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Human Herpesvirus 7 Infection Induces Profound Cell Cycle Perturbations
Coupled to Disregulation of cdc2 and Cyclin B and Polyploidization
of CD4 1 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
Human herpesvirus 7 (HHV-7) infection of both primary CD41
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 micros-
copy 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 G4n. Taken together, these data indicate that giant
cells, which represent the hallmark of in vitro HHV-7 infection, arise from single CD41 T cells undergoing a process of
polyploidization.
r 1998 by The American Society of Hematology.
H
infection on CD41 T cells and, in particular, on the formation of
giant polyploid cells.
UMAN HERPESVIRUS-7 (HHV-7) is a recently isolated
CD41 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 CD41 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 CD41 T lymphocytes, such as CD41 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
Blood, Vol 92, No 5 (September 1), 1998: pp 1685-1696
MATERIALS AND METHODS
Cells and HHV-7 infection. Enriched populations of CD41 T
lymphocytes were derived from the peripheral blood of healthy blood
donors. Primary CD41 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 CD41 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
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.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9205-0029$3.00/0
1685
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1686
SECCHIERO ET AL
Table 1. HHV-7 Infection of Primary CD41 Lymphocytes
and SupT1 Cells: Viral Expression Analysis
HHV-7–Positive Cells (%)
Days p.i.
Primary CD41 T Cells
SupT1 Cells
2
6
12
,5
49 6 7.5
77 6 9
,5
43 6 6
83 6 8.5
The percentage of HHV-7–positive cells was determined at serial
time points p.i. by indirect immunofluorescence, analyzing at least
four different fields and a total of approximately 200 cells per each
sample. Data are expressed as the mean 6 SD of four separate
experiments.
preactivated primary CD41 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 3 105
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 3 105 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.
Fig 1. PHA-stimulated primary CD41 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 G21M(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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HHV-7–MEDIATED CELL CYCLE PERTURBATIONS
1687
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 3 105 primary CD41 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 CD41 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
21mitosis (G21M) peaks were further considered. The proportions of
cells in the G1, S, and G21M phases of the cell cycle were calculated as
previously described.30 In particular, the S-phase events were divided
equally between the G1 and G21M phases, based on peak channels of
fluorescence intensity. For simplicity, the G1 and G21M values have
been provided.
For analysis of cdc2 and cyclin B expression, 10 3 106 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 220°C. Just before staining,
ethanol was removed by centrifugation at 400g for 10 minutes and, after
washings in wash buffer (PBS 1 1% FCS), the cells were incubated in
Fig 1
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–btubulin 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.
(Cont’d).
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1688
SECCHIERO ET AL
Table 2. HHV-7 Infection of Primary CD41 T Lymphocytes:
Cell Cycle Analysis
Table 3. HHV-7 Infection of SupT1 Cells: Cell Cycle Analysis
% of Cells
Cell and Day
Uninfected
2
4
6
8
10
12
Uninfected 1 PFA
2
4
6
8
10
12
HHV-7–infected
2
4
6
8
10
12
HHV-7–infected 1 PFA
2
4
6
8
10
12
G1(2n)
% of Cells
G2M(4n)
98.8
98.6
98.5
97.8
97.7
96.6
1.2
1.4
1.5
2.2
2.3
3.4
98.5
98.4
98.6
97.9
97.8
96.7
1.5
1.6
1.4
2.1
2.2
3.3
98.8
98.3
96.6
94.4
88.2
73.6
1.2
1.7
3.4
5.6
11.8
26.4
98.7
98.3
97.5
97.8
97.8
97.1
1.3
1.7
2.5
2.2
2.2
2.9
Data represent the means of four separate experiments. SD were
comprised within 15% of the means.
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 3 106 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 (g-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-28-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
Cell and Day
Uninfected
2
4
6
8
10
12
Uninfected 1 PFA
2
4
6
8
10
12
HHV-7–infected
2
4
6
8
10
12
HHV-7–infected 1 PFA
2
4
6
8
10
12
G1(2n)
G2M(4n)
78.6
77.5
76.2
78.6
77.4
78.2
21.4
22.5
23.8
21.4
22.6
21.8
75.3
74.5
76.0
76.9
74.2
75.9
24.7
25.5
24.0
23.1
25.8
24.1
78.0
76.6
73.1
69.1
64.6
62.0
22.0
23.4
26.9
30.9
35.4
38.0
73.1
75.6
73.8
75.3
75.5
75.7
26.9
24.4
26.2
24.7
24.5
24.3
Data represent the means of four separate experiments. SD were
comprised within 15% of the means.
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 3 104)
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
Table 4. BrdU Labeling Index in Uninfected and HHV-7–Infected
SupT1 Cell Cultures
Cell Type
BrdU Positivity (%)
Uninfected SupT1 cultures
HHV-7–infected SupT1 cultures
Cells negative for HHV-7 Ag
Cells positive for HHV-7 Ag
36 6 4.5
34 6 3.8
67 6 4.1
Data are expressed as the means 6 SD of four separate experments
performed at day 12 p.i. In each culture, approximately 300 to 400 cells
were scored. HHV-7–positive cells have been identified by double
staining with an anti–HHV-7 human serum.
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HHV-7–MEDIATED CELL CYCLE PERTURBATIONS
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
1689
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 6 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.
RESULTS
HHV-7 infection causes abnormalities of the cell cycle
progression in both primary CD41 T lymphocytes and SupT1
lymphoblastoid CD41 T-cell line. Both primary CD41 T
lymphocytes and SupT1 CD41 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
21mitosis (G21M).33 It is noteworthy that the calculated
proportion of primary CD41 T cells seen by flow cytometry in
the G1 peak also includes quiescent cells in G0.
;
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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1690
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.
Cell cycle analysis showed that, in mock-infected primary
CD41 T lymphocytes, the fraction of cells in G21M (4n) never
exceeded 3.4% (Table 2). On the other hand, the HHV-7–
infected primary CD41 T lymphocyte cultures had a DNA
profile that was skewed toward G21M (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 G21M (4n) with respect to uninfected cultures (Fig 1A
and Table 2).
With respect to primary CD41 T cells, a greater proportion of
SupT1 cells was found in the G21M (4n) phase of the cell cycle
during the culture time (Fig 1B and Table 3). However, whereas
the G1(2n)/G21M(4n) ratio remained rather constant in mockinfected SupT1 cells for the duration of the experiment,
HHV-7–infected cultures showed a progressive accumulation of
cells in G21M(4n) relative to those in G1(2n) (Fig 1B). The
differences between HHV-7–infected and uninfected cells be-
SECCHIERO ET AL
came 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 G21M (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).
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).
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 ,
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HHV-7–MEDIATED CELL CYCLE PERTURBATIONS
1691
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 G4n 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.
.05) reduced in HHV-7–infected cultures with respect to
uninfected cultures (16,926 6 1,870 cpm v 27,297 6 3,080
cpm, respectively; data were calculated as the means 6 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 virusinfected 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).
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 possibil-
ity, 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).
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
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1692
SECCHIERO ET AL
A
B
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 3 400. Representative fields
are shown.
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.
DISCUSSION
In this report, we have demonstrated that HHV-7 infection of
both primary CD41 T lymphocytes and SupT1 CD41 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
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HHV-7–MEDIATED CELL CYCLE PERTURBATIONS
1693
Fig 6. Morphological characterization of enlarged (diameter, G20 mm) 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 mm) 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 mm 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) 3 400; in (C) 3 1,000.
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 CD41 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 CD41 T
lymphocytes and SupT1 cells.18
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1694
SECCHIERO ET AL
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.
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 CD41 T cells suggests a novel mechanism for
potential HHV-7–induced immune disfunction.
ACKNOWLEDGMENT
The authors are very grateful to Prof G. Campadelli-Fiume for the
HHV-7 MoAb 5E1.
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1998 92: 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
Paola Secchiero, Lucia Bertolaso, Luca Casareto, Davide Gibellini, Marco Vitale, Kristi Bemis, Arrigo
Aleotti, Silvano Capitani, Genoveffa Franchini, Robert C. Gallo and Giorgio Zauli
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