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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1685-1696
Human Herpesvirus 7 Infection Induces Profound Cell Cycle
Perturbations Coupled to Disregulation of cdc2 and Cyclin B and
Polyploidization of CD4+ T Cells
By
Paola Secchiero,
Lucia Bertolaso,
Luca Casareto,
Davide Gibellini,
Marco Vitale,
Kristi Bemis,
Arrigo Aleotti,
Silvano Capitani,
Genoveffa Franchini,
Robert C. Gallo, and
Giorgio Zauli
From the Institute of Human Virology, University of Maryland at
Baltimore, Baltimore, MD; the Institute of Human Anatomy, University of
Ferrara, Ferrara, Italy; the Basic Research Laboratory, National Cancer
Institute, National Institutes of Health, Bethesda, MD; the Institute
of Microbiology, University of Bologna, Bologna, Italy; and the
Department of Biomedical Sciences and Biotechnology, Human Anatomy
Section, Brescia, Italy.
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ABSTRACT |
Human herpesvirus 7 (HHV-7) infection of both primary
CD4+ T lymphocytes and SupT1 lymphoblastoid T-cell line
induced a progressive accumulation of cells exibiting a gap 2/mitosis
(G2/M) and polyploid content coupled to an increased cell
size. The expression of both cyclin-dependent kinase cdc2 and cyclin B
was increased in HHV-7-infected cells with respect to the uninfected
ones. Moreover, the simultaneous flow cytometric analysis of cyclin B
and DNA content showed that cyclin B expression was not only increased
but also unscheduled with respect to its usual cell cycle pattern.
However, the levels of kinase activity associated to cdc2 were
decreased in HHV-7-infected cells with respect to
uninfected cultures. To elucidate the origin of the enlarged
HHV-7-infected cells, extensive electron and confocal microscopy
analyses were performed. Membrane fusion events associated to
cytoplasmic bridges, which characterize the formation of syncytia, were
never observed. On the other hand, analysis of serial sections of the
same cells strongly suggested that enlarged HHV-7-infected cells
contained a single polylobated nucleus. This was confirmed by flow
cytometry analysis performed on nuclei isolated from
HHV-7-infected cells, which showed multiple peaks with a DNA content
>4n. Taken together, these data indicate that giant cells, which
represent the hallmark of in vitro HHV-7 infection, arise from single
CD4+ T cells undergoing a process of polyploidization.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HUMAN HERPESVIRUS-7 (HHV-7) is a recently
isolated CD4+ T-lymphotropic herpesvirus1 whose
genetic content and organization are closely related to HHV-6 and
cytomegalovirus.2-4 HHV-7 was first isolated from the
peripheral blood lymphocytes of healthy individuals1,5,6
and it turned out to be a prevalent virus toward which the great
majority (>90%) of the population is seropositive by
adulthood.7-9 At present, the only clinical manifestation clearly associated to primary HHV-7 infection is exanthem
subitum.10-13 However, due to its high seroprevalence in
the general population,7-9 HHV-7 might represent a
potential opportunistic agent in immunocompromised hosts.14,15 Once reactivated, acute HHV-7 infection might
worsen the state of immunodeficiency due to its selective tropism for CD4+ T lymphocytes.1,16 In fact, in vitro
studies allowed to establish that the CD4 antigen acts as a critical
component of the receptor for HHV-7.16 Moreover, HHV-7
induces several cytopathic effects on cultured CD4+ T
lymphocytes, such as CD4+
downregulation,14,16,17 induction of cell death by necrosis as well as by apoptosis,18 and formation of giant
balloon-like cells,1,2,6,18 which represent the most
readily observed effect of acute HHV-7 infection.
So far, no studies have been performed to explore the impact of HHV-7
infection on cell cycle control/progression. Two classes of proteins
make up the protein-kinase complexes involved in the biochemical
control of the cell cycle. The serine/threonine cell division kinases
(cdk), also referred to as cyclin-dependent kinases, are the catalytic
subunits of these complexes,19 whereas the cyclins function
as the regulatory subunits. Cyclins are proteins that undergo dramatic
fluctuations in abundance as a function of cell cycle progression and
thus regulate the activation of the holoenzyme.20-22 There
are at least eight members of the cyclin gene family.23,24
Cyclin B is the best understood of the cyclins; it complexes with cdc2
to form M-phase promoting factor (MPF), the mitosis-initiating protein
kinase complex.21,25
This study was undertaken to evaluate the potential involvement of cell
cycle-associated proteins and protein kinase complexes in the
cytopathic effects induced by an acute HHV-7 infection on
CD4+ T cells and, in particular, on the formation of giant
polyploid cells.
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MATERIALS AND METHODS |
Cells and HHV-7 infection.
Enriched populations of CD4+ T lymphocytes were derived
from the peripheral blood of healthy blood donors. Primary
CD4+ T cells were purified by negative immunomagnetic
selection, using a mixture of monoclonal antibodies (MoAbs) to CD8,
CD14, CD19, CD20, and CD56 (all from Becton Dickinson, San José,
CA) and magnetic beads coated with goat antimouse IgG antiserum (Dynal, Great Neck, NY), as previously described.26 Primary cells
were cultured in RPMI (GIBCO BRL Life Technologies Inc, Gaithersburg, MD) containing 10% fetal calf serum (FCS; GIBCO BRL) and activated with 5 µg/mL of purified phytohemagglutinin (PHA; Sigma Chemicals, St
Louis, MO) plus 20 U/mL of human recombinant interleukin-2 (IL-2;
Boeringher Mannheim, Postfack, Germany). After 3 days of culture, cells
were washed twice and seeded again in complete medium plus 5 U/mL of
IL-2 alone, which was readded every 4 days.
SupT1 lymphoblastoid CD4+ T cells (AIDS Research and
Reference Reagent Program, National Institute of Health, Bethesda, MD) were routinely cultured in RPMI 1640 containing 10% FCS.
The HHV-7 isolate AL used in this study and the preparation procedures
of viral stock have been previously described.6 SupT1 or
preactivated primary CD4+ T cells were either mock-infected
or adsorbed with HHV-7 for 10 hours at 37°C at a multiplicity of
infection (MOI) of approximately 0.1. After adsorption, cells were
washed to remove viral inoculum and diluted to the concentration of 5 × 105 cells/mL with fresh medium in the absence or
presence of 100 µg/mL of phosphonoformic acid (PFA; Sigma). In other
experiments, HHV-7 infection was performed by coculture of productively
infected SupT1 cells with uninfected SupT1 cells at a ratio of 1:10,
respectively. The infection was allowed to proceed at 37°C. Viable
cells were scored at light microscopy by Trypan blue staining every
other day, when cell density was adjusted to 5 × 105
cells/mL.
The occurrence of a productive viral infection was monitored by
morphological analysis of formation of enlarged (diameter, >20 µm)
cells at light microscopy and by indirect immunofluorescence staining
by using either a human anti-HHV-7 antiserum (Advance Biotechnologies,
Columbia, MD) or a specific HHV-7 MoAb (5E1 MoAb; generously provided
by Prof G. Campadelli-Fiume, University of Bologna, Bologna,
Italy),27 as previously described.26,27
Preparation of isolated nuclei.
Nuclei were isolated from both uninfected and HHV-7-infected SupT1
cells, as previously described.28 Briefly, cells were centrifuged at 400g for 5 minutes; washed in phosphate-buffered saline (PBS); resuspended in a cell fractionation buffer containing 10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L NaCl, 2 mmol/L MgCl2,
and 1 mmol/L phenylmetylsulfonylfluoride (PMSF; all from Sigma) for 2 minutes at room temperature; and then cooled in an ice water bath at
0°C for 5 minutes. Nonidet P-40 (NP40) at 0.5% was then added, and
the suspension was passed through a syringe (22G needle, a single up
and down stroke). At this stage, all cells were disrupted, the
concentration of MgCl2 was adjusted at 5 mmol/L, and the
nuclear pellet was collected by centrifugation at 700g for 5 minutes. The nuclei were finally resuspended in a buffer containing
0.25 mol/L sucrose, 10 mmol/L Tris HCl, pH 7.4, 5 mmol/L
MgCl2, 10 mmol/L NaCl, and 0.5 mmol/L PMSF.
Flow cytometry.
For cell cycle analysis, samples containing either 5 × 105 primary CD4+ lymphocytes, SupT1 cells, or
nuclei isolated from SupT1 cells were harvested by centrifugion at
200g for 10 minutes at 4°C, fixed with cold 70% ethanol
for at least 1 hour at 4°C, and treated as previously
described.29 Briefly, samples were pelletted, treated with
0.5 µg RNAse (type I-A; Sigma), and resuspended in PBS containing 50 µg/mL propidium iodide (PI). Analysis of PI fluorescence
was performed on a FACStar Plus flow cytometer (Becton Dickinson) with
the FL2 detector in either a linear (cells) or a logarithmic (isolated
nuclei) mode using Lysis II software (Becton Dickinson). Ten thousand
to 30,000 events were collected for each sample. Data analysis was
performed with Lysis II software (Becton Dickinson). In most
experiments performed on primary CD4+ T lymphocytes or
SupT1 cells, samples were gated to exclude cells with a subdiploid
(<2n) or >4n DNA content and only the inferred gap 1 (G1), synthesis (S) and gap 2+mitosis (G2+M)
peaks were further considered. The proportions of cells in the
G1, S, and G2+M phases of the cell
cycle were calculated as previously described.30 In
particular, the S-phase events were divided equally between the
G1 and G2+M phases, based on peak channels of
fluorescence intensity. For simplicity, the G1 and
G2+M values have been provided.
For analysis of cdc2 and cyclin B expression, 10 × 106 cells were harvested by centrifugation at 200g
for 10 minutes at 4°C and fixed with cold 70% ethanol for at least
2 hours at  20°C. Just before staining, ethanol was removed
by centrifugation at 400g for 10 minutes and, after washings in
wash buffer (PBS + 1% FCS), the cells were incubated in cold 0.25%
Triton X-100 wash buffer for 5 minutes. After washings, staining was
performed by incubating aliquots of cell suspension with 10 µL of
anti-cyclin B (MoAb, clone GNS1; Santa Cruz Biotechnology, Santa Cruz,
CA) or anti-cdc2 MoAb (cdc2 p34; Santa Cruz) or isotype control
antibody (Pharmiger, San Diego, CA) for 30 minutes at room temperature. After two washings in PBS, 10 µL of goat antimouse IgG directly conjugated to fluorescein (GAM-FITC; Becton Dickinson) was added for 30 minutes at 4°C. For the simultaneous analysis of intracellular cyclin B and DNA content of cells, double staining was performed as
described.31
Western blot analysis.
SupT1 cells were lysed at 4°C in RIPA buffer (1% NP40, 1%
deoxycholate) containing 1 µg/mL aprotinin, 2 µg/mL leupeptin, 1 mmol/L PMSF, and 1 mmol/L sodium orthovanadate, and protein
concentrations were estimated by the Bio-Rad protein assay according to
the manufacturer's protocol (Bio-Rad, Hercules, CA).
Equivalent amounts of proteins per sample were subjected to
electrophoresis on a 12% sodium dodecyl sulfate
(SDS)-acrylamide gel. The gel was then electroblotted onto
a nitrocellulose membrane; equal loading of protein in each lane was
confirmed by brief staining of the blot with 0.1% Ponceau S followed
by destaining before reacting with the specific antibodies. Detection
of specific proteins was performed using the following antibodies:
anti-cyclin B MoAb (at 1:100 dilution; Santa Cruz), anti-cdc2 kinase
MoAb (at dilution 1:100; Santa Cruz), anti- -tubulin MoAb (at 1:200
dilution; Boeringher Mannheim), antiphosphotyrosine (P-Tyr) MoAb
(Upstate Biotechnology Inc, Lake Placid, NY). Immunoreactive bands were
visualized after incubation with a peroxidase-conjugated goat antimouse
IgG (at 1:5,000 dilution; Amersham Corp, Arlington, UK),
using the ECL Detection System (Amersham Corp) according to the
manufacturer's instructions.
Densitometric analysis of immunoreactive bands was performed with an
imaging densitometer (Model GS 670; Bio-Rad) using the Molecular Analyst software. The results were expressed in arbitrary units (a.u.).
Immunoprecipitations and histone H1 kinase assays.
Aliquots containing approximately 10 × 106 SupT1
cells were solubilized in RIPA buffer (1% NP40, 1% deoxycholate)
containing 1 µg/mL aprotinin, 2 mg/mL leupeptin, 1 mmol/L PMSF, and 1 mmol/L sodium orthovanadate for 30 minutes at 4°C. The suspension
was centrifuged at 14,000 rpm for 15 minutes and the proteic content was estimated by the Bio-Rad protein assay. A volume of supernatant containing 5 mg of proteins was incubated at 4°C with 50 µL of protein A-Sepharose together with 20 µL of anti-cdc2 IgG (Santa Cruz). The mixture was then centrifuged at 10,000 rpm for 15 minutes, washed at least three times with lysis buffer, and washed twice with
kinase buffer (40 mmol/L HEPES, 8 mmol/L MgCl2). Kinase
assays were performed with 18 µL of reaction mixture containing 40 mmol/L HEPES, 8 mmol/L MgCl2, 166 mmol/L ATP, 5 µCi of
( -32P)ATP (3,000 Ci/mmol; NEN, Boston, MA),
4 µg of histone H1 (Boehringer), and 10 µL of packed protein
A-Sepharose. After 20 minutes at 37°C, the reaction was stopped by
the addition of SDS sample buffer. The reaction mixture was loaded on
SDS-12% polyacrylamide gels, stained with Comassie brilliant blue,
dried, and autoradiographed. The spots corresponding to the substrate
were excised from the gel and radioactivity was counted in a liquid
scintillation counter.
In situ immunocytochemistry.
S-phase labeling was performed by evaluating
5-Bromo-2 -Deoxyuridine (BrdU) uptake, followed by
immunocytochemical analysis, as previously described.32
Briefly, uninfected and HHV-7-infected cultures were seeded at the
same density in fresh medium, followed by incubation with BrdU (final
concentration, 10 mmol/L; Sigma) at 37°C for 30 minutes. The medium
was then removed, and the cells were washed twice with PBS. Cells (8 × 104) were spun on slides with a Cytospin apparatus,
fixed for 10 minutes in 2% para-formaldehyde in PBS, and permeabilized
with a saponin buffer (0.1% saponin, 10% FCS in PBS) for 30 minutes at room temperature. DNA was denatured by a treatment with 4 mol/L HCl
for 10 minutes. After neutralization in 0.1 mol/L sodium tetraborate and washings in PBS, immunocytochemical detection of BrdU was performed
by using an anti-BrdU-FITC MoAb (Boehringer) for 1 hour at room
temperature. Identification of HHV-7-infected cells was performed by
additional staining with a human anti-HHV-7 antiserum (1:200 dilution;
Advance Biotechnologies) at room temperature for 15 minutes. Cells were
washed four times in PBS and incubated with Cy3-conjugated donkey
antihuman serum (1:2,000 dilution; Jackson ImmunoResearch, Bar Harbor,
ME). Ten to 20 fields per experiment (~300 to 400 cells
in total) were scored for labeled nuclei.
Immunocytochemical detection of cyclin B was performed by applying a
1:100 dilution of anti-cyclin B MoAb (Santa Cruz) for 1 hour at
37°C. Cells were washed in PBS and incubated for 1 hour at room
temperature with an FITC-conjugated sheep antimouse IgG serum (1:50
dilution; Boehringer).
Negative controls consisted of incubation with normal mouse serum
followed by identical second layer labelings as described above. Slides
were mounted with DABCO glycerol, observed, and photographed on a Zeiss Axiophot microscope (Zeiss, Oberchocken, Germany).
Morphological studies.
For the ultrastructural studies, SupT1 cells were fixed with 1%
glutaraldehyde in 0.1 mol/L PBS, postfixed with 1% osmium tetroxide,
and embedded in Epon according to routine techniques, as previously
described.18 Serial semithin sections were taken, stained
with 1% toluidine blue, and then observed and photographed at light
microscopy. Thin sections were mounted on nickel grids and examined by
transmission electron microscopy after staining with uranyl acetate and
lead citrate.
For confocal microscopy studies, samples were stained with propidium
iodide as described above and imaged by a Zeiss LSM410 inverted
confocal laser scanning microscope (Zeiss) coupled to a 25-mW Argon ion
laser as a light source. Image processing analysis on digitized optical
sections was performed on the graphics workstation Indy (Silicon
Graphics, Mountain View, CA).
Statistics.
The data are expressed as the mean ± standard deviation (SD) of the
mean of at least three separate experiments performed in duplicate.
Statistical analysis was performed using the two-tailed Student's
t-test.
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RESULTS |
HHV-7 infection causes abnormalities of the cell cycle progression in
both primary CD4+ T lymphocytes and SupT1
lymphoblastoid CD4+ T-cell line.
Both primary CD4+ T lymphocytes and SupT1 CD4+
lymphoblastoid T cells were infected with cell-free viral inoculum (MOI
0.1). Aliquots of cells, harvested from both HHV-7-infected and
uninfected cultures, were analyzed in a time-course experiment by
indirect immunofluorescence to monitor HHV-7 expression
(Table 1) and by flow cytometry, after
propidium iodide staining, to evaluate the DNA content
(Fig 1A and
B). Because the aim of this set of experiments was to analyze the
different cell cycle phases in HHV-7-infected versus uninfected cells,
samples were gated to exclude cells with a subdiploid (<2n) or
polyploid (>4n) DNA content. Moreover, the FL2 detector was used in a
linear mode, which gives some advantages in distinguishing among gap 1 (G1), synthesis (S), and gap 2+mitosis
(G2+M).33 It is noteworthy that the calculated proportion of primary CD4+ T cells seen by flow cytometry
in the G1 peak also includes quiescent cells in
G0.
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| Fig 1.
PHA-stimulated primary CD4+ T lymphocytes
(A) and SupT1 cells (B) were mock-infected or infected with HHV-7 in
the presence or absence of PFA, and cell cycle was analyzed by flow
cytometry, at 48-hour intervals, after staining of the DNA content with
propidium iodide. The X axis shows the DNA content in a linear scale,
determined based on fluorescence due to propidium iodide staining, and
the Y axis reflects the relative number of cells. The percentage of
cells in the G1(2n) and G2+M(4n) phases of
the cell cycle for each experimental point are reported in Tables 2 and
3. These results are representative of four separate experiments
performed in duplicate.
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Cell cycle analysis showed that, in mock-infected primary
CD4+ T lymphocytes, the fraction of cells in
G2+M (4n) never exceeded 3.4%
(Table 2). On the other hand, the
HHV-7-infected primary CD4+ T lymphocyte cultures had a
DNA profile that was skewed toward G2+M (4n) accumulation
(Table 2). Notably, at days 10 through 12 postinfection
(p.i.), a significantly (P < .01) higher
fraction of HHV-7-infected cells were in G2+M (4n) with
respect to uninfected cultures (Fig 1A and Table 2).
With respect to primary CD4+ T cells, a greater proportion
of SupT1 cells was found in the G2+M (4n) phase of the cell
cycle during the culture time (Fig 1B and
Table 3). However, whereas the
G1(2n)/G2+M(4n) ratio remained rather constant
in mock-infected SupT1 cells for the duration of the experiment,
HHV-7-infected cultures showed a progressive accumulation of cells in
G2+M(4n) relative to those in G1(2n) (Fig 1B).
The differences between HHV-7-infected and uninfected cells became
statistically significant (P < .01) at days 10 through 12 p.i. (Table 3), when the majority of the cells were infected (Table 1).
To elucidate the role of viral spread in the HHV-7-mediated induction
of G2+M (4n) accumulation, cell cycle was also analyzed in
cultures supplemented with PFA, a specific inhibitor of herpetic DNA
polymerase (Fig 1A and B and Tables 2 and 3). PFA-treated HHV-7-infected cultures exhibited a cell cycle profile very similar to
that of PFA-treated mock-infected cultures and significantly (P < .01) different from that of HHV-7-infected cultures at days 10 through 12 p.i.. These data demonstrate the need of HHV-7 replication for the progressive appearance of cell cycle abnormalities. To further
characterize this phenomenon, SupT1 cell line was used as a model
system for the next experiments.
In consideration of the promiscuity of infected and uninfected cells in
HHV-7-infected cultures, a dual-label staining was next used to more
specifically analyze the DNA synthesis (S) phase in combination with
the presence of viral antigens (Table 4). At day 12 p.i., among the cells staining positively for the expression of viral antigens, the majority stained positive also for BrdU incorporation, resulting in a significantly (P < .05) higher
percentage compared with the HHV-7-negative uninfected cells (Table
4). It is also noteworthy that, among the HHV-7-infected cells that incorporated BrdU, approximately 55% of these cells were small (diameter, 10 to 20 µm), whereas the remaining 45% showed an
increased size (diameter, >20 µm).
HHV-7 infection induces disregulation of the mitotic regulatory
proteins cdc2 and cyclin B.
One critical event that drives mammalian cells from G2 into
mitosis is the activation of the maturation-promoting
factor,19-21,25,34 whose components include cdc2 in
association with cyclin B. Therefore, the expression of both cdc2 and
cyclin B proteins was analyzed by Western blot
(Fig 2A) and flow cytometry
(Fig 2B). Because the cell size greatly varied among HHV-7-infected
and uninfected SupT1 cell cultures, Western blot experiments were
performed migrating equal amounts of proteins obtained from these
heterogeneous cell populations. At day 12 p.i., the levels of cdc2 and
cyclin B showed a clearly detectable increase in HHV-7-infected with
respect to uninfected SupT1 cultures (Fig 2A and B).
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| Fig 2.
Expression of the mitotic regulatory proteins,
cyclin B and cdc2, in uninfected and HHV-7-infected cells evaluated by
immunoblotting analysis of whole cell lysates (A) and by flow
cytometric analysis (B). (A) Equivalent amounts of protein lysates
obtained from HHV-7-infected and uninfected SupT1 cells were analyzed
by Western blot with an anti-cyclin B and with an anti-cdc2 MoAb. Equal
loading of protein in each lane was confirmed by staining with the
antibody to tubulin. The relative intensities of the bands were
densitometrically quantified and expressed in arbitrary units (a.u.).
(B) Intracellular expression of cyclin B and cdc2 proteins was analyzed
in uninfected and HHV-7-infected cells by indirect immunofluorescence
staining shown by flow cytometry. A representative analysis, performed
at 12 days p.i., is shown. A shift in the number of positively staining
fluorescent cells along the X axis (shaded histograms) shows the
increased level of cyclin B and cdc2 expression in HHV-7-infected
cultures. The control (open) histograms represent the background
intracellular fluorescence obtained from the staining of the same
cultures with a isotype control MoAb before FITC-conjugated secondary
Ab. Y axis, relative cell number. Data shown are from a single
experiment representative of four independent experiments with similar
results.
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Taking into account that tyrosine phosphorylation events are involved
in the regulation of several cell cycle kinases,19,20,35 we
also analyzed the P-Tyr content of intracellular proteins in HHV-7-infected and uninfected cultures
(Fig 3). The amount of tyrosine
phosphorylation of a 34-kD substrate appeared significantly increased
in the HHV-7-infected SupT1 cells, as compared with uninfected cells.
The size of this protein suggested that it might be cdc2p34, which was
confirmed by removing the anti-P-Tyr antibody and reprobing the same
membrane with a specific anti-cdc2 MoAb (Fig 3).
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| Fig 3.
Tyrosine phosphorylation of cdc2 in uninfected and
HHV-7-infected (12 days p.i.) SupT1 cells. Equivalent amounts of
protein lysates obtained from uninfected (lane 1) and HHV-7-infected
(lane 2) cells were analyzed by Western blot with an anti-P-Tyr MoAb.
Migration of a major tyrosine-phosphorylated substrate is indicated by
the asterix. Molecular size markers are indicated on the left (in
kilodaltons). The relative intensities of the bands were
densitometrically quantified and expressed in arbitrary units (a.u.).
These results are representative of three experiments performed.
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To further investigate the relationships between the HHV-7-associated
alteration of cell cycle progression and cdc2, immunoprecipitates were
prepared from equal amounts of proteins using anti-cdc2 MoAb, and
histone H1 kinase activity was measured in HHV-7-infected SupT1 cells
(days 10 through 12 p.i.) in comparison with uninfected cultures. The
kinase activity associated to cdc2 immunoprecipitates was significantly
(P < .05) reduced in HHV-7-infected cultures with respect to
uninfected cultures (16,926 ± 1,870 cpm v 27,297 ± 3,080 cpm, respectively; data were calculated as the means ± SD of
3 separate experiments).
Cyclin B expression is unscheduled during the cell cycle in HHV-7
cultures.
Cyclin B expression is typically induced in S phase, reaches maximal
levels in G2 phase, localizes to the nucleus in M phase, and is degraded before cell division.22
Figure 4 shows the relationship between
cyclin B expression and DNA content in both uninfected and
HHV-7-infected cells. As expected,31 only the
G2M (4n) fraction of cells showed high cyclin B levels in
the uninfected cultures. On the other hand, HHV-7-infected cells (day
12 p.i.) showed increase levels of cyclin B in all phases of cell
cycle, including G1S. These results demonstrate that cyclin
B expression is induced in virus-infected cells with G1S
DNA content and, of note, persists to high levels also in cells with a
DNA content of 4n or higher (Fig 4). Moreover, in situ
immunocytochemistry analysis of HHV-7-infected cultures showed that
aberrantly large (diameter, >20 mm) cells, invariably positive for
HHV-7 antigens (Fig 5A), also reacted strongly to the anti-cyclin B MoAb (Fig 5B).
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| Fig 4.
Bivariate cyclin B expression versus DNA content
distributions (scatter plots) in uninfected (top panels) and
HHV-7-infected (bottom panels) SupT1 cells. Cell cycle expression of
cyclin B was evaluated by simultaneous staining with propidium iodide
and anti-cyclin B MoAb followed by an FITC-conjugated MoAb. Staining of
uninfected and HHV-7-infected SupT1 cells with a isotype control MoAb
before FITC-conjugated secondary antibody is shown in the left panels.
Cyclin B-FITC fluorescence intensity is plotted on the X axis,
and fluorescence intensity of propidium iodide DNA staining is plotted
on the Y axis. The insets show the percentage of cells, calculated
after removal of the apoptotic cells, with a G1(2n), S,
G2M(4n), and >4n DNA content. The cell populations
showing high cyclin B expression, based on DNA content, are marked by
arrows. A representative analysis of three separate
experiments, performed at 12 days p.i., is shown.
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| Fig 5.
Immunofluorescence analysis of HHV-7 antigen (A) and
cyclin B (B) expression in uninfected and HHV-7-infected SupT1 cells.
The same cells are viewed by phase contrast microscopy (top panels) and
fluorescence microscopy (bottom panels). In (A), note that enlarged
cells, characteristic of HHV-7 infected cultures, are positively
stained for expression of viral antigens. In (B), note that, whereas
cyclin B expression is heterogenous among small cells, enlarged
HHV-7-infected cells always exhibit an intense staining for cyclin B. Original magnification × 400. Representative fields are shown.
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HHV-7 induces polyploidization of the infected cells.
Although it is usually assumed that enlarged polyploid cells appearing
in HHV-7-infected cultures are syncytia, resulting from the fusion of
individual HHV-7-infected cells with uninfected cells, this assumption
has never been proved. Because cyclin B degradation is necessary for
cell division to occur,22 the data given above suggest, as
an alternative possibility, that enlarged HHV-7-infected cells may
result from a process of polyploidization rather than from fusion
events. To address this issue, extensive light, electron, and confocal
microscopy examinations were performed. An example of the observations
performed is shown in Fig 6. At days 6 through 12 p.i., an heterogenous population of cells with a
progressively increasing size and polylobated nuclei was easily recognized (Fig 6A).
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| Fig 6.
Morphological characterization of enlarged (diameter,
>20 µm) cells from HHV-7-infected SupT1 cultures. (A) Light
micrographs showing cells with a progressively increasing size (a
through c) having prominent and lobulated nucleus. (B) Light
micrographs of serial semithin (1 µm) plastic sections of the same
cells. Note that the multiple nuclear sections evident in panel c
belong to the same irregular nucleus shown in panels a and b. (C)
Confocal microscope images of the nucleus of a giant HHV-7-infected
cell after staining of the DNA with propidium iodide. (a through c)
Confocal sections were taken 0.5 µm apart. Whereas two nuclear lobes
were clearly separated in panel a, they appeared connected by a nuclear
bridge in successive sections (panels b and c). In (B) and (C), the
arrow shows the same point in different sections. Original
magnification in (A) and (B) × 400; in (C) × 1,000.
|
|
Virus-induced cell fusion is characterized by three distinct stages:
(1) adhesion between two cells, (2) membrane fusion associated to
cytoplasmic bridges, and (3) enlargement of the bridges to yield what
would be recognized as a syncytium.36
Although adhesion between separate cells was easily noticed in
HHV-7-infected cultures (an example is shown in Fig 5A), extensive
transmission electron microscopy observations did not provide any
evidence of membrane fusion. Moreover, the analysis of serial 1-µm
semithin sections of the same cell strongly suggested that the various
nuclear lobes present in a given section likely belong to the same
nucleus (Fig 6B). This was also evident at confocal microscopy analysis
of serial 0.5-µm sections of HHV-7-infected cells examined after staining of the DNA with propidium iodide. Figure 6C shows a giant polyploid cell in which a nuclear bridge connecting two lobes of the
same nucleus was absent in a section (panel a), became clearly evident
in the section shown in panel b, and then almost disappeared in the
following sections of the same cell (panel c).
An additional approach to resolve this issue was to evaluate the DNA
content of nuclei isolated from HHV-7-infected (12 days p.i.) and
uninfected SupT1 cells. If giant HHV-7-infected cells resulted from
the fusion of previously separate cells (syncytia), one would expect to
observe a profile with only 2n/4n, whereas nuclei with a ploidy
>4n should appear only if these cells were true
polyploid nuclei. Taking into account that the ploidy follows a
log-normal distribution, 30,000 nuclei were analyzed using the FL2
detector in a logarithmic scale. As shown in
Fig 7, nuclei from HHV-7-infected cells
clearly showed distinct peaks of ploidy >4n, whereas uninfected SupT1
cells only showed a ploidy of 2 to 4n.
View larger version (16K):
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| Fig 7.
The DNA content was analyzed in nuclei isolated from
uninfected or HHV-7-infected (12 days p.i.) SupT1 cells by flow
cytometry after staining with propidium iodide. The X axis, in a
logarithmic scale, shows the DNA content determined based on
fluorescence due to propidium iodide staining, and the Y axis reflects
the relative number of cells. These results are representative of three
separate experiments.
|
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| |
DISCUSSION |
In this report, we have demonstrated that HHV-7 infection of
both primary CD4+ T lymphocytes and SupT1 CD4+
lymphoblastoid T-cell line causes cell cycle abnormalities resulting in
the accumulation of cells in the G2M phase of the cell
cycle in asynchronously dividing cell populations. Some of the
HHV-7-infected T cells eventually become polyploid, whereas those that
are unable to undergo polyploidization are likely to die by
apoptosis.18
One critical event that drives mammalian cells from G2 into
mitosis is the activation of the maturation-promoting factor, whose
components include cdc2 and cyclin B.21,34 cdc2 is highly regulated, requiring dephosphorylation on Tyr15 by CDC25
for its activation20,35; antagonizing this reaction is the
Wee 1 kinase, which phosphorylates cdc2 and thus inhibits the
transition into mitosis.37 The active cyclin B/cdc2 complex
promotes the dissolution of the nuclear membrane, chromatin
condensation, and spindle formation. At the end of mitosis, cyclin
B/cdc2 complexes disassemble and lose activity.
In HHV-7-infected CD4+ T cells, we noticed several
abnormalities of the mitotic promoting factor components, which may
account for the progressive accumulation of cells with a 4n or >4n
DNA content in these cultures. In fact, the total amount of cdc2, including its tyrosine phosphorylated inactive form, was upregulated, whereas the kinase activity associated to cdc2 was decreased in HHV-7-infected cultures with respect to the uninfected ones. Moreover, the total amount of cyclin B was increased in HHV-7 cells and its
expression was unscheduled, being observed also in cells with a
G1 or >4n DNA content.
Interestingly, some models of apoptosis have suggested a requirement
for disregulation of the maturation-promoting factor.38-42 It has been proposed that cyclin B/cdc2 might be involved in the nuclear changes (permeabilization to cytoplasmic nuclease, activation of nucleases, chromatin disruption, and condensation) that are observed
during apoptosis. Thus, although we cannot distinguish whether the
onset of G2 arrest is required for initiation of
HHV-7-induced apoptosis, the above described abnormalities of the
maturation-promoting factor may account for the progressive increase of
apoptosis associated with acute HHV-7 infection of both primary
CD4+ T lymphocytes and SupT1 cells.18
It has been recently demonstrated that aberrant expression of certain
kinases associated with cell cycle control can lead to
polyploidization,43,44 which is defined as the acquisition of elevated DNA content by a cell, regardless of the mechanism by which
such changes in ploidy occur. Although the mechanisms by which cells
achieve a polyploid DNA content are still largely unknown,
polyploidization physiologically occurs during the maturation of
megakaryocytes and also characterizes many virus-infected
cells.36,45,46 In particular, polyploid cells observed
during herpesvirus infections, also referred to as polykariocytes, are
thought to be formed by the fusion of previously separate cells and,
thus, represent syncytia.46 Electron microscopy studies
have shown that cell fusion starts with the formation of small
cytoplasmic bridges between adjacent cells at the points at which their
plasma membranes are in direct apposition. Subsequent enlargement of
these bridges leads to complete fusion of the cells.36
Analysis of HHV-7-infected SupT1 cells (performed at various days
p.i.) often showed apposition of the cell membranes of giant polyploid
and small cells, consistent with the notion that cell membrane
apposition represents the preferential route for cell-to-cell viral
transmission. However, we failed to detect fusion events in extensive
electron microscopy analysis. Although not conclusive, these findings
render unlikely the possibility that giant polyploid cells observed in
HHV-7-infected cultures are syncytia. The hypothesis that
HHV-7-infected polyploid cells are formed by repeated karyokinesis
without cytokinesis was also suggested by serial electron and confocal
microscopy analyses, indicating that HHV-7-infected giant cells
contain a single polylobated nucleus. A similar conclusion was obtained
analyzing isolated nuclei obtained from HHV-7-infected and uninfected
nuclei by flow cytometry. This implies that profound changes must occur
during M-phase to prevent nuclear division.47 In this
respect, the abnormalities of the maturation promoting factor observed
in our study are similar to those reported by the Datta et
al44 in the induction of megakaryocyte polyploidization.
A number of possibilities can be envisioned for the role that
G2M accumulation and polyploidization play in HHV-7
replication. (1) By prolonging the time that a host cell spends in the
activated state, the virus may maximize the output of progeny virus.
The opinion most widely held is that the same number of gene copies is
achieved through various polyploidization mechanisms as can be achieved
through mitosis and cytokinesis. The advantage of endopolyploidy to
HHV-7 might be that less time and energy is needed and that gene
transcription can continue uninterrupted by mitosis and cell
division.45 This interpretation agrees with the observation
that high levels of polyploidy are often characteristic of glands and
other cells which show intensive synthetic activity. (2) In addition to
reentering G0, an activated T cell can undergo apoptosis
and G2M accumulation may retard this event. (3) Cell-cycle arrest presents yet another advantage to the virus that is unrelated to
increased virion production: cytotoxic T cells kill target cells by
activation of cdc2, resulting in apoptosis. By preventing cdc2
activation, HHV-7 may render infected cells more resistant to cytotoxic
T-cell activity. A similar function has been proposed for the vpr gene
of human immunodeficiency virus-type 1 (HIV-1), which
induces G2 arrest.39,48-51 Because cultures of
HIV-1-infected cells arrested in G2 produced significantly
more virus than those in G1 phase, the cytostatic function
of vpr results in maximal virus production before the cell is
eliminated by the host immune response.52
Although the majority of the experiments presented here were performed
with the SupT1 lymphoid cell line, to facilitate detailed biochemical
analysis, the inhibition of cellular proliferation coupled to cell
cycle perturbations and polyploidization also in primary
CD4+ T cells suggests a novel mechanism for potential
HHV-7-induced immune disfunction.
| |
FOOTNOTES |
Submitted December 29, 1997;
accepted April 27, 1998.
Supported by the AIDS project of the Italian Ministry of Health.
Address reprint requests to Giorgio Zauli, MD, PhD, Institute of Human
Anatomy, Via Fossato di Mortara 66, 44100 Ferrara, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are very grateful to Prof G. Campadelli-Fiume for the HHV-7
MoAb 5E1.
| |
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Abstract
Introduction
Abstract