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Previous Article | Next Article 
Journal of Virology, May 2001, p. 4394-4398, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4394-4398.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vivo Accumulation of Cyclin A and Cellular
Replication Factors in Autonomous Parvovirus Minute Virus of
Mice-Associated Replication Bodies
Tarig
Bashir,
Jean
Rommelaere, and
Celina
Cziepluch*
Applied Tumor Virology Unit F0100 and
Institut National de la Santé et de la Recherche Médicale U
375, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany
Received 14 December 2000/Accepted 6 February 2001
 |
ABSTRACT |
Autonomous parvovirus minute virus of mice (MVM) DNA replication is
strictly dependent on cellular factors expressed during the S phase of
the cell cycle. Here we report that MVM DNA replication proceeds in
specific nuclear structures termed autonomous parvovirus-associated replication bodies, where components of the basic cellular replication machinery accumulate. The presence of DNA polymerases 
and 
in
these bodies suggests that MVM utilizes partially preformed cellular
replication complexes for its replication. The recruitment of cyclin A
points to a role for this cell cycle factor in MVM DNA replication
beyond its involvement in activating the conversion of virion
single-stranded DNA to the duplex replicative form.
| |
TEXT |
Autonomous parvoviruses and other
members of the Parvoviridae family are unique among animal
viruses in having linear single-stranded DNA genomes. The termini of
their approximately 5,000-nucleotide genomes contain palindromic
sequences which fold into stable hairpin structures and serve as
primers for viral DNA synthesis. The replicative cycle, which takes
place in the nucleus, is initiated by the synthesis of the cDNA strand
leading to the formation of double-stranded replicative-form DNA. This
reaction, also known as conversion, is exclusively dependent on
cellular factors. In addition, the subsequent amplification reactions
require the activity of the virus-encoded major nonstructural protein
NS1 (7).
Several cellular factors which are essential for DNA replication of the
prototype autonomous parvovirus minute virus of mice (MVM) have been
identified through cell fractionation and complementation assays in
vitro replication systems (5, 8, 20). However, very little
is known about the subnuclear organization of MVM DNA replication in
vivo. In contrast to viruses with double-stranded DNA genomes that
replicate in close proximity to preformed nuclear structures known as
promyelocytic leukemia (PML) bodies (for review see reference
18), autonomous parvovirus H-1 was shown to induce characteristic nuclear structures, termed H-1 parvovirus-associated replication (PAR) bodies, which are unrelated to PML bodies
(9). Similar structures were also characterized in
Aleutian mink disease virus-infected cells (21, 22). In
this study, we aimed to determine whether MVM also establishes PAR
body-like structures in the nuclei of infected cells and to analyze the
subnuclear distribution of cellular factors assumed to be involved in
MVM DNA replication in vivo. To this end, A9 mouse cells were infected with MVMp at a multiplicity of infection of 10 PFU per cell. At 15 h postinfection, cells were labeled for 20 min with bromodeoxyuridine (BrdU) at a concentration of 10 µM and then immediately fixed in 1%
formaldehyde for 10 min at room temperature. BrdU incorporation into
replicated viral DNA was detected with a BrdU-specific antibody (Becton
Dickinson) without prior denaturation, thus excluding the detection of
chromosomal DNA replication (21). Simultaneously, NS1 was
detected with the NS1-specific SP8 antibody (11). Analysis by confocal microscopy (LSM510 UV; Zeiss, Jena, Germany) revealed the
accumulation of NS1 in specific nuclear bodies (Fig.
1a) which were also found to be the sites
of ongoing viral DNA replication, as indicated by BrdU incorporation
(Fig. 1b and c). At this time postinfection, no signs of virus-induced
cytotoxicity were visible (Fig. 1d). From these data, we concluded that
MVM DNA replication proceeds in specific nuclear structures similar to
the ones previously described for other autonomous parvoviruses
(9, 21, 22). We would therefore like to propose the more
general term autonomous PAR (APAR) bodies for these virus-induced
structures.
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FIG. 1.
MVM DNA replication colocalizes with NS1 in APAR bodies
in the nuclei of infected A9 cells. Representative confocal optical
sections through the nuclei of infected cells are shown. NS1 was
localized with the SP8 polyclonal antiserum and a fluorescein
isothiocyanate (FITC)-conjugated secondary antibody (a). Replication
was monitored by incorporation of BrdU and indirect immunofluorescence
using a BrdU-specific antibody and a tetramethyl rhodamine
isothiocyanate (TRITC)-conjugated secondary antibody (b). In a merged
image, colocalized structures from panels a and b appear yellow (c). By
phase-contrast microscopy (Nomarski), the cells show no obvious sign of
NS1-induced cytotoxicity at the time of fixation (15 h postinfection)
(d).
|
|
MVM DNA replication starts only after host cells enter the S phase of
the cell cycle. Recently, it was shown that in vitro the conversion
reaction is activated by the cell cycle factor cyclin A, whose
production is induced at the G1/S transition
(1). Cyclin A is involved in the regulation of the S and
G2 phases (25) and has been reported to be
required for chromosomal (16) and simian virus 40 (10, 12) DNA replication. In order to test whether cyclin
A is present in APAR bodies, we performed double immunofluorescence
labeling and confocal microscopical analysis of MVM-infected A9 cells.
Cyclin A, which is homogeneously distributed throughout the nuclei of
mock-infected cells and absent from nucleoli (reference 2
and data not shown), was indeed found to massively accumulate together
with NS1 in the APAR bodies of infected cells (Fig. 2a to
c). Even if one assumes that the subnuclear location at which the conversion reaction takes place predetermines the site of APAR body formation, a sole role for cyclin A
in the conversion reaction is difficult to reconcile with such a strong
accumulation of cyclin A in APAR bodies at 15 h postinfection,
when conversion is thought to be complete. This result could indicate
that the continuous presence of cyclin A is also required for
subsequent NS1-dependent replicative steps. One can furthermore
speculate that the sequestration of this factor is responsible for the
previously described parvovirus-induced cell cycle arrest (23,
24) because deprivation of cyclin A may lead to a failure of
cdk1 activation in the G2 phase, thus preventing cell cycle
progression.
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FIG. 2.
Cyclin A, but not cyclin E or cdk2, accumulates in APAR
bodies. Representative confocal images of nuclei from double-labeled
MVM-infected A9 (a to c) or NBE (d to i) cells are shown. NS1 was
detected with FITC using the NS1-specific 3D9 antibody (a generous gift
from N. Salomé and D. Pintel) (a, d, and g). Images of cyclin A
(b), cyclin E (e), and cdk2 (h) were detected with TRITC in the same
confocal plane as shown in the left column using the respective primary
antisera for cyclin A (a generous gift from M. Pagano), cyclin E (Ab-1;
Neomarkers, Fremont, Calif.), and cdk2 (Ab-3; Neomarkers). The
superimposition of both channels from the left and middle columns
reveals either the colocalization (yellow signal [c]) or the lack of
colocalization (distinct red and green signals [f and i]) of the
respective factors in relationship to APAR bodies.
|
|
Besides cyclin A, cyclin E is involved in the regulation of the
G1/S transition and is known to play a role in chromosomal DNA replication (14, 16). In contrast to cyclin A,
however, cyclin E did not accumulate in APAR bodies (Fig. 2d to f).
Since the cyclin E-specific antibody best recognized the human protein, in these experiments we infected human NBE cells and verified that MVMp
indeed induces the formation of APAR bodies in this human cell line, as
detected by NS1-specific (Fig. 2d) and BrdU-specific (data not shown)
antibodies. The failure to detect an accumulation of cyclin E does not
exclude the presence of minute amounts, of this factor in APAR bodies.
Our data are, however, in agreement with recent in vitro experiments
showing no contribution of cyclin E or cdk2 in the initiation of
conversion (1).
Cyclin A forms an active complex with cdk2 at the beginning of the S
phase. This complex is assumed to be involved in the regulation of DNA
replication, since both cyclin A and cdk2 were found to be associated
with replication foci in mammalian cells (3) as well as
with replicating DNA in the simian virus 40 in vitro replication system
(13). We therefore attempted to detect cdk2 in APAR
bodies. Using several different cdk2-specific antibodies, cdk2 could
not be shown to accumulate in APAR bodies (Fig. 2g to i). The observed
accumulation of cyclin A in APAR bodies (Fig. 2b) would then point to a
structural role for cyclin A in these bodies, independent of complex
formation with cdk2. However, it is still possible that only catalytic
amounts too small to be detected by the method used here are required
for MVM DNA replication in APAR bodies.
Parvovirus DNA replication proceeds according to a
leading-strand-synthesis mechanism and has been shown to be dependent
on proliferating cell nuclear antigen (PCNA) in vitro (5).
These and other lines of evidence point to DNA polymerase as the
enzyme responsible for parvovirus DNA replication (6).
Here, we show that DNA polymerase indeed accumulates in APAR bodies
in MVM-infected NBE cells (Fig. 3a to c)
and therefore at the sites of ongoing MVM DNA replication. DNA
polymerase requires for processive DNA synthesis the presence of
the cofactor PCNA, which is also involved in other processes, like DNA
recombination and repair (15). PCNA is distributed
throughout the nucleus except the nucleoli, but it specifically
accumulates in the APAR bodies of MVM-infected NBE cells (Fig. 3d to
f). This finding corroborates earlier in vitro data demonstrating that
PCNA is indispensable for MVM DNA replication (1, 5).
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FIG. 3.
Cellular replication factors DNA polymerases and
, PCNA, and RPA accumulate in APAR bodies. Representative confocal
images of nuclei from double-labeled MVM-infected NBE cells are shown.
NS1 was detected with FITC using the SP8 antiserum (a, d, g, and j).
Replication factors were detected with TRITC in the same confocal plane
as shown in the left column, using the respective antibodies against
DNA polymerase (Transduction Laboratories) (b), PCNA (PC10; Upstate
Biotechnology) (e), RPA (Ab-1; Oncogene) (h), and DNA polymerase (SJK132-20 [26]) (k), respectively. A merged image of both channels
from the left and center columns provides evidence for colocalization
of these factors in APAR bodies (c, f, i, and l).
|
|
The single-strand-DNA-binding protein replication protein A (RPA) is
required for MVM DNA replication in vitro (5) and cannot
be replaced by other proteins with single-strand-DNA-binding activity
(J. Christensen, personal communication). Furthermore, a direct
interaction between the 70-kDa subunit of RPA and NS1 was demonstrated
in vitro (4). In agreement with these in vitro findings we
have observed that RPA massively accumulates at the sites of ongoing
parvovirus DNA replication in infected cells (Fig. 3g to i), which
strongly suggests its involvement in this process in vivo.
Surprisingly, DNA polymerase was also found at the sites of MVM DNA
replication (Fig. 3j to l). So far, there is no evidence for a
contribution of DNA polymerase to parvovirus DNA replication, since
neutralizing antibodies against this enzyme do not impair viral DNA
replication in vitro (5, 19). Furthermore, the presence of
preformed hairpin primers makes a DNA polymerase -dependent primase
activity dispensable. However, it was shown that DNA polymerase can
be isolated together with DNA polymerase and the PCNA loading
factor, replication factor C, from mammalian cells as a stable complex
that is replication competent when template DNA, PCNA, and nucleotides
are added (17). It could be demonstrated that in this
complex, DNA polymerase is active on single-stranded-DNA templates
only when primer synthesis is a precondition for replication. On a
primer-template junction, as present in parvovirus DNA, the DNA-binding
affinity of replication factor C is higher than that of DNA polymerase
, thereby rendering the latter inactive (17, 27). Since
DNA polymerase is required for chromosomal DNA replication, it is
tempting to speculate that MVM utilizes cellular replication complexes
that are at least partially preformed, exploiting only the DNA
polymerase activity and thus evading the need for an
energy-consuming dissociation of preformed cellular complexes. This
would again highlight the opportunistic and minimalistic strategies
used by these viruses.
In summary, the data presented here show for the first time the
formation of APAR bodies in MVM-infected cells and the accumulation of
various cellular replication factors in these structures. Our data
support the idea that the basic cellular replication machinery is used
by MVM for viral DNA replication, including DNA polymerase , PCNA,
RPA, and cyclin A. DNA polymerase , which is part of the cellular
replication machinery, was unexpectedly found to accumulate in APAR
bodies but is most likely not active in parvovirus DNA replication. For
cyclin A, it will be of interest to identify the role this factor plays
in parvovirus DNA replication, in addition to its role in the
activation of the conversion reaction in vitro.
| |
ACKNOWLEDGMENTS |
We acknowledge the generous gift of antibodies from D. Pintel and
N. Salomé (University of Missouri
Columbia) and antiserum from
M. Pagano (New York University, New York, N.Y.). The expert support of
H. Spring (DKFZ, Heidelberg, Germany) with acquisition of data by
confocal microscopy is gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Applied Tumor
Virology, Abteilung F0100 and INSERM U 375, Deutsches
Krebsforschungszentrum, Postfach 101949, 69009 Heidelberg, Germany.
Phone: 49 6221 424977. Fax: 49 6221 424962. E-mail:
C.Cziepluch{at}dkfz-heidelberg.de.
| |
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Journal of Virology, May 2001, p. 4394-4398, Vol. 75, No. 9
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4394-4398.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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