nuclear envelope. The association
of the HSV-1 genome with telomeres and centromeres was
assessed by dual-color DNA-FISH. No co-localization of the HSV-
1 genome with telomeres was observed when assessing single or
multiple patterns (Figure 3A). In mouse cells, the centromeres are
positioned at the surface of pericentromeric aggregates (also called
chromocenters), which are commonly detected by Hoechst
staining  (Figure 3A and S2). By dual DNA-FISH, performed
using specific probes to detect minor and major satellites, we
confirmed that the organization observed in the cultured cells was
similar to that in TG neurons, and that heterochromatic
aggregates detected by DNA staining represent pericentromeres
(Figure S2). Latent HSV-1 co-localized with centromeric repeats
in 10.160.8% of the single-pattern neurons and in 39.468.8% of
the multiple-pattern neurons (Figure 3B). In contrast, the
frequency of association with pericentromeres remained low for
both single- and multiple-pattern neurons (4.5163.4% and
8.4364.9%, respectively, n=1,249 neurons in two mice). Within
individual neuron nuclei, only a subset of HSV-1 FISH spots was
associated with centromeres, showing that they are not the only
residence sites of latent genomes. An immuno-FISH staining of the
centromeric protein (CENP)-A, which is essential for the stability
and functionality of the centromere  further confirmed the
localization of a subset of HSV-1 genome onto the centromeres
(Figure 3C–D). Additionally, these data demonstrate that the
HSV-1-associated centromeric loci are likely to be functional
centromeres. The association of HSV-1 genomes with centro-
meres did not appear to be an artifact caused by a strong HSV-1
signal in the multiple pattern for the following reasons: (i) the
centromere and HSV-1 signals were largely co-localized (Figure 3A
and 3D, bottom right image); (ii) the positioning of HSV-1
genomes adjacent to pericentromeres did not increase concomi-
tantly with an increase in HSV-1 signal density (Figure 3B and S2);
(iii) and co-detection of HSV-1 and telomeres did not result in
signal co-localization, even though each cell contained twice as
many telomeres as centromeres and telomeres are proximal to
centromeres in acrocentric mouse chromosomes.
Latent HSV-1 genomes associate with PML-NBs
Because the single HSV-1 pattern did not frequently coincide
with centromeres, and because a tight interplay between HSV-1
and PML-NBs exists in vitro, we developed an immuno-FISH
approach to analyze whether PML-NBs could be involved in
HSV-1 latency. In non-infected tissues, PML was detected by
immuno-FISH in both non-neuronal cells and neurons. Neurons
contained 1–10 PML spots, although a subpopulation of neurons
did not display any detectable signal in the nucleus (Figure 4A). In
mice at 28 d.p.i., a qualitative assessment of the number of PML-
NBs in infected neurons did not reveal any obvious change, by
comparison with uninfected neurons. In latently infected mice,
PML protein invariably associated with single-pattern HSV-1
genomes, whereas it associated with HSV-1 genomes in only 61%
of multiple-pattern neurons (n=201 neurons). In the latter case,
only some HSV-1 spots were associated with PML, revealing
heterogeneity among the genomes regarding their association with
To determine whether HSV-1 genomes associated with bona fide
PML-NBs, immuno-FISH and 3D microscopy were used to detect
two stable signature components of PML-NBs, ATRX and Daxx.
Both ATRX and Daxx were found to be associated with single-
pattern HSV-1 genomes (Figure 4B); in multiple-pattern genomes,
ATRX and Daxx co-localized with at least one HSV-1 genome
focus, consistent with the observed frequency of the association of
these genomes with PML. A triple-labeling experiment confirmed
that PML and Daxx (Figure 4C) or PML and ATRX (not shown)
simultaneously associated with the HSV-1 genome. Careful
inspection of PML-NBs associated with HSV-1 revealed that
PML protein had a ring-like shape, with HSV-1 genome in its
center (Figure 4D). The presence of HSV-1 DNA within PML-
NBs was intriguing because PML-NBs have been generally found
to be devoid of nucleic acids and to be localized adjacent to or
within 2 mm of genomic loci [37,55]. High-resolution 3D confocal
microscopy confirmed that in the case of the HSV-1 latent
genome, the DNA was clearly inside the PML ring (Figure 4D),
and that PML was wrapped around the viral genome. This
organization was also observed during the early phase of mouse
infection (Figure 5C) and in vitro in cells infected with replication-
defective HSV-1 . Thus, our observations show that PML
assembles around HSV-1 genomic DNA, forming an atypical
DNA-containing PML-NB (DCP-NB).
Association of the HSV-1 genome with PML-NBs is
initiated during early stages of the latency process and
participates in HSV-1 genome pattern formation
PML-NB reorganization and co-localization with HSV-1
genomes were observed as early events in lytic infection in
cultured cells [43,45,56,57], raising the possibility that the
association we observed during latency could be initiated early
during the establishment of latency (the acute phase). In immuno-
FISH analyzes performed on sections from mice sacrificed at
6 d.p.i., we observed that the PML protein signal within PML-
NBs was stronger, and the PML-NBs were generally larger and
more numerous in acute-phase tissues compared with latently
infected and non-infected tissues (Figure 5A). Increases in the PML
signal were observed in both neurons and accessory cells and,
importantly, were restricted to infected TG (Figure 5A–B and S3;
see Materials and methods for details), demonstrating that the
increase in the PML signal resulted from the on-going infection.
The increase in the PML signal could be attributable to the
recruitment of nucleoplasmic PML (which accounts for 90% of
nuclear PML;  into PML-NBs, or to an increase in the overall
amount of PML. Western blotting of whole TG from mice at
6 d.p.i., showed increases in total PML and PML isoform levels in
acutely infected TG compared with non-infected TG (see
Materials and methods) and TG from non-infected mice
(Figure 5B), demonstrating that the change in PML protein
pattern results from an increase in total cellular PML protein and
not only from a more efficient recruitment of nucleoplasmic PML
into PML-NBs. These data support the stimulation of PML
expression during the acute phase of HSV-1 infection, probably as
a result of IFN pathway activation . PML and HSV-1 formed
1–12 DCP-NBs per infected neuron nucleus, and most of them
also contained ATRX, and Daxx (Figure 5C). Thus HSV-1
genome and PML patterns were significantly different from those
HSV-1 Nuclear Positioning and LAT Expression
PLoS Pathogens | www.plospathogens.org5 August 2012 | Volume 8 | Issue 8 | e1002852
observed in latently infected neurons. We conclude that acute
infection provokes a PML response, leading to the formation of
HSV-1 DCP-NBs, and that the association between PML and the
viral genome is initiated during the very early stages of the latency
The above observations raised the possibility that the HSV-1/
PML interaction may play a role in the formation of the latent
HSV-1 patterns. To address this, we quantified HSV-1 latent
genome patterns in latently infected PML-deficient mice. Both
PML+/2and PML2/2mice displayed a significant decrease in the
number of single-pattern neurons and a concomitant increase in
the number of neurons with the super-multiple pattern (Figure 5D–
E). These data show that PML protein and/or PML-NBs
influence the intra-nuclear pattern adopted by the viral genome
within latently infected neurons. Additionally, based on the
number of viral genome foci detected within individual neurons,
Figure 3. Latent HSV-1 genomes co-localize with centromeres in neuron nuclei. (A) Co-detection of HSV-1 genomes with telomeres and
centromeres. TG sections from mock-infected and latently infected (SC16/lip) mice were stained by dual-color DNA-FISH using HSV-1-, telomere- and
centromere-specific (Minor satellite, MiSat) probes. Tissues were counterstained with Hoechst 33258, which revealed aggregated pericentromeres in
mouse cells (see text for details). The boxed areas show single-channel images. Arrowheads show co-localization of the HSV-1 signal with the minor
satellite signal. Scale bar=5 mm. (B) TG sections from two mice were stained with HSV-1 genome- and centromere-specific probes as in (A), and
neurons in which the HSV-1 genome signal was associated with centromeres and pericentromeres were counted. Bars show the standard error of the
mean. (C–D) TG sections from latently infected (SC16/lip) mice were stained by immuno-FISH with an HSV-1 genome-specific probe and anti-CENP-A
antibody. Pericentromeres were counterstained with Hoechst. The images show that HSV-1 genome spots precisely co-localize with the CENP-A
signal at the surface of pericentromeres. Scale bar=5 mm.
HSV-1 Nuclear Positioning and LAT Expression
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HSV-1 Nuclear Positioning and LAT Expression
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Figure 4. PML-NBs form around HSV-1 genomes in neurons. (A) Co-localization of HSV-1 genomes with PML protein. TG sections from mock-
infected and latently infected (SC16/lip) mice (28 d.p.i.) were stained by immuno-FISH to co-detect the HSV-1 genome and PML protein. The boxed
areas show single-channel images. Scale bar=5 mm. (B) The HSV-1 genome associates with major PML-NB components. TG sections from latently
infected mice were stained by immuno-DNA-FISH using anti-ATRX or anti-Daxx antibody and HSV-1 probe. HSV-1 genomes associated with ATRX or
Daxx are indicated by arrows and enlarged in the insets. (C) TG sections from the same mouse as in (B) were stained by dual immuno-FISH using anti-
PML and anti-Daxx antibodies and HSV-1 probe. The arrow shows an HSV-1-containing PML-NB stained with anti-Daxx antibody. (D) Confocal
microscopic analysis of an HSV-1-containing PML-NB, showing (by fluorescence line-scan measurement and 3D reconstruction of a confocal Z-stack)
that the HSV-1 DNA-FISH signal is localized within the PML-NB.
Figure 5. PML/PML-NBs control the distribution of incoming and latent HSV-1 genomes. (A–B) PML signals increase in neuronal and non-
neuronal cells during the acute phase. (A) TG sections from mock-infected and infected mice sacrificed during the acute phase (6 d.p.i.) or latency
(28 d.p.i.) were stained by immuno-FISH using HSV-1 probe and anti-PML antibody. In the SC16/lip model, inoculation of the virus on the upper left side
induced asymmetrical acute and latent infection (see Materials and methods). All images were collected using identical gain and exposure settings. For
acute infection, images of the left and right TG were collected from the same section. (B) Western blot analysis of PML protein in left and right TG
harvested from mock-infected and infected mice during acute infection (6 d.p.i.). NIH3T3 cells were used as a positive control. A pan-HSV-1 serum was
used to confirm ongoing acute infection in the left TG, through detection of lytic HSV-1 protein (arrows). Lytic infection is known to occur in early acute
phase in some neurons andaccessory cells, andis cleared as latency is being established [20,107] andseefigure S3). Actin was used as a loading control.
(C) HSV-1-containing PML-NBs were detected during acute phase. TG sections from infected (SC16/lip) mice sacrificed at 6 d.p.i. were stained by
immuno-FISH using HSV-1 probe and anti-PML (left), anti-ATRX (middle), and anti-Daxx (right) antibodies. The fluorescence plot profiles (dashed lines)
areconsistentwitharing-shapedPML-NBcontainingtheHSV-1genomeandATRXandDaxxproteinsinitscenter. a.u.=arbitrary units.Scalebar=5 mm.
(D) The HSV-1 genome pattern is altered in PML-knockout mice. Wild-type, heterozygous, and PML-knockout mice were infected by corneal inoculation
with the 17syn+ HSV-1 strain and were sacrificed at 28 d.p.i. HSV-1 genome patterns were quantified in serial sections spanning the whole ganglion.
n=4 mice per genotype (,1500 infected neurons per genotype). Bars show the standard error of the mean. (E) Same data set as in (D). Fractions of
neurons displaying very strong HSV-1 DNA-FISH signals (‘‘super-multiple’’ pattern, Figure S1) within the multiple-pattern neurons in (D) are shown.
HSV-1 Nuclear Positioning and LAT Expression
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we conclude that in absence of PML/PML-NBs, the number of
genome copy in latent TGs is higher, suggesting that PML/PML-
NBs play a role in limiting the number of viral genomes that
Heterogeneity in HSV-1 genome transcriptional status in
The above observations establish strong links between LAT
expression, HSV-1 intra-nuclear distribution, and the association
of the HSV-1 genome with PML-NBs and centromeres. This
raised the possibility that the association of the HSV-1 genome
with PML-NBs and centromeres may regulate LAT transcription.
In support of this hypothesis, in single-pattern neurons, HSV-1 is
systematically associated with PML-NBs, and LAT RNA is rarely
present. To test whether association of genomes with PML NBs in
multiple pattern cells had any effect on LAT expression in those
cells, we performed a triple labeling experiment to simultaneously
detect the HSV-1 genome, 2-kb LAT RNA, and PML/
centromeres. Preliminary observations suggested that the 2-kb
LAT signal was not correlated with the association of the HSV-1
genome with PML-NBs or centromeres. To confirm this, we
traced LAT expression and the association of HSV-1 with PML
and centromeres for each neuron across the entire TG of one
mouse. The data clearly showed that there was no correlation
(Table 2). This suggests that within a nucleus containing multiple
copies of the HSV-1 genome, the association of some of the HSV-
1 genomes with PML-NBs or centromeres does not have a
dominant negative effect on the expression of LAT from the other
copies of the viral genome.
An individual neuron contains a heterogeneous population of
HSV-1 genomes (‘‘free’’ or associated with a nuclear domain).
Thus, the transcriptional status of these genomes may also be
heterogeneous. To explore this possibility, we utilized the primary
(nascent) 8.3-kb LAT transcript (Figure 1B) as a marker of the site
of active transcription, in order to identify genomes that were
being transcribed. Figure 6A illustrates the co-detection of HSV-1
genomes (red), the nascent 8.3-kb LAT transcript (blue), and the
stable 2-kb LAT RNA (as a control). The nascent LAT RNA
appeared as a set of large dots (1 to 7 per nucleus), each dot being
associated with at least one HSV-1 genome spot (Figure 6). Such
dotted pattern has been previously observed by ISH using
peroxidase and alkaline phosphatase staining . This suggests
that in a single neuron, LAT can be transcribed from several
copies of the HSV-1 genome. Notably, these neurons also
contained several HSV-1 spots that were not associated with any
8.3-kb LAT RNA signal. Although we cannot exclude the
possibility that these genomes are transcribed at a level below
the sensitivity of our FISH method, the data suggest that they are
not transcribed. Overall, these results show that only a fraction of
the HSV-1 genomes withina single infected neuron are significantly
transcribed and that the transcriptional status of HSV-1 genomes is
highly heterogeneous in individual neurons.
Absence of LAT transcription from PML-NB- and
centromere-associated HSV-1 genomes
We next analyzed whether LAT is actively transcribed from
PML-NB-associated latent HSV-1 genomes. In mouse TG
sections, we detected the HSV-1 genome, its associated nascent
LAT RNA product, and PML-NBs by a triple-labeling approach.
In the LAT-expressing neurons, the nascent 8.3-kb LAT RNA was
never associated with viral genomes that co-localized with PML-
NBs (Figure 6B, bottom panel). We paid particular attention to
8.3-kb LAT-positive neurons with the single and single+ patterns,
and observed that the LAT positive neurons were all neurons with
a single+ pattern, and that the genome that was transcribed was
the one not associated with PML. The larger HSV-1 genome spot
surrounded by PML protein was never associated with an 8.3-kb
LAT RNA spot (Figure 6B, top and middle panels). These data
further support the idea that PML-NBs repress transcription of the
associated HSV-1 genome. However, in the 8.3-kb LAT-positive
neurons with the multiple pattern, several non-transcribed HSV-1
genomes were not associated with PML-NBs. It is likely that 8.3-kb
LAT is not transcribed from many of the genomes and that factors
other than PML-NBs also regulate LAT transcription.
We extended the analysis to centromere-associated HSV-1
genomes. Similarly to the findings for PML-NBs, centromere-
associated viral genomes were never adjacent to the nascent 8.3-kb
LAT RNA, suggesting that centromeres may also inhibit LAT
transcription (Figure 6C).
If PML were to inhibit LAT transcription from the HSV-1
genome, one would expect to see an increase in LAT expression in
a PML-deficient background. The 2-kb LAT RNA-FISH analysis
of latently infected PML+/2and PML2/2mice revealed that the
percentage of 2-kb LAT-positive neurons was higher in PML2/2
mice compared with wild-type and heterozygous mice (Figure 6D).
The increase in the LAT-positive neuron percentage was not
simply related to greater numbers of neurons with multiple/super-
multiple pattern. Indeed, within this category of neurons, LAT
expression was twofold higher in PML2/2animals (Figure 6E).
These data demonstrate that PML/PML-NBs play a role in the
regulation of LAT expression and support their transcriptional
Here, we report the structural and functional interactions of the
genomes of a persistent virus, HSV-1, with the host-cell nuclear
environment. Our data reveal two new features of the viral
genomes that characterize the latency state. First, the intra-nuclear
Table 2. Correlation between PML-NBs or centromeres HSV-1 genome association and LAT expression.
Association with PML in multiple pattern neuronsAssociation with centromeres in multiple pattern neurons
Associated 33.7% 27.9%Associated27.0%16.4%
Not associated 19.2% 19.2%Not associated 26.0% 30.6%
To evaluate the impact of HSV-1 genome association with PML-NB (left) and centromeres (right), on LAT expression, LAT RNA and HSV-1 association with PML-NB or
centromere was determined in individual neurons displaying multiple pattern of HSV-1 genome, using an immuno-RNA/DNA-FISH approach. The proportion of neurons
in which an association of at least one HSV-1 spot and either PML or centromeres was determined in LAT+ and LAT2 neurons. The values are in percentage of the total
infected neurons within an entire TG of one latently infected mouse.
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Figure 6. Absence of LAT transcription from PML-NB- and centromere-associated HSV-1 genomes. (A) Detection of nascent 8.3-kb
primary LAT RNA. TG sections obtained from SC16/lip mice at 28 d.p.i. were stained by RNA/DNA-FISH using an HSV-1 genome DNA probe and LAT-2
(2-kb LAT) and LAT-5 (8.3-kb LAT) RNA probes (Figure 1B). (B) HSV-1 genomes producing nascent 8.3-kb LAT are not associated with PML-NBs. TG
sections obtained from infected (SC16/lip) mice at 28 d.p.i. were subjected to dual immuno-FISH labeling with HSV-1 genome DNA probe, LAT-5 RNA
probe, and anti-PML antibody. Examples of single, single+, and multiple patterns are shown. Dashed line images are single-channel images (right).
Scale bar=5 mm. (C) HSV-1 genomes producing nascent 8.3-kb LAT are not associated with centromeres. TG sections obtained from infected (SC16/
lip) mice at 28 d.p.i. were subjected to dual RNA/DNA-FISH labeling with HSV-1 genome DNA probe, LAT-5 RNA probe, and minor satellite DNA
probe. Scale bar=5 mm. (D–E) PML wild-type, heterozygous, and knockout mice were infected (17syn+/eye) and sacrificed at 28 d.p.i. TG sections
were stained by RNA/DNA-FISH using LAT-2 RNA probe and HSV-1 DNA probe. Graphs show (D) total numbers of neurons expressing 2-kb LAT RNA
and (E) numbers of multiple/super multiple HSV-1 pattern neurons expressing 2-kb LAT RNA in PML+/+, PML+/2and PML2/2mice. n=7 mice (,2200
neurons) per genotype. Bars show the standard error of the mean.
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distribution of the latent genome is not random and correlates with
viral gene expression, and second, the host-cell nuclear domains
play a role in viral genome pattern acquisition and in the control
of viral gene expression. Thus, the interaction between the viral
genomes and host-cell nuclear components represents a new level
of host–virus interaction, which is likely to participate in the
process of latency and reactivation.
The ability to explore the cell host–virus interaction is of
outmost importance in our understanding of persistent viral
infections because regulation of the latent HSV-1 genome relies
mainly on cellular components. A substantial benefit of the in situ
immuno-DNA/RNA-FISH developed in this study lies in the
simultaneous detection of the viral genome, virally encoded
transcripts, and cellular components in the same cell. This will
enable us to address important issues of cell host–virus interactions
in tissues obtained from physiologically infected animal models but
also in emerging in vitro HSV-1 latency models . The FISH
approach provides high-resolution individual cell data without
sacrificing the access to a global view of the virus and of the host-
cell population. This appears as a major advantage given that
HSV-1 latency is highly heterogeneous. FISH and immuno-FISH
will be essential assets to study latency and will complement the
currently used biochemical and molecular approaches.
We confirmed that LAT was expressed in a fraction of infected
neurons and that viral copy numbers varied among neurons.
Based on the HSV-1 genome pattern and our estimate of genome
copy number per FISH spot, the single and multiple patterns likely
represent the low-copy and high-copy virus genome-containing
neurons, respectively, identified by contextual analysis .
Additionally, we found that the HSV-1 latent genomes were
heterogeneously distributed within neuron nuclei and preferen-
tially associated with PML-NBs and centromeres. LAT expression
is positively correlated with HSV-1 genome pattern and negatively
correlated with its association with PML-NBs and centromeres,
demonstrating that the intra-nuclear distribution of HSV-1
genomes is a major feature of the latency process. LAT detection
was almost exclusively associated with the multiple genome
pattern, demonstrating that LAT expression is restricted to
neurons with high viral genome copy numbers. Single-cell
contextual analysis has also revealed that a high genome copy
number per neuron is associated with a higher probability of virus
reactivation , suggesting that this parameter may be a key
aspect of latent genome status. Importantly, the various copies of
HSV-1 within a single nucleus are not transcriptionally equivalent,
with LAT being transcribed only from a subset of genomes. This
suggests that latent HSV-1 genomes are comparable to, and
behave like, multi-allelic cellular genes, raising the possibility that
only a subset of these genomes are susceptible to sustain full
reactivation (i.e., to reach expression of lytic genes). The dotted
pattern observed with the 8.3 kb LAT probe was previously
reported , and was proposed to be sites of early processing of
the LAT transcript. Our data confirm this hypothesis by
demonstrating that these clouds of LAT primary RNA are
associated with HSV-1 genomes.
PML-NBs are probably the most thoroughly studied nuclear
domains in the context of virus infection for their involvement in
the innate antiviral response and in the interferon (IFN) response
pathway. Our data from the acute phase and from PML2/2mice
support a role for PML-NBs in limiting the extent of viral
replication during acute-phase, and thus the number of HSV-1
genomes that establish latency in each neuron. These data are
consistent with the known role of PML-NBs, through the activity
of several of their major components such as PML, Sp100, Daxx,
ATRX, and small ubiquitin-like modifier (SUMO) protein, as
repressors of HSV-1 onset of lytic infection in cultured cells
[57,60–63]. We provide a clear demonstration that PML
expression increased in vivo in acutely infected mouse TG, and
that the PML protein, through the formation of HSV-1-containing
PML-NBs, associated with the HSV-1 genome during the early
phase of latency. Additionally, we showed that the HSV-1
genomes remain associated with PML-NBs during latency in over
80% of infected neurons, suggesting that PML-NBs play an
antiviral role influencing latency and probably reactivation.
PML-NBs have been proposed to create a specific local nuclear
environment by concentrating proteins and hosting biochemical
reactions within the PML shell. PML-NBs reorganize in response
to various stressors, potentially to relocate their activity at selected
nuclear sites . The reorganization of the PML-NBs resulted in
the formation of new PML-NBs around the HSV-1 genome at
early stages of latency, thus altering the immediate nuclear
environment of the incoming viral genome. Importantly, we
showed that PML-NBs remain associated with viral genomes long
after replicative infection has ceased, indicating that maintenance
of this particular type of DNA-containing PML-NB (DCP-NB)
requires neither on-going viral replication nor the associated
antiviral and IFN signaling pathways. The pattern of both HSV-1
genome and PML-NBs are dramatically different between acute
phase and latency, indicating that a profound remodeling of these
patterns takes place during establishment of latency. Ongoing
studies will provide pattern analysis at intermediate time between
6 d.p.i. and 28 d.p.i. The formation of DCP-NBs can be seen as a
response to the presence of chromatinized foreign DNA , or
more broadly, the presence of pathology-associated abnormal
chromatin [65,66]. Moreover, PML-NBs repress the synthesis of
the LAT primary transcript through their association with HSV-1
genomes, from which microRNAs are produced . The atypical
assembly of a PML-NB around a genetic locus may thus be
considered a distinct form of PML-NB controlling the expression
of noncoding RNA in pathological situations. Indeed, PML-NBs
assemble around pericentromeric satellite sequences and telo-
meres, two cellular loci known to give rise to noncoding RNA
We showed that HSV-1 genomes are also associated with host
neuron centromeres during latency. Bishop and colleagues
previously showed that foreign DNA delivered by polyomavirus-
like particles was localized to centromeres . Our data from a
biologically relevant context and an in vivo model support the idea
that centromeres represent docking sites for virus genomes.
Centromeres and the adjacent pericentromeres are among the
best-characterized nuclear domains that silence nearby genes [29–
31]. Consistent with this, we showed that centromere-associated
HSV-1 genomes did not express LAT RNA. We want to
emphasize that the association with HSV-1 occurs at the
centromere itself, which distinguishes the current set of data from
most other published data related to associations of cellular genes
with pericentromeres [67–75]. Only a subset of HSV-1 genomes
within a nucleus is found associated with centromeres, indicating
that this association is not the main mechanism repressing
transcription of latent genomes. Of note, both PML-NBs and
centromeres (because of their proximity with pericentromeres) are
enriched in ATRX and Daxx. In addition, hDaxx has been shown
to co-localize with centromeres in human cells . This raises the
possibility that both nuclear domains exert their repressive effect
on HSV-1 transcription through common factors .
Interestingly, HSV-1 has developed strong ‘‘anti-centromere’’
activity through the combined activities of the viral E3 ubiquitin
ligase ICP0 protein  and the proteasome. In cultured cells,
ICP0 induces the degradation of at least 10 CENPs, which results
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in the alteration of centromeric chromatin and destabilization of
the centromeres [78–81] (S. Gross and P. Lomonte, personal
communication). The biology of HSV-1 does not favor ICP0-
induced centromere destabilization, prompting the mitotic arrest
of infected cells [82,83]. Indeed, HSV-1 is able to replicate
independently of the cell cycle , and the lytic cycle does not
depend on cell arrest at the mitotic phase. This suggests that HSV-
1 targets centromeres not to control their effect on chromosome
segregation, but rather to control an activity more relevant of
differentiated, non-dividing cells. On the other hand, it is
suspected that ICP0, which does not bind DNA and is not a
transcription factor per se , inhibits the activities of numerous
repressive nuclear factors in order to favorably modify the nuclear
environment to stimulate the virus replicative cycle, at both the
onset of a new infection and during the course of reactivation
[60,86–93]. ICP0 is known to be essential for full reactivation of
HSV-1 in latently infected quiescent cells [94–96]. We therefore
propose that centromeres, although they seem to act as repressors
during latency, may offer a favorable nuclear environment for
transcriptional events during reactivation, providing their protein
composition and structure are modified by ICP0. This agrees with
data showing that centromere/pericentromere regions are sites of
intense transcriptional activity following the exposure of cells to a
variety of stressors such as heat shock, UV, and heavy metals,
which potentially induce HSV-1 reactivation [97–99]. On-going
work should provide evidence to support this hypothesis.
Materials and Methods
For animal experiments performed in France: all procedures
involving experimental animals conformed to ethical issues from
the Association for Research in Vision and Ophthalmology
(ARVO) Statement for the use of animals in research, and were
approved by the local Ethical Committee of UPR-3296-CNRS, in
accordance with European Community Council Directive 86/
609/EEC. All animals received unlimited access to food and
For animal experiments performed in the USA: animals were
housed in American Association for Laboratory Animal Care-
approved housing with unlimited access to food and water. All
procedures involving animals were approved by the Children’s
Hospital Animal Care and Use Committee and were in
compliance with the Guide for the Care and Use of Laboratory
Virus strains, mice and virus inoculation
Wild-type HSV-1 strains SC16 and 17syn+ were used. Stocks
were generated in rabbit skin cell monolayers, and viral titers were
determined as described previously . Briefly, six-week-old
inbred female BALB/c mice (Janvier Breeding, Le Genest Saint
Ile, France), were inoculated with 106PFU of the SC16 virus,
injected into the upper-left lip of the mice. Mice were observed
daily for clinical signs of ocular infection from 0 to 28 d.p.i. The
sided inoculation of the lip results in an asymmetrical infection,
which is characterized by an extremely low load of virus on the
right TG compared to the left TG. Thus, data presented in this
study were collected on the left TG, except in Figure 3A and 3D,
as indicated . Data presented in this study were collected from
the left TG, except for those shown in Figure 3A and 3D. For the
17syn+/eye model, inoculation was performed as described
previously . Briefly, prior to inoculation, mice were
anesthetized by intra-peritoneal injection of sodium pentobarbital
(50 mg/kg of body weight). A 10 mL drop of inoculum containing
105PFU of 17syn+ was placed onto each scarified corneal surface.
This procedure results in ,80% mice survival and 100% infected
TG. PML wild-type, heterozygous, and knockout mice were
obtained from the NCI Mouse Repository (NIH, http://mouse.
ncifcrf.gov; strain, 129/Sv-Pmltm1Ppp) . Genotypes were
confirmed by PCR, according to the NCI Mouse Repository
P009: 59-cTG cGc TGc ccG AGc TGc cAG G -39
P010: 59-cAG cGc AGG GTT GcG GTG GTT GG -39
P011: 59-cTc ccG ATT cGc AGc GcA TcG cc -39
Frozen sections of mouse TG were performed as previously
described . Mice were anesthetized at 6 or 28 d.p.i., and
before tissue dissection, mice were perfused intra-cardially with a
solution of 4% formaldehyde, 20% sucrose in 16PBS. The whole
head, or individual TG were prepared as previously described,
and 10 mm frontal sections were collected in three parallel series,
and stored at 280uC.
DNA-FISH probes were Cy3 labeled by nick-translation as
described previously . Briefly, cosmids 14, 28 and 56 
comprising a total of ,90 kb of HSV-1 genome (see Figure 1A)
were labeled by Nick translation (Roche Diagnostic) with dCTP-
Cy3 (GE Healthcare), and stored in 100% formamide (Sigma-
Aldrich). The DNA-FISH procedure was adapted from Solovei
et al. .
Frozen sections stored at 280uC were thawed, rehydrated in
16 PBS and permeabilized in 0,5% Triton X-100. Heat based
unmasking was performed in 100 mM citrate buffer, and sections
were post-fixed using a standard methanol/acetic acid procedure,
and dried for 10 min at RT. DNA denaturation of section and
probe was performed for 5 min at 80uC, and hybridization was
carried out overnight at 37uC. Hybridization mix contained 30 ng
of each probe in 10% dextran, 16 denhardt, 2XSSC, 50%
formamide. Sections were washed 3610 min in 2XSSC and
3610 min in 0.2XSSC at 37uC, and nuclei were stained with
Hoechst 33258 or ToPro3 (Invitrogen). All sections were mounted
under coverslip using Vectashield mounting medium (Vector
Laboratories) and stored at +4uC until observation.
Frozen sections were treated as described for DNA-FISH up to
the antigen-unmasking step. Tissues were then incubated for 24 h
with the primary antibody (diluted at 1/100). After three washes,
secondary antibody (1/200) was applied for 1 h. The secondary
antibodies (Invitrogen) were either AlexaFluor-conjugated (PML,
CENP-A), or HRP conjugated (ATRX and Daxx), which were
subsequently detected by enzymatic amplification according to
manufacturer’s guideline (TSA, Invitrogen). Following immuno-
staining, the tissues were post-fixed in 1% PFA, and DNA-FISH
was carried out from the methanol/acetic acid step onward.
RNA-FISH probe labeling and RNA-FISH procedures were
performed as described previously . Biotinylated single-strand
RNA probes were prepared by in vitro transcription (Ambion) using
plasmids pSLAT-2, pSLAT-4 and pSLAT-6 as template (see
Figure 1) (Kind gift of S. Efstathiou, University of Cambridge,
UK). Frozen sections were treated as described for DNA-FISH up
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to the antigen-unmasking step using solutions containing 2 mM
the RNAse inhibitor Ribonucleoside vanadyl complex. The
sections were pre-hybridized in 50% formamide/26 SSC and
hybridized overnight at 65uC with 50 ng of RNA probe a 50%
formamide buffer. Sections were washed in50% formamide/26
SSC at 65uC, and in 26SSC at room temperature. Detection was
performed using streptavidin-HRP conjugate, followed by TSA
amplification (Invitrogen) with an AlexaFluor 350 conjugated
substrate, according to the manufacturer’s guidelines. The DNA-
FISH procedure was performed starting from the methanol/acetic
acid post-fixation step.
The following primary antibodies were used: anti-mouse PML
(mAb3739, Millipore), anti-mouse CENP-A (rabbit mAb C51A7,
Cell Signaling Technologies), anti-ATRX H-300 (Santa Cruz
Biotechnology), anti-Daxx M-112 (Santa Cruz Biotechnology),
anti-pan-HSV-1 (LSBio), and anti-mouse actin (Sigma-Aldrich).
All secondary antibodies were Alexa Fluor-coupled and were
raised in goats (Invitrogen). HRP-coupled secondary antibodies
were provided with the TSA kit (Invitrogen).
Microscopy and imaging
Observations and most image collections were performed using
an inverted Cell Observer microscope (Zeiss) with a Plan-
Apochromat 6100 N.A. 1.4 objective and a CoolSnap HQ2
camera from Molecular Dynamics (Ropper Scientific). When
indicated, images were collected on a Zeiss LSM 510 confocal
microscope using a Plan-Apochromat 663 N.A. 1.4 objective,
except for those shown in Figure 2E, which were collected using a
Zeiss LSM 780 microscope. Line scans, 3D projections, and
surface rendering were performed using AIM and Zen software
TGs were collected at 6 or 28 d.p.i. and snap-frozen. Frozen
tissues were ground, thawed in lysis buffer (10 mM Tris-EDTA,
pH 8.0) containing a protease inhibitor cocktail, and briefly
sonicated. Protein extracts were homogenized using QiaShredders
(Qiagen). Protein concentration was estimated by the Bradford
method. Extracted proteins were analyzed by Western blotting
using anti-mouse PML antibody (mAb3739, Millipore) .
mouse TG sections. (A) Control experiment demonstrating the
specificity of the HSV-1 DNA-FISH signal. TG section of 28 d.p.i.
infected mice (SC16/lip infection), were stained by dual color
DNA-FISH using the Cy3 labeled Cos64 control probe (Cos64
Cy3 probe) or a mix of HSV-1 Cos14, Cos28 and Cos56 probes
(HSV-1 Cy3 probe) and a commercially available biotinylated
HSV-1 probe (Enzo Life Sciences). The Cos64 probe was
prepared from the empty cosmid backbone present in the
Cos14, Cos28 and Cos56 vectors. The biotinylated HSV-1 probe
was detected using TSA technology (Invitrogen). (B) Images of
neurons containing 2 types of underrepresented HSV-1 genome
patterns. Same experiment as in figure 1D. The ‘‘single+’’ pattern
contains one spot similar to the spot of the ‘‘single’’ pattern, and an
additional 1 or 2 smaller spots. The ‘‘super-multiple’’ pattern
corresponds to neurons containing a large amount of viral DNA
that fills the entire nucleus, as numerous spots or very large
aggregates (4 mm or more). Such pattern is rarely observed in the
SC16/lip inoculation model, and is present in a few percent of
In situ detection of HSV-1 genome by DNA-FISH on
neurons in the 17syn+/eye inoculation model (see Figure 5E).
centromeres at the surface of pericentromeres. (A) Intranuclear
organization of centromeres and pericentromeres in mouse
neuronal tissues. Dual color DNA-FISH was performed on TG
section of a non-infected mouse, using a biotinylated Minor
satellite probe (revealed with AlexaFluor 488 conjugated strepta-
vidin) and a Cy3 labeled Major satellite probe. Pericentromeres
(major sat.) of several chromosomes aggregate into 2 to 5 clusters
that are frequently found next to the nucleolus. Centromeres
(minor sat.) are located at the surface of the pericentromeres. This
organization is similar to what has been described in cultured cells
, and demonstrates that DNA staining by Hoechst is a
relevant approach for the detection of pericentromeres. The
number and organization of pericentromeric aggregates are
consistent with previous studies . (B) Colocalization of
HSV-1 genomes with centromeres in multiple pattern is not due
to high density of HSV-1 genome spots. Same experiment and
data set that are presented in figure 3B. We re-analyzed the data
and sub-divided the ‘‘multiple pattern’’ neurons into 2 categories:
neurons with distinct spots of 1–2 mm in diameter (example:
multiple pattern in figure 1D), and neurons with large spots and/
or a cloud of fine spots (example: multiple pattern in figure 3A).
The results showed that the denser the HSV-1 spots, the higher
the localization at centromeres. However, the association with
pericentromeres remained low even in nuclei harboring an
abundant HSV-1 signal. Data are from 3 mice (1865 neurons).
Specific localization of HSV-1 latent genomes on the
acute infection. (A) Asymmetrical acute infection in the SC16/lip
model. HSV-1 DNA-FISH was performed on sections of a 6 d.p.i.
infected mouse using the HSV-1 Cy3 labeled probe. The section
was imaged with a 406objective on a widefield microscope using
a tiling scan module. At this magnification, the high auto-
fluorescence of the tissue provides a map of the TGs. A close up
view of the left TG reveals neurons in which FISH signal is very
high, and marks ongoing acute infection. Scale=100 mm. (B)
Asymmetrical increase of PML and PML-NB signal during acute
infection. Same experiment as in figure 5A. Shown are low
magnification images of the left and right TG of an acutely
PML and PML-NBs abundance is related to ongoing
We thank S. Efstathiou (University of Cambridge, UK) and James Hill
(LSU Health Sciences Center, New Orleans, USA) for providing samples
from HSV-1-infected mice and rabbits, respectively, and for reagents; A.
London ˜o (Institut Curie, Paris, France) for telomere FISH probes; H.
Masumoto (Kazusa DNA Research Institute, Chiba, Japan) and S.
Khochbin (Institut Albert Bonniot, Grenoble, France) for helpful
discussions; and L. Francelle and N. Ahmar for technical support. Images
using the Zeiss LSM 510 confocal microscope were collected at the Centre
Technologique des Microstructures, Plateforme de l’Universite ´ Claude
Bernard Lyon 1.
Conceived and designed the experiments: FC PL. Performed the
experiments: FC CP KH SG AR NS ML PL. Analyzed the data: FC
CB NS ML PL. Contributed reagents/materials/analysis tools: FC DT NS
ML PL. Wrote the paper: FC PL.
HSV-1 Nuclear Positioning and LAT Expression
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