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INTRODUCTION |
Herpes simplex virus type 1 (HSV-1)1 is a major human
pathogen whose lifestyle is based on a long-term hide-and-seek
interaction with the infected host. After initial infection at the
periphery, the virus enters sensory neurons and establishes a lifelong
latent infection (for review, see Ref. 1). Periodic reactivation from latency usually results in mild illness symptoms such as cold sores.
These episodes of reactivation have enabled the virus to evolve
optimally in parallel with the host. During lytic infection, the HSV-1
152-kilobase pair double-stranded DNA genome expresses at least
74 temporarily regulated genes. These genes are classified as
immediate-early (IE), early, and late, depending on the time course of
their synthesis and requirement for prior viral gene expression and DNA
replication. During latency, the viral genome undergoes dramatic
changes resulting in an almost complete silencing of transcription (for
review, see Ref. 2). After successful penetration of the cell and
release of viral DNA in the nucleus, the first step of a productive
infection is the synthesis of IE proteins. Four of the five IE proteins
encoded by HSV-1 regulate gene expression. ICP4 and ICP27 are essential
for virus replication (3, 4), whereas ICP22 is dispensable for virus
viability in most cell types (5). The requirement for ICP0 (also called Vmw110) for the onset of lytic infection is not absolute. Viruses either deficient for its expression or expressing an inactivated form
of the protein, rather than being noninfectious in cultured cells, show
a cell type- and multiplicity-dependent growth
defect (6). ICP0 appears to increase the probability of the initiation of productive infection. This feature assumes greater significance during reactivation of the virus from latency, because it has been
shown that in the absence of ICP0 the probability for mutant viruses to
reactivate is dramatically decreased both in cultured cells and in
mouse models (7, 8). This defect can be overcome in cultured cells by
providing exogenous ICP0 (9-12).
ICP0 is a RING finger zinc-binding protein that was initially studied
because of its transactivation activity in transfection assays
(reviewed in Ref. 13). The RING finger domain has been shown to be
essential for the biological activities of ICP0 outlined above (for
review, see Ref. 14). New fields of interests arose after the discovery
that ICP0 localizes to, and then disrupts, nuclear domains called ND10,
nuclear dots, PML nuclear bodies, or promyelocytic oncogenic
domains (15-17). More recently, it was observed that centromeres are
also targeted by ICP0 early in infection. Centromeres contain a number
of specific proteins (reviewed in Ref. 18), several of which are
recognized by anti-centromere autoimmune antibodies in the sera of
patients with a variety of conditions (19). CENP-B (80 kDa) is a
sequence-specific
-satellite DNA-binding protein that localizes
throughout the centromeric heterochromatin located beneath the
kinetochore (20, 21). CENP-C (140 kDa) is an essential component of the
inner kinetochore plate of active centromeres (22, 23) that is required
for maintaining proper kinetochore size and has been implicated in the
metaphase-to-anaphase transition in mitotic cells by stabilizing microtubule attachments (24). Because the 17-kDa CENP-A protein localizes in centromeres and copurifies with nucleosome core particles, it was suggested to function as a centromere-specific core histone (25). Indeed, CENP-A contains a histone H3-related histone fold domain
and has been described as histone H3-like centromere protein (26).
CENP-A is a homodimer (27) that localizes in the inner kinetochore
plate and associates with
-satellite DNA. These observations suggest
that there is a distinct nucleosome structure within the centromere
(22, 28). Recently, it has been shown that, in contrast to CENP-B (29)
but similarly to CENP-C, mouse CENP-A (termed Cenpa) is essential for
the viability of embryos, which show in its absence severe mitotic
problems early in their development (30).
We have previously shown that during HSV-1 infection CENP-C is
degraded in an ICP0-dependent manner, and infected mitotic cells are arrested with condensed chromosomes distributed in a prometaphase-like arrangement termed pseudoprometaphase (31-33). However, it had been previously reported that anti-CENP-C antiserum was
not sufficient to generate this phenotype in microinjected cells, and a
pseudoprometaphase arrest was only observed after microinjection of
cells with human anti-centromere autoimmune sera (24). Therefore we
suggested that although degradation of CENP-C would undoubtedly
contribute to the mitotic arrest of HSV-1-infected cells, the observed
pseudoprometaphase phenotype is an indication of a destabilization of
other centromere proteins (33). In this report we show that the 17-kDa
CENP-A protein is degraded in HSV-1-infected cells in an ICP0- and
proteasome-dependent manner. The use of cells expressing
transiently and constitutively an influenza hemagglutinin (HA1)
epitope-tagged derivative version of CENP-A shows that CENP-A is
effectively degraded either during infection or as a result of the
expression of fully functional ICP0 alone. These data are supported by
immunofluorescence experiments that show the disappearance of CENP-A
from centromeres in both infected and transfected cells. Because CENP-A
is thought to be involved in the assembly of transcriptionally silent
heterochromatin, this finding suggests a direct method by which the
HSV-1 genome might be silenced during latency and quiescence and how
this could be alleviated by ICP0.
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EXPERIMENTAL PROCEDURES |
Viruses, Cells, and Plasmids--
HSV-1 strain 17 syn+ (17+) was
the parental strain used in this study. Temperature-sensitive
(ts) mutant virus tsK expresses nonfunctional
ICP4 at 38.5 °C (3). Mutant in1330 is a derivative of
tsK from which the IE1 gene encoding ICP0 has been deleted. ICP0 mutant viruses dl1403 and FXE (6, 34) were also used. All viruses were grown and titrated in baby hamster kidney cells propagated in Glasgow modified Eagle's medium containing 10 units/ml penicillin and 100 µg/ml streptomycin and supplemented with 10% newborn calf serum and 10% tryptose phosphate broth.
Hep2 cells and HeLa tTA-CAHA cells expressing an influenza
hemagglutinin (HA1) epitope-tagged derivative version of CENP-A, CENP-A-HA (27), were grown at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics as
above. HeLa tTA-CAHA cultures were supplemented with 400 µg/ml G418,
330 ng/ml puromycin, and 1 µg/ml tetracyclin. Human fetal lung (HFL)
cells were grown at 37 °C in Dulbecco's modified Eagle's medium
supplemented with 5% fetal bovine and 5% new born calf sera, 2%
glutamine, 1% nonessential amino acids, and antibiotics as above.
Plasmids pCI110 and pCIFXE express ICP0 and its RING finger mutant,
respectively (35). Plasmid pcDLCA-HA expresses the protein CENP-A-HA
and has been described previously (26).
Immunofluorescence and Confocal Microscopy--
Hep2 cells were
seeded at 1.5 × 105 cells per well in 24-well
Linbro multiwell plates containing one coverslip and either infected or
transfected the following day. After infection or transfection, cells
were fixed for 10 min with formaldehyde (5% v/v of the 30% stock
solution in PBS containing 2% sucrose), washed three times with PBS,
then permeabilized for 5 min in a PBS solution containing 0.5% Nonidet
P-40 and 10% sucrose. Primary antibodies were diluted in PBS
containing 1% newborn calf serum. After incubation at room temperature
for 1 h, coverslips were washed at least three times with PBS plus
1% newborn calf serum, and then treated with appropriate secondary
antibodies. After a further 30-min incubation, coverslips were washed
three times with PBS plus 1% newborn calf serum and then washed once
with water before being mounted using Citifluor. Cell samples
were examined using a Zeiss LSM 510 confocal microscope with
three lasers giving excitation lines at 488, 543, and 633 nm. The data
from the channels were collected separately to avoid channel overlap,
with 8-fold averaging at a resolution of 1024 × 1024 pixels using
optical slices of ~1 µm. The microscope was a Zeiss Axioplan
utilizing either × 40 or × 63 oil immersion objective lenses, numerical aperture 1.4. Data sets were processed using the LSM 510 software and then exported for preparation for printing using Adobe Photoshop.
Antibodies--
Antibodies used for immunofluorescence were as
follows: monoclonal antibodies 11060 (dilution 1:1,000) against ICP0
and 58S (dilution 1:50) against ICP4, respectively (see Refs. 36 and 37), human anti-centromere autoimmune (huACA) serum (dilution 1:20,000) (19), and monospecific rabbit anti-CENP-A (dilution 1:500)
(38). The secondary antibodies used were fluorescein isothiocyanate-conjugated sheep anti-mouse and goat anti-rabbit IgG
(Sigma, 1:100), Cy3-conjugated goat anti-mouse (1:500) and anti-rabbit
(1:5000) IgG (Amersham Pharmacia Biotech), and Cy5-conjugated goat
anti-human IgG (1:500) (Amersham Pharmacia Biotech).
Western Blotting--
HFL cells were seeded at 1.5 × 105 cells per well in 24-well Linbro multiwell
plates and infected 48 h later. HeLa tTA-CAHA cells were seeded at
0.5 × 105 cells per well in medium not
containing tetracycline to induce synthesis of CENP-A-HA. Forty-eight h
later the medium was replaced by fresh medium containing 1 µg/ml
tetracycline to repress synthesis of CENP-A-HA for 24 h. Cells
were then infected and harvested for Western blotting. For the
preparation of nuclear fractions, HFL cells were harvested after
infection in 100 µl of a hypotonic buffer (20 mM HEPES
(pH 7.4), 1 mM MgCl2, 10 mM
KCl, 0.5% Nonidet P-40, 0.5 mM dithiothreitol) and
incubated on ice for 30 min. After centrifugation at 6000 × g for 4 min, supernatants were removed, and pellets
containing nuclei were resuspended in 70 µl of Laemmli buffer and
boiled for 5 min. SDS-polyacrylamide gels (12.5%) were prepared and
run in the Bio-Rad MiniProtean II apparatus, and then proteins were
electrophoretically transferred to nitrocellulose membranes (BA85,
Schleicher & Schüll) according to the manufacturer's
recommendations. After blocking in PBS containing 0.1% Tween 20 (PBST)
and 5% dried milk overnight at 4 °C, the membranes were incubated
with the primary antibody in PBST, 5% dried milk at room
temperature and then washed in PBST at least three times before
incubation with horseradish peroxidase-conjugated secondary antibody in
PBST, 2% dried milk at room temperature for 1 h. After extensive
washing, filters were soaked in improved bioluminescent reagent
(PerkinElmer Life Sciences) and exposed to film. Primary
antibodies were as follows: huACA serum (dilution 1:10,000), mAb 11060 (dilution 1:10,000), and mAb 12CA5 (Roche Molecular
Biochemicals) against HA-1 epitope (dilution 1 µg/ml).
Cotransfection Experiments--
Hep2 cells were seeded at 1 × 105 cells per well in 24-well Linbro multiwell
plates. The following day, plasmid pcDL CA-HA (40 ng) and
plasmid pCIneo, pCI110, or pCIFXE (350 ng) were cotransfected according to the manufacturer's recommendations (LipofectAMINE PLUS,
Life Technologies, Inc.). Twenty-four h later, cells were harvested for
Western blotting. Alternatively, the medium was changed and replaced by
fresh complete medium containing the proteasome inhibitor MG132 at a
concentration of 5 µM. Cells were then harvested 0, 2, 4, and 8 h post-addition of MG132 for Western blotting.
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RESULTS |
A 17-kDa Centromere Protein Previously Reported as Being CENP-A Is
Degraded as a Result of Infection with a Virus Expressing Functional
ICP0, Provided That the Proteasome Pathway Is Active--
To analyze
the putative degradation of centromere proteins other than CENP-C
during infection, we performed Western blotting of HSV-1-infected cell
extracts using a huACA serum that can recognize centromere proteins
CENP-A, -B, and -C having molecular masses of 17, 80, and 140 kDa, respectively (19). However, it is not unusual that only one or two
of these proteins are predominantly detected by Western blotting, with
CENP-A seemingly being the most antigenic. A complex signal was
detected at the high molecular weight range because of the recognition
of multiple viral proteins by anti-herpesvirus protein antibodies often
present in human sera (data not shown). We thus restricted the number
of viral proteins synthesized in infected cells using the
temperature-sensitive virus tsK and its ICP0-deficient
derivative in1330, which express nonfunctional ICP4 at
38.5 °C and therefore synthesize only the viral IE proteins (3).
Coupled with the use of nuclear extracts, this approach limited the
number of viral antigens detected by the huACA serum. HFL cells were
infected at a multiplicity of infection of 20 plaque-forming units per
cell and harvested 1.5, 4, and 6 h post-infection. Analysis by
Western blotting demonstrated that a 17-kDa (hereafter referred to
as 17K/CENP-A) protein was noticeably diminished by 4 h
post-infection in cells infected by tsK (see Fig.
1B). The detection of this
protein by the huACA serum and its electrophoretic mobility were
consistent with it being CENP-A. 17K/CENP-A was not affected in cells
infected by in1330, suggesting that ICP0 was implicated in
the effect. The expression of ICP0 in cells infected by tsK
but not in1330 was confirmed by stripping and reprobing the
membrane with mAb 11060 anti-ICP0 (data not shown). Parallel analysis
demonstrated that, as expected, the ICP0 target proteins CENP-C and
PML (32, 41) were also degraded in tsK- but not
in1330-infected cells (data not shown). A 15-kDa protein
detected beneath 17K/CENP-A by huACA was also detected by rabbit 554 and rabbit-L sera against CENP-C and CENP-B, respectively (21, 24),
making it a protein nonspecifically recognized by at least three
different sera directed against centromeric proteins (data not
shown).

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Fig. 1.
ICP0-induced degradation of the 17K/CENP-A
centromeric protein in HSV-1-infected cells. A, HFL
cells were infected with 20 plaque-forming units/cell of wild type
(17+) or ICP0-deficient (dl1403 and FXE) viruses.
The membrane was stripped and reprobed with mAb 11060 against
ICP0 ( -ICP0) to show ICP0 synthesis. M
represents mock infected cells. B, HFL cells were infected
with 20 plaque-forming units/cell at the restrictive temperature of
38.5 °C with viruses tsK (ICP4ts) or
in1330 (ICP0 , ICP4ts) in the presence
(+) or absence (-) of the proteasome inhibitor
MG132. Proteins from cell nuclear extracts were harvested 1.5, 4, and 6 (B) or 8 (A) h post-infection and analyzed by
Western blotting using huACA serum. hpi, hours
post-infection. MW corresponds to molecular mass markers
220, 97.4, 66, 46, 30, 21.5, and 14.3 kDa indicated by
dashes down the left-hand side of the
panels.
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Because we did not expect to detect too many viral antigens at the low
molecular weight range that would interfere with the detection of
17K/CENP-A, we then performed Western blotting on HFL cells infected by
HSV-1 viruses expressing the wild type (17+) or the RING finger mutant
(FXE) version of ICP0 and the ICP0-null mutant dl1403.
Although the effect was usually less efficient than that with
tsK, wild type HSV-1 still induced the degradation of
17K/CENP-A, whereas dl1403 and FXE did not (Fig.
1A). The efficiency of 17K/CENP-A degradation in
tsK-infected cells correlates with the large amount of ICP0
synthesized in those cells compared with 17+-infected cells (about 10 times more) at the same time post-infection and at the same
multiplicity of infection (data not shown). Given the better efficiency
of degradation obtained using the tsK virus, we decided to
perform subsequent Western blots using this virus for cell infections.
Recently several cellular proteins have been reported to undergo
degradation in an ICP0- and proteasome-dependent manner
during HSV-1 infection. We thus checked whether 17K/CENP-A was degraded via the same pathway. We performed infections of HFL cells in the
presence or absence of proteasome inhibitor MG132 under conditions that
do not interfere with viral gene expression (41). Fig. 1B
shows that, as expected, the 17K/CENP-A degradation was sensitive to
the proteasome inhibitor MG132. These results show that a 17-kDa protein recognized by huACA antibodies, which is most likely CENP-A, is
degraded in infected cells with an efficiency correlating with the
amount of ICP0 synthesized, and this effect is dependent on the
presence of ICP0 and its RING finger domain and on an active proteasome pathway.
CENP-A Is Lost from Centromeres in Wild Type but Not FXE- or
dl1403-infected Cells--
To visualize the effects of infection on
the nuclear distribution of CENP-A, immunofluorescence was performed on
Hep2 cells infected for 2 or 8 h with viruses 17+,
dl1403, or FXE. CENP-A, centromeres, and infected cells were
detected using a monospecific rabbit serum, huACA, and monoclonal
antibodies against ICP0 or ICP4, respectively. Two h post-infection,
CENP-A was still present at centromeres of infected cells (data not
shown). Eight h post-infection, CENP-A was no longer detected at
centromeres of cells infected by wild type virus (Fig.
2A, panels
A-D), whereas those infected by dl1403 (Fig.
2A, panels E-H) or FXE (Fig.
2A, panels I-L) still retained CENP-A
in centromeres. Of note is the decrease in centromere labeling by the
huACA serum in wild type virus-infected cells, which is probably
because CENP-A is one of the major antigens recognized by this serum
(19). The addition of MG132 at the start of the infection abrogated the
disappearance of CENP-A in 17+-infected cells, which correlates with
the absence of degradation of 17K/CENP-A observed by Western blotting
under the same experimental conditions (data not shown).

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Fig. 2.
The loss of the protein CENP-A from
centromeres of infected cells is dependent on the expression of
functional ICP0. Subconfluent Hep2 cells were infected by 17+,
dl1403, or FXE viruses. Eight h post-infection, cells were
fixed and treated for immunofluorescence using mAbs 11060 against ICP0
(red in A, green in B) and
58S against ICP4 (red), monospecific anti-CENP-A rabbit
serum (green in A, red in
B), and huACA serum (blue). A,
panels A-D, cells infected with the wild type 17+ virus.
The upper right cell is not infected and shows the
colocalization of CENP-A with centromeres. Panels E-H,
cells infected with the ICP0-null mutant dl1403 detected by
the expression of ICP4 (the upper left cell is not
infected). Panels I-L, cells infected with the ICP0 RING
finger mutant FXE. The presence of CENP-A in centromeres is shown by
the light blue dots in the merge figures resulting from the
colocalization of CENP-A and centromere proteins. B,
panel A, a wild type virus-infected cell in
pseudoprometaphase showing the loss of CENP-A from centromeres in cells
with this phenotype. Panel B, a rare wild type
virus-infected cell in metaphase, with CENP-A still present at
centromeres. Panel C, a dl1403 infected cell in
the common anaphasetelophase phenotype, with CENP-A still present
at centromeres. The presence of CENP-A at centromeres is shown by the
pink dots resulting from the colocalization of CENP-A and
centromere proteins. The bars represent 5 µm.
Centr, centromeres.
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Although wild type-infected cells attempting mitosis are highly likely
to be blocked at pseudoprometaphase, a very small percentage can still
be found in metaphase or anaphase (see Ref. 33). Because CENP-A is
undoubtedly implicated in some aspects of mitosis (30), we checked the
distribution of CENP-A in infected cells at different stages of
mitosis. All wild type virus-infected mitotic cells with a
pseudoprometaphase phenotype (determined by the distribution of
unstained chromosomes in the background staining of infected mitotic
cells; for more details see Ref. 32) showed no CENP-A present at
centromeres (Fig. 2B, panel A).
Interestingly, the very small number of infected cells found in
metaphase still possessed CENP-A at the centromeres (Fig.
2B, panel B). Virus FXE- and
dl1403-infected mitotic cells could be found at the same
frequency at the different stages of the cell cycle, with no alteration
in the CENP-A distribution (see the telophase
dl1403-infected cell as an example (Fig. 2B, panel C)). These data show that the expression of
ICP0 in infected cells results in the disappearance of CENP-A from
centromeres, an effect that is dependent on the RING finger domain of
ICP0. Moreover, it is likely that CENP-A is required at centromeres for
a mitotic cell to reach metaphase, because in its absence the phenotype
most commonly observed in wild type HSV-1-infected mitotic cells is pseudoprometaphase.
ICP0 Specifically Induces the Loss of CENP-A from
Centromeres--
ICP0 alone was able to induce the loss of CENP-A from
centromeres, because Hep2 cells transfected with plasmids expressing wild type ICP0 or its RING finger mutant FXE gave results similar to
those observed in infected cells (Fig.
3). These results confirm the essential
role played by ICP0 and rule out any requirement for other viral
proteins. As expected, the RING finger mutant protein FXE (which does
not localize in centromeres) had no effect on the distribution of
CENP-A.

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Fig. 3.
ICP0 alone is able to induce the loss
of the protein CENP-A from centromeres. Subconfluent Hep2 cells
were transfected with plasmids pCIneo (A), pCI110
(B), or pCIFXE (C) expressing no ICP0, wild type
ICP0, or the RING finger mutant protein FXE, respectively. Twenty four
h post-transfection, cells were fixed and treated for
immunofluorescence using mAb 11060 against ICP0 (green),
monospecific anti-CENP-A rabbit serum (red), and huACA serum
(blue). The presence of CENP-A at centromeres is shown by
the pink dots resulting from the colocalization of CENP-A
and centromere proteins. The bars represent 5 µm.
Centr, centromeres.
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ICP0 Specifically Induces the Degradation of CENP-A--
The
results obtained so far have shown that HSV-1 is able to induce the
degradation of the 17K/CENP-A protein recognized by huACA serum in an
ICP0- and proteasome-dependent manner. These results
correlate with the loss of CENP-A from centromeres both in infected and
transfected cells. The rabbit anti-CENP-A serum used in the
immunofluorescence studies gave poor results in our Western blotting
experiments; so to demonstrate unequivocally that CENP-A is degraded
during HSV-1 infection and to show that ICP0 alone was responsible for
that effect, we used either HeLa tTA-CAHA cells, which constitutively
express an influenza hemagglutinin (HA1) epitope-tagged derivative of
CENP-A (CENP-A-HA), or cells transfected with plasmid pcDLCA-HA, which
expresses the same protein (26, 27). Prior to infection, HeLa tTA-CAHA
cells were treated as described under "Experimental Procedures" to
induce CENP-A-HA expression. Fig.
4A shows a significant
reduction of the amount of CENP-A-HA protein in cells infected by the
tsK virus expressing wild type ICP0. This effect was
abrogated in the presence of MG132 (data not shown) and did not occur
in in1330-infected cells. The relatively less efficient
degradation of CENP-A-HA compared with 17K/CENP-A could be explained by
the somehow exogenous nature of CENP-A-HA, whose synthesis from a
constitutive promoter could increase its amount compared with
endogenous 17K/CENP-A. Immunofluorescence experiments confirmed the
loss of CENP-A-HA from centromeres in tsK- and wild
type-infected cells but not in in1330-, dl1403-, or FXE-infected cells, results identical to those observed for endogenous CENP-A (data not shown, but see Fig. 2A). These
results perfectly match those observed for the degradation of
17K/CENP-A and demonstrate that CENP-A is effectively degraded during
infection in an ICP0- and proteasome-dependent manner.

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Fig. 4.
ICP0 induces the degradation of CENP-A via
the proteasome pathway. A, HeLa tTA-CAHA cells were
infected at the restrictive temperature of 38.5 °C with 20 plaque-forming units/cell of viruses tsK or
in1330. Total cell proteins were harvested 6 h
post-infection and analyzed by Western blotting using mAb 12CA5
( -HA) against the HA-1 epitope of the CENP-A-HA-tagged
protein. B and C, Hep2 cells were cotransfected
with 40 ng of the plasmid pcDLCA-HA and 350 ng of pCIneo,
pCI110, or pCIFXE plasmids. B, the cells were harvested
24 h later for Western blotting using mAb 12CA5, and the membrane
was sequentially reprobed to detect ICP0 and actin. NT
corresponds to nontransfected Hep2 cells. C, cells were
harvested 24 h post-transfection (lanes 0) or the
medium was replaced with that containing MG132 (5 µM) for
2-8 h (lanes 2, 4, and 8) or without
MG132 for 8 h (lanes 0/8) before harvesting the cells.
Total cell proteins were analyzed by Western blotting using mAb 12CA5,
and then the same membrane was stripped and reprobed sequentially for
ICP0 and actin. MW corresponds to molecular mass
markers 220, 97.4, 66, 46, 30, 21.5, and 14.3 kDa indicated by
dashes down the left-hand side of the
panels.
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To demonstrate that ICP0 could induce the degradation of CENP-A by
itself, Hep2 cells were cotransfected with plasmids pcDLCA-HA and
pCIneo, pCI110, or pCIFXE and harvested for Western blotting 24 h
later. Fig. 4B shows the decrease in the intensity of the CENP-A-HA band in cells cotransfected with pCI110 but not pCIFXE, which
express wild type ICP0 and the RING finger mutant protein FXE,
respectively. To confirm that the CENP-A-HA ICP0-induced degradation
occurred via the active proteasome pathway, MG132 was added to
the samples 24 h post-transfection for a period of time between 0 and 8 h. The idea was that inhibition of the proteasome activity
with MG132 should stabilize CENP-A-HA, resulting in a re-increase of
the CENP-A-HA signal even in the presence of ICP0. Fig. 4C
shows that the amount of CENP-A-HA in cells coexpressing ICP0 increases
as early as 2 h post-addition of MG132 to reach a maximum after
8 h. The amount of CENP-A-HA never returned to the level observed
in cells cotransfected with pCIneo, but this might be expected
considering the time period of the experiment. MG132 had no effect on
the expression of CENP-A in cells cotransfected with pCIneo. These
results confirm the essential role of ICP0 and its RING finger domain
in the induced degradation of CENP-A and correlate with the
immunofluorescence data showing that ICP0 alone is able to induce the
loss of CENP-A from centromeres.
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DISCUSSION |
We recently observed that ICP0 blocks the progression of cells
through mitosis, and we suggested that this effect correlates with the
ICP0-induced degradation of at least one centromere protein, CENP-C
(33). However, based on previous data (24), we also suggested that the
observed pseudoprometaphase phenotype indicated that effects on other
centromere functions were also occurring. The present study
demonstrates that the 17-kDa centromere protein CENP-A is also degraded
in an ICP0- and proteasome-dependent manner both in
infected and transfected cells, and this correlates with the loss of
CENP-A from centromeres. These new data confirm the previous
interpretations about the effects of ICP0 on the stability of several
cellular proteins and give new insights into the possible cellular
mechanisms acting on viral chromatin structure during virus infection.
ICP0: From Regulator of Gene Transcription to Regulator of Cellular
Protein Stability--
ICP0 was initially characterized as a broad
transactivator of viral and cellular genes and was found to regulate
the balance between latent and lytic infection by promoting the onset
of lytic gene expression. In its absence, low multiplicity infection of some cultured cell lines leads to viral genomes preferentially maintained in a quiescent or latent state. Early in the infection ICP0
localizes to subnuclear domains called ND10 and then induces their
destruction (39). Interestingly, parental HSV-1 genomes (as well as
those of several other DNA viruses) preferentially migrate to the
periphery of ND10 at the earliest stages of infection (40), which
suggests a possible link between the ICP0-induced loss of ND10 and
activation of transcription from the viral genome. The destruction of
ND10 following infection occurs because PML and Sp100 (two of
the major ND10 components), and their SUMO-1 modified isoforms, are
degraded in response to ICP0 activity (41-44). ICP0 thus controls the
stability of at least two major ND10 proteins via the ubiquitin
degradation pathway, an idea consistent with the induction by
ICP0 of colocalizing conjugated ubiquitin (45).
More recently, ICP0 has been shown to disrupt centromeres and induce
the degradation of the essential centromere protein CENP-C in a similar
manner (32). The presence of ICP0 in both ND10 and centromeres could be
related to the presence of certain proteins in both structures. These
include the heterochromatin-associated protein HP1, which binds to
Sp100 (46, 47), and hDaxx, which interacts with both CENP-C and
SUMO-1-modified PML (48, 49); both these proteins are
dynamically associated with both ND10 and centromeres (50). Because
Sp100, HP1, and hDaxx have all been implicated in the repression of
gene expression (47, 51, 52), the association of parental viral genomes
in the vicinity of structures rich in these proteins assumes
considerable significance, especially because they or their binding
partners are degraded in response to ICP0 expression. The idea that
these structures and proteins may in some way be involved in repression
of viral gene expression and that ICP0 therefore relieves this
repression is very compelling.
New Implications from the ICP0-induced Degradation of
CENP-A--
Our previous study describing the ICP0-induced degradation
of CENP-C raised a number of questions concerning the mechanism and
implications of this effect. Because two of the first ICP0 target
proteins to be identified were both modified by SUMO-1, we previously
suggested that SUMO-1-modified proteins may in some way be targeted by
ICP0. Because of previous evidence linking proteins related to CENP-C
and SUMO-1 in yeast, we considered it possible that CENP-C may be
modified by SUMO-1 and thus be an ICP0 target. However, given that the
CENP-A calculated molecular mass is about 16 kDa (26) and that
a monomer of SUMO-1 has a gel mobility of about 22 kDa (53), it is
highly unlikely that the 17K/CENP-A band is modified by SUMO-1.
Therefore SUMO-1 modification is unnecessary for a protein to be
degraded in response to ICP0 expression, a conclusion consistent with
the lack of evidence that the catalytic subunit of DNA-PK, another ICP0
target protein, is modified by SUMO-1 (54).
We also suggested that the ICP0-induced loss of CENP-C would inevitably
have serious consequences for kinetochore structure and function during
mitosis. The additional degradation of CENP-A underlines this previous
conclusion and confirms the prediction that the stability of centromere
components other then CENP-C would also be affected by HSV-1 infection.
Our results are consistent with the suggestion of Tomkiel et
al. (24) that the pseudoprometaphase arrest phenotype could be due
to the inactivation of CENP-A. The observations of Fig. 2B
provide additional support for this hypothesis, because they show that
CENP-A is absent from centromeres of HSV-1-infected cells in
pseudoprometaphase, whereas in the very rare infected cells in
metaphase, CENP-A was still present.
The Significance of CENP-A Degradation--
CENP-A is a
particularly intriguing target for ICP0-induced degradation. It is
highly unlikely that CENP-A (or CENP-C) is an "innocent bystander"
that happens to be degraded because it is present in the wrong place at
the wrong time. Indeed, whereas CENP-A (or CENP-C; 32) is removed from
centromeres of HSV-1-infected cells, previous data showed that CENP-B
seems not to be affected (32), a result consistent with the detection
of centromere antigens by huACA serum. On the other hand, it has been
shown that whereas most of the viral DNA found in the nervous system of
acutely infected mice is not assembled into a regular nucleosomal
structure (55), during latency the viral genome is closely associated
with nucleosome structures (56). These studies suggested that this
association could be part of the mechanism by which viral genomes are
repressed during latency. It is an exciting prospect to determine
whether CENP-A could be a component of these latent viral
genome-associated nucleosomal structures. This is particularly
pertinent because, firstly, CENP-A can associate not only with
-satellite DNA independently of its sequence (22, 25, 26, 28), but
also with other chromosomal loci that are not normally used for
centromere assembly (for reviews, see Ref. 57), and secondly, the
association of CENP-A with heterochromatic DNA is not restricted to
human DNA (58). These recent observations suggest at least the
possibility that the chromatin structure of latent viral DNA could
include CENP-A-containing nucleosomes correlating with a
heterochromatic state that would silence viral gene expression.
This study shows for the first time that a nucleosome-associated
heterochromatin protein is targeted for degradation by a viral protein
known for its key role in the reactivation of the virus from latency.
In addition to the potential use of ICP0 for a better understanding of
the role of CENP-A in the cell, this study might be of considerable
significance with respect to several aspects of viral and/or cellular
gene expression, including association of DNA with alternative
nucleosome-like structures, repression of gene transcription,
heterochromatin assembly, mechanisms of cellular defense against viral
infection, and latency.