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Journal of Virology, May 2000, p. 4807-4815, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
H-1 Parvovirus-Associated Replication Bodies: a
Distinct Virus-Induced Nuclear Structure
Celina
Cziepluch,1,*
Stefan
Lampel,2
Annabel
Grewenig,1
Christine
Grund,3
Peter
Lichter,2 and
Jean
Rommelaere1
Applied Tumor Virology Unit, F0100 and
Institut National de la Santé et de la Recherche Médicale U
375,1 Organization of Complex Genomes
Unit,2 and Division of Cell
Biology-A0100,3 Deutsches
Krebsforschungszentrum, D-69120 Heidelberg, Germany
Received 13 December 1999/Accepted 17 February 2000
 |
ABSTRACT |
We have identified a nuclear structure that is induced after
infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of
H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition,
two cellular proteins were shown to accumulate in H1 PAR-bodies: (i)
the proliferating cell nuclear antigen (PCNA) which is essential for
chromosomal and parvoviral replication and (ii) the NS1-interacting
small glutamine-rich TPR-containing protein (SGT), suggesting a role
for the latter in parvoviral replication and/or gene expression. Since
many DNA viruses target preexisting nuclear structures, known as
PML-bodies, for viral replication and gene expression, we have
determined the localization of H-1 PAR- and PML-bodies by
double-fluorescence labeling and confocal microscopy and found them to
be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize
with other prominent nuclear structures such as nucleoli, coiled
bodies, and speckled domains. Electron microscopy analysis revealed
that NS1, as detected by indirect immunogold labeling, was localized in
ring-shaped electron-dense nuclear structures corresponding in size and
frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear
bodies for DNA replication but rather induces the formation of a novel
structure in the nucleus of infected cells.
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INTRODUCTION |
Autonomous and adeno-associated
parvoviruses have linear, single-stranded DNA genomes of approximately
5,000 nucleotides flanked with terminal palindromes. They target the
nucleus for replication, gene transcription, and finally assembly of
progeny virions (1, 4, 18, 48). To mount a lytic infection,
the adeno-associated viruses (AAV) depend on helper virus functions
provided by viruses of either the adenovirus or herpesvirus family (for
a review see reference 5). This dependence is also
reflected by the recruitment of AAV into adenovirus replication
centers, where they are able to utilize cellular as well as helper
virus proteins for their replication (65). In the absence of
helper virus and genotoxic stress, AAV integrate into the host genome
in a site-specific manner (6).
Infections with autonomous parvoviruses, to which the well-studied
minute virus of mice (MVM) and the closely related H-1 virus belong, is
strictly dependent on host cell functions expressed during the S-phase
of the cell cycle but does not require helper virus factors (18,
62, 68). The major nonstructural protein NS1, a phosphoprotein
with mainly nuclear localization, is essential for viral DNA
replication (19, 50) and also transactivates transcription
from the viral P38 capsid gene promoter (22, 34, 49).
Infection with H-1 virus or MVM finally leads to cell death, and
accumulation of NS1 was shown to be the major cause for this effect
(10). The precise molecular mechanism of NS1-associated cytotoxicity is not yet understood; however, several virus- or NS1-induced cellular disturbances that could cause cytotoxicity, including cell cycle arrest (44, 45), altered expression and phosphorylation patterns of cellular proteins (2), and for the human lymphoblastoid cell line U937, induction of apoptosis (47), have been reported.
Our study aimed at identifying the subnuclear compartment utilized by
autonomous parvovirus H-1 for replication. Since NS1 is known to be
essential for the initiation of virus replication, we have used NS1 as
an initial marker for sites of virus replication and analyzed the
nuclear localization of NS1 in infected human NBE cells. NS1 was
distributed nonhomogenously throughout the nucleus. A fraction of the
protein was present diffusely in the nucleoplasm yet excluded from the
nucleoli, whereas another fraction accumulated strongly in body-like
structures. We further demonstrated that these bodies are the sites of
H-1 DNA replication and coined the term H-1 parvovirus-associated
replication bodies (H-1 PAR-bodies).
Many DNA viruses target PML-bodies for viral replication and thereby
often induce changes in the protein composition of these bodies (for
reviews, see references 23 and
36). In noninfected cells, PML-bodies are thought to
be involved in transcriptional regulation (for a recent review, see
reference 35). The question of why viruses target
PML-bodies is still open. It was suggested that PML-bodies might
function as a depository or reservoir for proteins needed for viral
replication. Evidence for the involvement of PML-bodies in putative
antiviral defense mechanisms came from the observation that PML-bodies
increase in size and number when cells are treated with interferon
(25, 26, 32, 38, 60). It was thus suggested that viruses
modify PML-bodies in order to adapt these structures to their needs
and/or to avoid these putative defense mechanisms (36).
Through extensive confocal microscopic analysis of double-fluorescence
labeling experiments, we have therefore determined the spatial
relationship of H-1 PAR-bodies and the following nuclear structures:
(i) PML-bodies, (ii) nucleoli, known to be involved in rRNA
transcription, processing, and more (53); (iii) coiled
bodies, thought to be involved in snRNP biogenesis (35); and
(iv) speckled domains, which serve as storage sites for splicing
factors such as SC-35 (39). Analysis of H-1 PAR-bodies at
the ultrastructural level revealed a ring-shaped electron-dense structure found only in infected cells. Thus, our results show that H-1
PAR-bodies represent a distinct nuclear structure that is induced after
parvovirus infection and serves as site of H-1 virus DNA replication.
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MATERIALS AND METHODS |
Viruses and mammalian cell lines.
Parvovirus H-1 was
collected after infection of NBE cells (56) at a
multiplicity of infection of 5 PFU per cell, and purified by cesium
chloride gradient centrifugation, as previously described (11). Otherwise, all infections were performed with a
multiplicity of 10 PFU per cell.
The cell line NBE was grown in modified Eagle's medium supplemented
with 5% fetal calf serum (Life Technologies) at 37°C in a 10%
CO2 atmosphere. Synchronization of NBE cells was achieved by an isoleucine-aphidicolin double-block protocol as previously described (13, 46). In brief, cells were seeded in medium lacking isoleucine. After 48 h, the medium was replaced by fresh complete medium containing 12 mg of aphidicolin (Sigma) per ml. After
10 h, the cells were infected or mock treated for 1 h and further incubated in the presence of aphidicolin. Another 9 h later, the cells were released from the aphidicolin block by addition of fresh aphidicolin-free medium.
Immunofluorescence.
Cells were grown on coverslips, fixed in
1% formaldehyde for 10 min at room temperature, dehydrated with
methanol for 5 min and acetone for 2 min, and permeabilized with 1%
saponin for 15 min at room temperature. After being washed with
phosphate-buffered saline (PBS), the cells were preincubated with 1%
goat serum in PBS, followed by two washing steps, one with 350 mM
NaCl-0.2% Tween 20-0.2% NP-40 in PBS and the other with 2 mM
MgCl2 in PBS. Primary and secondary antibodies were
successively incubated for 1 h each, followed by three PBS washes.
After staining with DAPI (4',6'-diamino-2-phenylindole; 250 µg/ml in
H2O) for 1 min, coverslips were mounted onto glass slides
in Elvanol (polyvinyl alcohol; molecular weight, 77,000 to 79,000;
ICN). For the detection of PCNA, cells were treated as described above
except that incubation in formalin was omitted.
For immunolocalization, the following antibodies were used: for NS1,
the polyclonal antiserum SP8 (
8) and the monoclonal
antibody
3D9 (a generous gift from N. Salomé and D. Pintel);
for
PML-bodies, the commercially available monoclonal PML antibody
(Santa
Cruz Biotechnology), a monoclonal SP100 antibody (a generous
gift from
G. Maul), and a polyclonal SP100 antiserum (
61); for
SGT,
the affinity-purified polyclonal antiserum AC1.2 (
21);
for
PCNA, a commercially available monoclonal antibody (Upstate
Biotechnology, Lake Placid, N.Y.); for No38, a polyclonal antiserum
from guinea pig (a generous gift from M. Schmidt-Zachmann)
(
55);
for speckled domains, the commercially available
monoclonal antibody
SC-35 (Sigma, Munich, Germany) (
59); for
coiled bodies, a polyclonal
p80/coilin antiserum (a generous gift from
A. Lamond); for NS2,
the SP6 antiserum (
8); and for capsid
proteins, a polyclonal
serum (
29).
Visualization of DNA replication and in situ localization of
viral nucleic acids.
To detect ongoing DNA replication, cells were
labeled with bromodeoxyuridine (BrdU) at a final concentration of 10 µM in the medium for 20 min, fixed with 3.7% formaldehyde for 10 min, and then washed with PBS and H2O. To denature
double-stranded DNA, the preparations were incubated with 7.4% HCl for
10 min and then washed with H2O and PBS. Replicating DNA
was visualized by immunofluorescence after incubation with a
BrdU-specific antibody (Becton Dickinson) and a secondary antibody
coupled to tetramethyl rhodamine isocyanate (TRITC) as described above.
To localize viral nucleic acids, fluorescence in situ hybridization
(FISH) was performed essentially as described previously
(
31). Briefly, after immunolocalization of NS1, the
preparations
were denatured with 70% formamide (pH 7) in 2× SSC (1×
SSC is
0.15 M NaCl plus 0.015 M sodium citrate) at 73°C for 3 min,
followed
by incubation in 50% formamide (pH 7) at 73°C for
equilibration.
The infectious H-1 virus DNA clone pSR19 (
24)
was biotinylated
via nick translation as previously described
(
33). Labeled DNA
(200 ng) was denatured with 50% formamide
(pH 7) in 2× SSC at
75°C for 5 min and applied onto the specimen
followed by renaturation
overnight at 37°C. Hybridization signals
resisting stringent washing
conditions (65°C in 0.1× SSC) were
visualized using streptavidin-fluorescein
isothiocyanate (FITC)
(Dianova).
Fluorescence microscopy.
After immunolabeling and mounting,
specimens were analyzed by conventional epifluorescence microscopy
(Leica; 40× and 63× objectives with immersion oil), as well as
confocal laser scanning microscopy (Zeiss LSM310 or LSM510 UV; 63×
Plan-Apochromat objective). For the colocalization studies, optical
sections were acquired simultaneously in order to avoid voxel shift.
For the detection of FITC and TRITC signals, the 488- and 543-nm
excitation lines of a argon-krypton laser were applied. For data
acquisition and imaging, the LSM510 UV software version 2.3 and Adobe
Photoshop 4.4 were used.
Electron microscopy.
Synchronized and infected NBE cells
were fixed for 15 min with 2% formaldehyde (freshly prepared from
paraformaldehyde), rinsed several times with PBS containing 50 mM
NH4Cl, permeabilized with 0.5% Triton X-100 in PBS for 5 min, briefly washed with PBS, and incubated for 1 h with the
anti-NS1 polyclonal serum SP8 at a dilution of 1:1,000 or the
monoclonal antibody 3D9 at a dilution of 1:5. After three washes with
PBS, the cells were incubated for 2 h with nanogold-coupled
secondary antibodies. Silver enhancement, fixation, and embedding in
Epon were carried out as described previously (52). Data
were acquired with an electron microscope (EM910; LEO Optics,
Oberkochen, Germany).
| |
RESULTS |
To identify the subnuclear compartment in which parvovirus H-1 DNA
replication proceeds, we have analyzed the distribution of the
virus-induced protein NS1, which is known to be essential for
replication of the viral genome. For this, NS1 was detected by indirect
immunofluorescence in time course experiments using synchronized NBE
cells infected with H-1 virus. Cells were infected while being blocked
at the G1/S border through isoleucine depletion followed by
aphidicolin treatment and subsequently released into the mitotic cycle.
The earliest time point at which NS1 could be detected by
immunofluorescence using the monoclonal antibody 3D9 was 8 h after
removal of aphidicolin. In the course of infection, increasing amounts
of NS1 accumulated in the nucleoplasm of infected cells. Using
epifluorescence or confocal microscopy, we noticed that NS1 strongly
accumulated in distinct nuclear bodies. In addition, NS1 was also
distributed diffusely throughout the nucleoplasm at lower
concentrations but absent from nucleoli (Fig.
1a). This particular nuclear distribution
of NS1 was observed in cells starting at 12 h after release from
aphidicolin block (Fig. 1a) up to the latest time point tested (18 h
postrelease [data not shown]). NS1 accumulation continued during the
course of infection (Fig. 1b). Since we did not monitor the fate of
individual sites of NS1 accumulation, it is not clear whether
neighboring bodies merged over time or whether single bodies increased
more in size than others in a given time interval.
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FIG. 1.
NS1 localizes in the nucleoplasm, accumulates in nuclear
bodies, and is absent from nucleoli. Immunofluorescence experiments
were performed with synchronized and H-1-infected NBE cells at 12 h (a) and 16 h (b) after release of the aphidicolin block. Panels
represent confocal sections showing NS1 immunolocalized with the
monoclonal antibody 3D9 and detected via an FITC-conjugated secondary
antibody. Bar, 20 µm in both panels.
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H-1 viral DNA sequences and ongoing replication are confined to the
same nuclear structure in which NS1 accumulates.
Since NS1 is
essentially required for viral genome amplification and viral gene
transcription, we tested whether viral nucleic acid sequences were
localized within the nuclear structures in which NS1 accumulated. For
this, synchronized, H-1 virus-infected NBE cells were subjected to a
combination of FISH and immunolocalization. Examination of the
specimens using confocal laser scanning microscopy revealed a strict
and exclusive localization of viral nucleic acid sequences within the
NS1-containing nuclear bodies for all cells analyzed (Fig.
2). It is likely that most of the signal represents hybridization of the probe to amplified viral DNA, but
contribution of viral transcripts cannot be excluded. In mock-infected cells, no signals corresponding to NS1 or H-1 viral sequences were
detected.
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FIG. 2.
H-1 sequences colocalize with NS1 in nuclear bodies.
Synchronized and infected NBE cells were simultaneously subjected to
immunolocalization using the anti-NS1 polyclonal serum SP8 and FISH
with the biotinylated probe pSR19. NS1 was detected via an
FITC-conjugated secondary antibody (a), and H-1 genomes were visualized
using streptavidin-Cy3 (b). The equivalence in size and shape of the
signals present in both channels of one typical confocal section
indicate a complete colocalization of NS1 and H-1 genomes. Bar, 20 µm
in all panels.
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|
To ascertain the presence of viral DNA in the identified nuclear
structure and to test whether viral DNA is replicated there,
we
performed BrdU labeling experiments using synchronized and
infected NBE
cells that were released for 17 h from the aphidicolin
block and
pulse-labeled with BrdU for 20 min prior to fixation.
The specimens
were incubated with the anti-NS1 serum SP8 and the
corresponding
secondary antibody for immunolocalization of NS1.
After acid treatment
for denaturation of DNA, incorporated BrdU
was detected by indirect
immunofluorescence using a primary BrdU-specific
antibody. In all
infected cells, however, a strict colocalization
of DNA replication
with NS1-containing bodies was observed (Fig.
3). Strict colocalization of NS1 and BrdU
incorporation was also
observed when no DNA denaturation step was
performed (data not
shown). The protocol used here to visualize BrdU
incorporation
also permitted the detection of chromosomal DNA
replication (Fig.
3b). In infected cells, however, DNA replication was
observed
only in the nuclear bodies which accumulated NS1; no
incorporation
of BrdU into chromosomal DNA was detected. These results
indicated
that in cells where parvovirus DNA replication took place,
chromosomal
DNA replication had been either completed or blocked.
Biochemical
evidence for the downregulation of cellular DNA replication
during
parvoviral infection has been reported previously
(
18). Our
results demonstrate this effect for the first time
on a single-cell
level. On the basis of these results, we conclude that
the bodies
in which NS1 accumulated are also the nuclear structure in
which
H-1 virus DNA replication proceeds and thus refer to this
structure
as H-1 PAR-bodies.
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FIG. 3.
Parvoviral DNA replication colocalizes with NS1 in
nuclear bodies. NS1 was localized via the polyclonal antiserum SP8 and
an FITC-conjugated secondary antibody (a). Replication was monitored by
incorporation of BrdU and indirect immunofluorescence using a
TRITC-conjugated secondary antibody (b). The cell in the center of the
confocal section (arrowhead) exhibits a colocalization of NS1 and viral
DNA replication. Arrows point to cells in which chromosomal replication
is still in process. Bar, 20 µm.
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|
We also analyzed the subcellular localization of the smaller
nonstructural protein NS2 using the SP6 antibody; we found NS2
to be
mainly present in the cytoplasm but also homogenously distributed
in
the nucleus of infected cells. We could not detect a specific
accumulation of NS2 in H-1 PAR-bodies (U. Bodendorf and C. Cziepluch,
unpublished results). This pattern of NS2 distribution is in agreement
with results reported by others (
17) and ourselves
(
7) for
the NS2 protein expressed in cells infected by the
closely related
virus MVM. When we analyzed the localization of capsid
proteins
using a polyclonal serum raised against recombinant capsid
protein,
we observed a predominantly diffuse nuclear staining in
immunofluorescence
experiments. It is, however, known that progeny
single-stranded
DNA accumulation is linked to capsid formation
(
51,
63). We
therefore expect that capsid assembly also
takes place in close
proximity to H-1 PAR-bodies. We are currently
attempting to raise
an antibody directed against assembled capsids,
which might allow
the detection of assembled capsids close to H-1
PAR-bodies.
PCNA accumulates in H-1 PAR-bodies.
PCNA is required for
polymerase 
-driven chromosomal (27, 28) and most likely
also parvoviral (12) DNA replication and can therefore be
used as a representative cellular marker for DNA replication. This
prompted us to test whether PCNA was present in H-1 PAR-bodies.
Confocal analysis of indirect double-immunofluorescence experiments
showed that PCNA was distributed throughout the nucleoplasm of infected
cells excluding nucleoli. In addition PCNA was concentrated in specific
nuclear structures, showing a clear colocalization with NS1 and
therefore H-1 PAR-bodies (Fig. 4). Since
PCNA is an essential component of the cellular replication machinery, this finding confirms the results obtained from the BrdU incorporation experiments (Fig. 3) and strongly supports the idea that H-1 PAR-bodies represent the sites of autonomous parvovirus DNA replication. It is not
known whether PCNA accumulates in H-1 PAR-bodies through direct
interaction with the viral genome or with other cellular constituents
such as replication factor C, which is known to load PCNA onto DNA
(9).
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FIG. 4.
The essential replication factor PCNA accumulates with
NS1 in H-1 PAR-bodies. Double immunofluorescence was performed with
synchronized and H-1-infected NBE cells 14 h after release from
aphidicolin block. NS1 was immunolocalized with the anti-NS1 monoclonal
antibody 3D9 and an FITC-conjugated secondary antibody (a), and PCNA
was localized with a polyclonal anti-PCNA antibody and a
TRITC-conjugated secondary antibody (b). Confocal images of both
channels from the same confocal plane clearly indicate complete
colocalization of the signals. Bar, 20 µm.
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The cellular NS1-interacting protein SGT colocalizes with NS1 in
H-1 PAR-bodies.
Rat and human SGT proteins were previously
identified as cellular interacting partners for NS1 of H-1 virus.
Neither the function of SGT nor the function of the NS1-SGT complex is
understood. It has been speculated that SGT might be involved in
housekeeping processes, since sgt transcripts are present in
all rat and human tissues tested. In noninfected cells, SGT is
distributed diffusely in the cytoplasm as well as the nucleus but is
essentially absent from nucleoli (21, 30). We determined the
nuclear localization of SGT in infected cells by immunofluorescence
microscopy. SGT was present throughout the nucleoplasm and excluded
from nucleoli, and most strikingly accumulated with NS1 in H-1
PAR-bodies (Fig. 5). Since NS1 and SGT
are able to interact directly in vitro, it is tempting to speculate
that SGT is also recruited to H-1 PAR-bodies through direct interaction
with NS1 in vivo. When we investigated the subcellular distribution of
SGT in adenovirus-infected cells with or without AAV coinfection, we
found that the distribution of SGT was not altered (data not shown). It
therefore appears that SGT is specifically recruited only to the DNA
replication sites of autonomous parvovirus H-1. The accumulation of SGT
in H-1 PAR-bodies point to a specific role for SGT in replication and/or gene expression of autonomous parvoviruses.
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FIG. 5.
NS1 and the cellular NS1-interacting protein SGT
colocalize in H-1 PAR-bodies. Double immunofluorescence was performed
with synchronized and H-1-infected NBE cells 14 h after release
from aphidicolin block. NS1 detected with FITC results in a green
signal (a). SGT was immunolocalized with the polyclonal anti-SGT serum
AC1.2 and was stained using a TRITC-conjugated secondary antibody (b).
(c) Merged confocal images of panels a and b, showing the
colocalization of fractions of SGT and NS1. Bar, 20 µm.
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H-1 PAR-bodies represent a distinct nuclear structure.
Viruses
with double-stranded DNA genomes such as adenovirus, simian virus 40, or herpes simplex virus type 1 were shown to replicate in association
with PML-bodies, and it was therefore of interest to determine the
relationship between H-1 PAR- and PML-bodies. To do so, confocal
analysis of double-immunofluorescence experiments was performed, using
synchronized, H-1 virus-infected NBE cells. At 14 h after release
from aphidicolin block, the PML-bodies were clearly visible in both
uninfected and infected cells as detected with a monoclonal PML
antibody (Fig. 6a and c). H-1 PAR-bodies were detected with the SP8 serum directed against NS1 (Fig. 6b). The
nuclear localization of PML- and H-1 PAR-bodies indicated that the two
structures were essentially distinct from each other and did not
colocalize (Fig. 6c). The same results were obtained with an
independent monoclonal antibody directed against SP100 (data not
shown). However, it cannot be excluded that other protein components
besides PML and SP100 are common to both structures.
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FIG. 6.
H-1 PAR-bodies form a distinct nuclear compartment. A
set of different nuclear bodies were colocalized with NS1 in infected
NBE cells 14 h after release from aphidicolin block. Shown are PML
bodies recognized by an anti-PML antibody (a and c), coiled bodies
recognized by an anti-p80/coilin antibody (d and f), speckled domains
recognized by an anti-SC-35 antibody (g and i), and nucleoli recognized
by an anti-No38/B23 antibody (j and l). (b, e, h, and k)
Immunolocalization of NS1 in the same confocal plain as shown in the
left column, detected with FITC. Overlays of the channels from the left
and center columns (c, f, i, and l) exhibit differential staining of
red and green signals, providing evidence for spatial separation of H-1
PAR-bodies from all nuclear bodies investigated in this study. Bars: a,
15 µm; d, g, and j, 20 µm.
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Up to 18 h after release from aphidicolin block, we did not
observe a significant change in the number of PML-bodies in
virus-infected
cells compared to noninfected cells (data not shown). We
did,
however, observe that the number of PML-bodies was decreased in
asynchronous NBE cells at 48 h postinfection or in cells that
expressed high amounts of NS1 after transient transfection when
DNA
condensation and other signs of induced cell death were visible
(data
not shown). It is therefore possible that NS1, though not
localized in
PML-bodies, disturbs PML and SP100 protein accumulation
and/or PML-body
stability through its known cytotoxic effects
(
10,
64).
Furthermore, we investigated the spatial distribution of H-1 PAR-bodies
with respect to a set of different nuclear structures
such as coiled
bodies, speckled domains, and nucleoli (Fig.
6d
to l). Antibodies
directed against p80/coilin, SC-35, and No38/B23
were used for the
detection of coiled bodies (Fig.
6d and f),
speckled domains (Fig.
6g
and i), and nucleoli (Fig.
6j and l),
respectively. As illustrated in
Fig.
6f, i, and l, H-1 PAR-bodies
did not show any colocalization with
either of the nuclear structures
tested. Taken together, our results
show that H-1 PAR-bodies represent
a distinct nuclear structure that
can be distinguished spatially
from those previously described,
including PML-bodies, coiled
bodies, speckled domains, and
nucleoli.
Ultrastructure of H-1 PAR-bodies.
To investigate whether the
H-1 PAR-bodies detected by light microscopy correspond to structures
visible by electron microscopy in ultrathin sections, we performed
immunogold localization of NS1. NBE cells blocked at the
G1/S transition were infected with H-1 virus or mock
treated, released for 17 h into the mitotic cycle, fixed in
paraformaldehyde, and processed for sectioning. The preparations were
then incubated with an NS1-specific antibody, either the antibody 3D9
(Fig. 7a) or the antiserum SP8 (Fig. 7b), and the corresponding gold-conjugated secondary antibodies. The results
obtained with both NS1 antibodies were very similar and in agreement
with the data obtained by confocal laser scanning analysis of
immunofluorescence experiments. NS1 was found both diffusely in the
nucleoplasm and also strongly accumulated in electron-dense nuclear
structures, with diameters of approximately 1 to 5 µm (Fig. 7a and
b). At 17 h after release from aphidicolin block, the nucleoli
appeared to be morphologically intact and essentially free of gold
particles (Fig. 7b). Using the 3D9 antibody to detect NS1, the overall
staining with gold was less intense and the electron-dense structure of
the H-1 PAR-bodies could readily be seen (Fig. 7a).
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FIG. 7.
Electron micrographs of ultrathin sections through
infected NBE cells showing the ultrastructure of H-1 PAR-bodies. (a and
b) Immunogold localization of NS1 with the monoclonal antibody 3D9 (a)
and the polyclonal antibody SP8 (b) to electron dense structures
identified as H-1 PAR-bodies; (c and d) ultrastructure of H-1
PAR-bodies. Bars: 0.5 µm (a and c), 1 µm (b), and 0.1 µm (d and
e).
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When infected cells were directly analyzed by electron microscopy
without indirect immunogold labeling, we detected ring-shaped
structures composed of a less electron-dense core surrounded by
material of higher electron density which corresponded in size
and
number to H-1 PAR-bodies (Fig.
7c). Since the ring-shaped
structure was
apparent in the preparations that were not treated
with antibodies,
this most probably reflects the native structure
of H-1 PAR-bodies more
closely. Our analysis did not yield evidence
that H-1 PAR-bodies were
generally localized in close proximity
to other nuclear structures that
could be identified by electron
microscopy. In preparations of
uninfected cells, no structures
resembling H-1 PAR-bodies were
detectable, indicating that H-1
PAR-bodies are induced by virus
infection.
At higher magnifications, we detected in the internal space of H-1
PAR-bodies structures which were very similar in size and
shape to
empty viral particles (Fig.
7d and e). The magnifications
achieved
here, however, do not allow an unequivocal identification
of single
viral capsids. Further investigations with antibodies
specifically
directed against assembled capsids are therefore
required to acertain
whether progeny viruses concentrate in H-1
PAR-bodies.
H-1 PAR-bodies appears to be very similar, if not identical, in
structure to electron-dense entities previously observed in
H-1
virus-infected NBE cells and termed "nucleolar inclusions"
(
1) and "hollow spheres" (
58). In contrast
with their original
interpretation as functionally irrelevant
structures, the present
work clearly points to these structures as the
sites at which
parvovirus DNA replication takes place. Furthermore, our
data
do not support the original assignment of these structures as
nucleolar
derivatives.
| |
DISCUSSION |
Virus-encoded and cellular proteins form H-1 PAR-bodies.
This
study led to the identification of a nuclear structure that is distinct
from known nuclear structures such as coiled bodies, speckled domains,
nucleoli, and PML-bodies by both its spatial distribution and at least
some of its protein constituents. This new type of nuclear body was
identified by analyzing the localization of NS1, which is known to be
essential for parvovirus replication (16, 19). A fraction of
NS1 accumulated in specific nuclear bodies which we identified as the
sites of viral DNA replication since H-1 virus nucleic acid sequences
and ongoing DNA synthesis were colocalized there. These structures were
termed H-1 PAR-bodies. Our findings are reminiscent of recent
observations concerning Aleutian mink disease virus-infected Crandell
feline kidney cells which show that NS1 localizes to discrete sites of
viral DNA replication. While NS1 from Aleutian mink disease virus
appeared to be exclusively localized at these sites (42,
43), we have reproducibly detected a fraction NS1 from H-1 virus
outside of H-1 PAR-bodies by both immunofluorescence and immunogold
labeling using two different NS1 antibodies. The function of this NS1
pool is unknown, as is the mechanism which regulates the nuclear
distribution of NS1. It has previously been shown that biochemical
activities of NS1 are regulated by phosphorylation in vitro (40,
41) and in vivo (15). It is thus possible that the
nuclear distribution of NS1 is also regulated through differential phosphorylation.
H-1 PAR-bodies contain not only viral proteins but also cellular
factors. PCNA is an essential factor of the cell DNA replication
machinery thought to be required for autonomous parvoviral DNA
replication (
12). In agreement with this finding, we have
observed
an accumulation of this factor in H-1 PAR-bodies. It may be
expected,
but remains to be shown, that other cellular factors which
are
equally important for parvovirus DNA replication such as the
parvovirus
initiation factor, replication protein A (
12),
and the high-mobility-group
proteins (
20) also accumulate in
H-1 PAR-bodies. The present
study further demonstrated that SGT, a
cellular protein previously
identified for its ability to interact with
NS1 in vitro and in
the yeast two-hybrid system (
21),
accumulates in H-1 PAR-bodies.
This finding suggests a functional role
for SGT in autonomous
parvoviral DNA replication and/or gene
expression, and we are
therefore investigating whether the presence of
SGT in H-1 PAR-bodies
is essential for virus replication. Since SGT and
NS1 can interact
directly in vitro, it is possible that recruitment of
SGT to H-1
PAR-bodies is mediated by NS1. It was previously observed
that
a fraction of SGT is modified in the presence of NS1, most likely
by phosphorylation (
21), and we are therefore testing
whether
recruitment of SGT into H-1 PAR-bodies is regulated by
phosphorylation.
H-1 PAR-bodies have a distinct ultrastructure.
H-1 PAR-bodies
were identifiable in ultrathin sections through immunogold labeling of
NS1 and displayed a characteristic ultrastructure with a less
electron-dense core surrounded by material of higher electron density.
These structures are not artifacts due to immunogold labeling since
they could also be detected in unstained preparations. The
ultrastructure of H-1 PAR-bodies fits nicely with a recently suggested
unified model for cellular transcription and DNA replication factories.
In these factories, the enzymatic functions are proposed to be
concentrated in the center, while the nucleic acids, i.e., genes and
transcripts, are found surrounding the core (14). Accordingly the outer ring of H-1 PAR-bodies, in which NS1 accumulates, might contain newly synthesized viral DNA and transcripts. In this
respect, H-1 PAR-bodies could serve as a model for cellular transcription and replication factories, allowing the investigation of
parameters which regulate the assembly and stability of such centers.
In previous studies, ultrastructural changes induced by parvovirus H-1
in NBE cells have been thoroughly analyzed (
1,
58).
The most
pronounced ultrastructural alterations observed were
reported to be (i)
margination of the nuclear chromatin, (ii)
modifications and eventual
breakdown of nucleoli, and (iii) appearance
of doughnut-shaped bodies
of assumed nucleolar origin (
1).
Subsequent studies
performed with H-1 virus-infected NBE cells
synchronized by a double
methotrexate block revealed that the
earliest morphological changes
occurred in nucleoli at 12 h postinfection,
when fragmentation and
loss of nucleolar fibrous matter were detected.
Between 18 and 36 h postinfection, electron-dense hollow spheres
thought to be composed
of fibrous and/or granular nucleolar elements
and to contain incomplete
H-1 virus particles were observed. These
structures were interpreted as
being by-products of nucleolar
breakdown and were considered irrelevant
for H-1 virus propagation
(
58). The ultrastructure of H-1
PAR-bodies resembles the doughnut-shaped
nucleolar inclusions observed
by al-Lami and coworkers (
1)
and the hollow spheres
described by Singer and Toolan (
58) so
closely that we
consider them to be identical. Additional evidence
for the identity of
H-1 PAR-bodies and these previously observed
structures comes from
experiments in which the nuclear sites of
ongoing viral DNA replication
were localized. Our results show
that BrdU incorporation is confined to
H-1 PAR-bodies (Fig.
4).
When cells were pulse-labeled with
[
3H]thymidine, Singer and Rhode also observed
incorporated label
only in the electron-dense structures and not within
intact nucleoli
(
57). The present work questions the
conclusions drawn from
these previous studies by showing that the
nuclear bodies induced
by parvovirus infection are unlikely to be of
nucleolar origin.
Indeed, we could detect H-1 PAR-bodies when nucleoli
appeared
to be still intact, and we found no evidence for a
relationship
between H-1 PAR-bodies and nucleoli (Fig.
6 and
7). Our
data,
however, do not exclude the possibility that some proteins
present
in nucleoli are also recruited to H-1 PAR-bodies. A good
candidate
protein is nucleolin, which has indeed been shown to form a
specific
complex with the minus strand of MVM DNA (
3).
Finally, our
finding that cellular (PCNA) and viral (NS1) factors known
to
be essential for parvovirus growth accumulate with replicating
DNA
in H-1 PAR-bodies argues against the previously accepted view
that
parvovirus-induced nuclear structures are not relevant to
the virus
life
cycle.
Why are H-1 PAR-bodies induced after H-1 infection?
Viruses
with double-stranded DNA genomes target PML-bodies for the
transcription of their early gene products and also initiate DNA
replication close to these sites (36). Data presented in this study demonstrate that H-1 PAR-bodies are the sites for H-1 virus
DNA replication and that they represent a novel nuclear structure that
is distinct from PML-bodies and other known nuclear structures. These
results were unexpected and raise the question of why this virus
induces specific nuclear structures instead of utilizing preexisting ones.
One possible reason for the fact that the growth of H-1 virus does not
require their targeting to PML-bodies may be traced
back to the
proposed role of these bodies in cellular antiviral
defense mechanisms.
Viruses with double-stranded DNA genomes express
so-called early
proteins shortly after infection. These viral
products often alter host
cell physiology and in particular drive
quiescent cells into a
replicative phase in order to support viral
DNA synthesis. It may be
speculated that an infection with PML-associated
viruses is rapidly
sensed by target cells, which then have the
opportunity to mount an
antiviral response. This defense mechanism
needs to be circumvented by
the virus for it to replicate, which
could be achieved through the
targeting and modification of PML-bodies.
Indeed, the immediate-early
gene products encoded by some but
not all DNA viruses induce the
dispersion of proteins present
in PML-bodies (
36).
Furthermore, mutant herpes simplex 1 viruses
that are not able to
disrupt PML-bodies were shown to be impaired
in expression of viral
genes and also in the reactivation of latent
viral genomes
(
37). It remains to be determined whether the
dispersion of
PML-body proteins is required to adapt these sites
to specific viral
needs or to modify putative cellular antiviral
responses
(
36). In contrast to double-stranded DNA viruses,
autonomous
parvoviruses cause opportunistic infections because
their genes
are unable to be expressed immediately after infection
due to the
single strandedness of their genome and have to wait
until host cells
reach S-phase to undergo genomic conversion to
duplex replicative forms
that can be expressed and amplified (
19).
This dependence
may delay the time at which infected cells are
induced to mount an
antiviral response, saving parvoviruses the
need for early targeting
and modification of PML-bodies. In this
respect it is worth pointing
out that parvoviruses were indeed
reported to be poor inducers of
antiviral effectors such as interferon
(
54,
66,
67).
Nevertheless, it should be stated that at
late stages of infection, H-1
virus had an effect on PML-bodies,
which were found to decrease in
number. It is tempting to relate
this late alteration to the
intracellular accumulation of the
NS1 protein, which is known to have a
cytotoxic function (
10).
The precise molecular mechanism of
NS1-associated cytotoxicity
is poorly understood, although several
parvovirus- or NS1-induced
cellular disturbances, including cell cycle
arrest, DNA cleavage
(
44,
45), dysregulation of cellular
promoters (
64), and
altered expression and phosphorylation
of specific cellular proteins
(
2), have been reported. It is
therefore possible that late
in infection, PML and SP100 proteins are
also targets for these
pleiotropic cytopathic effects of
NS1.
In conclusion, we have identified through immunofluorescence
experiments H-1 PAR-bodies as the site of H-1 virus DNA replication
and
have characterized these nuclear bodies also at the ultrastructural
level. More recent results obtained in our laboratory show that
similar
bodies are found in MVM-infected mouse cells (T. Bashir
and C. Cziepluch, unpublished results). This finding might indicate
that H-1
PAR bodies represent a prototype for structures also
induced in the
nucleus of cells infected by other autonomous
parvoviruses.
| |
ACKNOWLEDGMENTS |
We acknowledge the generous gift of antibodies from A. Lamond
(University of Dundee), G. Maul (Wistar University, Philadelphia, Pa.),
D. Pintel (University of Missouri, Columbia), and H. Will (HPI,
Hamburg, Germany) and from N. Salomé (University of Missouri, Columbia), M. Schmidt-Zachmann (DKFZ, Heidelberg, Germany), and T. Sternsdorf (HPI). The expert support of H. Spring (DKFZ) with acquisition of data by confocal microscopy is gratefully acknowledged. We thank T. Bashir for help with FACS analyses in the initial phase of
the project. We are very grateful to W. W. Franke (DKFZ) for
critical discussion of the electron microscopic data and comments on
the manuscript. We also thank T. Bashir, U. Bodendorf, and N. Salomé (DKFZ) for comments on the manuscript. Finally, we thank
H. zur Hausen for continuous encouragement and support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Applied Tumor
Virology Unit, Abteilung F0100 and INSERM U 375, Deutsches
Krebsforschungszentrum, Postfach 101949, 69009 Heidelberg, Germany.
Phone: 49 6221 424973. Fax: 49 6221 424962. E-mail:
C.Cziepluch{at}dkfz-heidelberg.de.
| |
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Journal of Virology, May 2000, p. 4807-4815, Vol. 74, No. 10
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
