Journal of General Virology (2007), 88, 1446–1453
DOI 10.1099/vir.0.82589-0
Murine gammaherpesvirus 68 ORF20 induces
cell-cycle arrest in G2 by inhibiting the Cdc2–
cyclin B complex
R. Nascimento and R. M. E. Parkhouse
Correspondence
Instituto Gulbenkian de Ciência, Apartado 14, Oeiras, Portugal
R. M. E. Parkhouse
[email protected]
Received 22 September 2006
Accepted 17 January 2007
The objective of this work was to identify novel viral ‘evasion’ genes without homology in the
database through functional assays. Using this approach, the ‘unassigned’, conserved murine
gammaherpesvirus ORF20 gene was shown to localize in the nucleus and to induce cell-cycle
arrest followed by apoptosis in both mouse and human cells. Such growth-arrested cells did not
express phospho-histone H3, demonstrating that the virus protein caused arrest at the G2 stage of
the cell cycle. To characterize the mechanism further, Western blots of ORF20-recombinant
lentivirus-infected cells were developed with antibodies to cyclin B1, Cdc2 and phospho-Tyr-15Cdc2. This analysis revealed a relative increase in cyclin B and phospho-Tyr-15-Cdc2, from 24 to
72 h after infection with recombinant lentivirus. The demonstration that Cdc2 is in its inactive
phosphorylated form and the clearly increased levels of cyclin B indicated that the virus gene blocks
the progression of cells into mitosis by acting at the level of the Cdc2–cyclin B complex. To confirm
this result, the Cdc2–cyclin B complex in ORF20-expressing cells was shown to be essentially
without kinase activity. As the ORF20 gene is conserved in all herpesvirus, it may be presumed to
have evolved to fulfil an important, as yet undefined, biological role in host-cell modification.
INTRODUCTION
Manipulation of cell-cycle regulation is a commonly
employed strategy of viruses for achieving a favourable
cellular environment. Indeed, the study of such strategies
not only has contributed to our understanding of virus
replication, but may also have the potential to reveal novel
details of cellular protein interactions in the regulation of
the eukaryotic cell cycle. For example, there are early and
immediate-early herpesvirus proteins that delay cell-cycle
progression or cause arrest at the G1 stage (Cayrol &
Flemington, 1996; Izumiya et al., 2003; Jean et al., 2001; Lu
& Shenk, 1999). A number of viruses target the critical
regulators of G2/M progression, the cyclins and cyclindependent kinases or their regulators (Friborg et al., 1999;
Groschel & Bushman, 2005; He et al., 1995; Park et al.,
2000). The expression of cyclin B oscillates throughout the
cell cycle, beginning to accumulate during S phase and
reaching its maximum at the G2/M phase. Activation of the
Cdc2–cyclin B complex for entry into mitosis is regulated
by dephosphorylating Cdc2 on Thr-14 and Tyr-15. In this
work, we describe a ‘non-assigned’ gene of murine gammaherpesvirus 68 (MHV68), which has evolved into a hostmodifying gene causing inactivation of the Cdc2–cyclin B
complex.
MHV68 is a particularly useful laboratory model for the
study of gammaherpesviruses such as Epstein–Barr virus
and Kaposi’s sarcoma associated herpesviruses (Nash et al.,
1446
2001; Simas & Efstathiou, 1998). Originally isolated from
wild rodents, it is capable of infecting laboratory strains of
mice, thus providing a system to study gammaherpesvirus
infection and pathogenesis (Sunil-Chandra et al., 1992a).
After intranasal infection, MHV68 establishes a productive infection in lung epithelial cells followed by a latent
infection of spleen B cells, macrophages, dendritic cells and
lung epithelial cells (Flano et al., 2000; Sunil-Chandra et al.,
1992b; Weck et al., 1999). In addition, MHV68 replicates
efficiently in vitro, thereby permitting the study of early
events in virus infection and replication.
Although the genome of MHV68 contains genes unique
to this virus (Virgin et al., 1997), there are many others
that are conserved among the herpesvirus subfamilies. The
majority of these have identified functions, but there are a
significant number of shared, non-homologous herpesvirus
genes without a known function. One of these is ORF20
from MHV68, which has a predicted N-terminal domain
homologous to herpesvirus UL24 gene products (Alba
et al., 2001). The universal presence of UL24 in herpesviruses genomes from the three subfamilies, alpha-, betaand gammaherpesviruses, suggests a fundamental and
conserved role in the biology of herpesviruses.
Here, we have demonstrated that, as reported for its
human herpesvirus homologues, ORF20 localizes in the
nucleus. More importantly, expression of MHV68 ORF20
induced G2/M cell-cycle arrest followed by apoptosis of
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MHV68 ORF20 induces G2 cell-cycle arrest
both mouse and human cells. The mechanism of its action
is to maintain Cdc2 in its phosphorylated inactive state, so
that Cdc2–cyclin B complexes in cells expressing ORF20
exhibit almost no kinase activity.
METHODS
Cells. HeLa, 293T and murine NIH3T3 cells were cultured in 5 %
CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10 %
fetal calf serum at 37 uC.
Plasmids. MHV68 ORF20 was cloned into plasmid pCDNA3 fused
in frame with an N-terminal influenza haemagglutinin (HA) peptide
tag. The human cytomegalovirus (CMV)–vesicular stomatitis virus G
envelope protein, the packaging plasmid pCMVR8.9 and the vector
pHR-CMV-eGFP constructs have been described previously (Ikeda
et al., 2002).
For construction of a recombinant lentivirus vector (pHR-CMVORF20eGFP), ORF20 was excised from pCDNA3, together with
the HA tag, by BamHI/XhoI digestion and cloned into the vector
pHR-CMV-eGFP upstream of an internal ribosome entry site-driven
enhanced green fluorescent protein gene (eGFP).
Production and measurement of lentivirus. Lentivirus was
produced by transient transfection of 293T cells with a weight ratio
of 3 : 1 : 1 of vector to packaging to envelope plasmids using Fugene 6
(Roche) according to the manufacturer’s instructions. Control
lentivirus was produced by co-transfection of the packaging and
envelope plasmid together with the empty pHR-CMV-eGFP plasmid.
For production of recombinant lentivirus expressing ORF20, the
plasmid pHR-CMV-ORF20eGFP was used. Supernatants containing
the lentivirus were collected at 48, 72 and 96 h post-transfection and
clarified by low-speed centrifugation, and the lentivirus was collected
by ultracentrifugation (25 000 r.p.m. in an SW28 rotor in a Beckman
centrifuge). Virus pellets were resuspended in fresh culture medium
and frozen at 280 uC. Lentivirus titres were measured by infection of
293T cells with a dilution factor of 4. Analysis of lentivirus-infected
cells was done by detecting eGFP-positive cells by flow cytometry at
48 h post-infection (p.i.).
Immunofluorescence. NIH3T3 and HeLa cells were infected with
recombinant lentivirus containing the MHV68 ORF20 gene (pHRCMV-ORF20eGFP) or the control ‘empty’ lentivirus (pHR-CMVeGFP) at an m.o.i. of 10. At the time points indicated, cells seeded
onto coverslips were washed in PBS and fixed with 4 % paraformaldehyde in PBS (20 min, room temperature). Fixed cells were
permeabilized using PBS/0.1 % Triton-X 100 (20 min, room temperature). After washing with PBS, the fixed cells were blocked in
PBS/5 % BSA/0.05 % Tween 20 (30 min, room temperature) prior to
incubation with antibodies.
High-affinity anti-HA (Roche) and anti-phospho-histone H3
(Upstate Biotechnology) antibody were diluted in PBS/0.05 % Tween
20/1 % BSA and incubated with the specimen (1 h, room temperature). After three 5 min washes in PBS/0.05 % Tween 20, coverslips
were incubated with Alexa Fluor anti-rat secondary antibody (Jackson
ImmunoResearch) diluted in PBS/1 % BSA/0.05 % Tween 20 (1 h,
room temperature). After washing with PBS/0.05 % Tween 20,
coverslips were counterstained with the nuclear stain 4-6-diamino2-phenylindole (DAPI) at 20 ng ml21 in PBS (2 min, room temperature), mounted in Vectashield (Vector Laboratories) and observed
under a fluorescence microscope.
Cell-cycle analysis. NIH3T3 cells were infected with recombinant
pHR-CMV-ORF20eGFP or control pHR-CMV-eGFP lentivirus at an
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m.o.i. of 10. At the indicated time points, cells were trypsinized
(Gibco-BRL), washed once with PBS and fixed with 90 % ethanol
overnight at 4 uC. After fixation, cells were washed once with PBS
and resuspended in PBS/0.1 % Triton X-100 and incubated with
50U DNase-free RNase A (Calbiochem) (30 min, room temp). After
incubation, cells were stained with propidium iodide (Sigma)
(20 mg ml21 in PBS, 15 min, room temperature) before analysis.
Flow cytometry analysis was performed using a FACSCalibur (Becton
Dickinson) and cell-cycle analysis was performed using CELLQUEST
software.
TdT-mediated dUTP nick end labelling (TUNEL). To determine
the percentage of apoptosis, a FlowTACs kit (R&D Systems) was used
as specified by the manufacturer. Briefly, at 72 h p.i., cells were
trypsinized, washed with PBS and fixed in 3.7 % formaldehyde solution (10 min, room temperature). After fixation, cells were washed in
PBS, permeabilized in Cytonin and incubated in labelling reaction mix (1 h, 37 uC). The reaction was terminated by adding ‘stop’
buffer, cells were centrifuged and visualization of the apoptotic cells
was achieved by incubation with streptavidin–allophycocyanin (BD
Biosciences Pharmingen) (15 min, room temperature, in the dark).
Positive and negative controls were prepared using the same procedure and as provided by the supplier. Cells were washed in PBS and
analysed using a FACSCalibur (Becton Dickinson) and data analysis
was performed using CELLQUEST software.
Immunoblotting. Cells cultured in 60 mm diameter Petri dishes
were infected with different lentivirus constructs at an m.o.i. of 10
and harvested at various times p.i. The dishes were placed on ice and
washed in PBS before scraping off the cells.
Cells were lysed in 50 ml lysis buffer containing 0.1 M potassium
phosphate, 1 % Triton X-100, 2 mM EDTA, 1 mM dithiothreitol,
1 mM PMSF and a mixture of protease (Sigma) and phosphatase
(Roche) inhibitors (30 min, 0 uC). Protein concentrations were
determined by the Bradford assay (Bio-Rad). For SDS-PAGE, 50 mg
protein was loaded per lane and the separated proteins were transferred to PVDF membrane (Bio-Rad) and blocked with 5 % non-fat
milk (1 h, room temperature). Development of the blots was
performed with a monoclonal antibody for Cdc2 and polyclonal
antibodies for cyclin B1 (Santa Cruz Biotechnology), anti-phosphoTyr-15 Cdc2 (Cell Signalling Technology) and anti-a-tubulin (SigmaAldrich). Horseradish peroxidase-conjugated secondary antibodies
were purchased from Dako. Finally, the identified molecules were
revealed with SuperSignal chemiluminescent reagents (Perbio
Science) according to the manufacturer’s instructions.
In vitro kinase assays. A histone H1 kinase assay was performed to
measure the kinase activity of the Cdc2–cyclin B complex. NIH3T3
cells infected with lentivirus constructs at an m.o.i. of 10 were lysed at
48 h p.i. in a buffer from Cell Signalling Technology containing
1 mM b-glycerophosphate, 1 mM sodium orthovanadate and a mix
of protease inhibitors from Roche. Lysates containing 600 mg protein
were incubated for 1 h at 4 uC in 300 ml lysis buffer with 5 mg anticyclin B1 antibody (Santa Cruz Biotechnology) and immune
complexes were collected on 30 ml of protein G–Sepharose beads.
The beads were washed three times with lysis buffer and once with
kinase buffer containing 20 mM Tris/HCl (pH 7.5), 10 mM MgCl2
and 0.25 mM dithiothreitol, before being incubated for 10 min
at 30 uC in 30 ml kinase buffer containing 2.5 mCi [c-32P]ATP
(Amersham-Pharmacia), 10 mg histone H1 (Sigma) and 50 mM
ATP. The reaction mixture was centrifuged and SDS sample buffer
was immediately added to the supernatant prior to 12 % SDS-PAGE.
The gel was dried and exposed for autoradiography for 1 h at 280 uC.
The protein G beads were collected from the kinase reaction, washed
three times in kinase buffer and denatured in 30 ml SDS sample buffer
for separation by SDS-PAGE and subsequent transfer to PVDF
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1447
R. Nascimento and R. M. E. Parkhouse
0 h 24 h 48 h 72 h
37 kDa
HA_ORF20
25 kDa
a-Tubulin
50 kDa
Fig. 1. The expressed ORF20 gene product migrates with a
molecular mass of 28 kDa in SDS-PAGE. NIH3T3 cells were
infected with recombinant lentivirus. At the indicated time points,
cells were lysed for immunobloting and the ORF20 protein was
developed with anti-HA (top panel) antibody. Anti-a-tubulin antibody was used as a loading control (bottom panel). Molecular
mass markers are indicated.
membrane (Bio-Rad). The membrane was blocked with 5 % nonfat milk (1 h, room temperature), developed with a monoclonal
antibody for Cdc2 (Santa Cruz Biotechnology) and a secondary
horseradish peroxidase-conjugated antibody, and revealed with
SuperSignal chemiluminescent reagents (Perbio Science) according
to the manufacturer’s instructions.
RESULTS
Modification of cell-cycle progression in stable
cell lines expressing the MHV68 ORF20 gene
In order to study the function of ORF20, the virus gene was
expressed in mouse and human cells through a lentivirus
expression vector encoding eGFP. Transduced cells were
GFP
DAPI
Anti-HA
first tested for expression of ORF20 by Western blot analysis of the HA tag. As can be seen in Fig. 1, the expressed
protein migrated with a molecular mass of 28 kDa.
High rates of infection (~90 %) were demonstrated by GFP
expression (FACS analysis) and most of the infected cells
(80–90 %) expressed the ORF20 gene, as judged by counterstaining for the HA peptide tag introduced in the Nterminal region of the ORF20 protein. In these cells clearly
expressing the virus protein, however, immunofluorescence
revealed a nuclear localization of the virus protein in the
typically enlarged cells at 24–48 h p.i. (Fig. 2). Significantly,
mouse NIH3T3 cells infected with ORF20-recombinant
lentivirus failed to divide and died after 3–5 days, showing
the typical fragmented nuclear morphology of apoptosis.
Prior to this, the cells became enlarged and flat, suggesting
that ORF20 might interfere with cell-cycle progression.
Induction of cell-cycle arrest in human and murine
cell lines by MHV68 ORF20
To determine the impact of ORF20 on cell-cycle progression, murine NIH3T3 cells were infected with the lentivirus
construct containing the ORF20 gene. Cells were harvested at different time points for cell-cycle analysis by flow
cytometry after staining with propidium iodide. Cells
expressing MHV68 ORF20 showed a significantly increased
G2/M : G1 ratio from 24 to 48 h p.i. (Fig. 3, second row).
As molecules involved in the cell cycle are highly conserved
in mammalian cells, we repeated the same experiment
with human 293T and HeLa cells and obtained a similarly
increased G2/M : G1 ratio in both cell types (Fig. 3 bottom
row; this only shows the experiment in the HeLa cells).
Merge
24 h
ORF20
48 h
24 h
eGFP
48 h
1448
Fig. 2. Nuclear localization of MHV68 ORF20
in NIH3T3 cells. NIH3T3 cells infected with
lentivirus expressing MHV68 ORF20 (top two
rows) or eGFP (bottom two rows) were
seeded onto glass coverslips (see Methods).
At 24 and 48 h, cells were fixed, permeabilized
and subjected to double staining with DAPI to
visualize nuclei (blue) and with anti-HA monoclonal antibody (red) to visualize ORF20
expression in the GFP–lentivirus (green)infected cells (top row, 24 h; second row,
48 h). An identical experiment was conducted
with the eGFP ‘empty’ non-recombinant lentivirus (third row, 24 h; fourth row, 48 h).
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Journal of General Virology 88
MHV68 ORF20 induces G2 cell-cycle arrest
150
0
0
30
1000
0
FL2-A
1000
G0/G1: 39.0
S: 14.9
G2/M: 46.1
0
0
FL2-A
1000
0
FL2-A
1000
Infection with a control, non-recombinant lentivirus did
not change the cell-cycle profile (Fig. 3, top and third
rows).
Induction of apoptosis by MHV68 ORF20
Staining cells with DAPI at 72 h p.i. revealed that NIH3T3
cells expressing ORF20 did not progress in the cell cycle,
but instead underwent apoptosis, showing typical apoptotic nuclear bodies (Fig. 4a, top and middle rows). To
confirm this observation, the cells were also examined
by FACS analysis at 72 h p.i. for levels of lentivirus infection (eGFP expression) and apoptosis by the TUNEL procedure. The level of infection was 80–90 % and almost
50 % of recombinant lentivirus-infected NIH3T3 (Fig. 4b)
and HeLa (data not shown) cells were apoptotic. There
were no significant levels of apoptosis in control cells
infected with the lentivirus containing the empty vector.
Identical results were obtained with human (HeLa) cells
infected with control and recombinant lentivirus (data not
shown).
Cells expressing MHV68 ORF20 enter apoptosis in
G2 phase
To define whether the arrest of cells by ORF20 was in the
G2 phase of the cell cycle or in mitosis, and to determine
whether the cell entered mitosis prior to apoptosis, expression of the mitosis marker phosphorylated histone H3 was
FL2-A
1000
G0/G1: 79.27
S: 8.62
G2/M: 12.11
0
DNA content
http://vir.sgmjournals.org
1000
0
G0/G1: 76.94
S: 6.93
G2/M: 16.13
0
Counts
1000
0 20 40 60 80 100
FL2-A
Counts
Counts
0 20 40 60 80 100
1000
G0/G1: 68.0
S: 8.56
G2/M: 23.44
Counts
0
0 10 20 30 40 50
200
FL2-A
FL2-A
G0/G1: 19.63
S: 17.57
G2/M: 62.80
Counts
80
FL2-A
G0/G1: 31.22
S: 13.88
G2/M: 54.9
80
Counts
0 20 40 60 80 100
1000
0
ORF20
FL2-A
G0/G1: 71.76
S: 8.39
G2/M: 19.85
0
HeLa
0
0
G0/G1: 61.17
S: 10.5
G2/M: 28.33
0
eGFP
1000
Counts
Counts
0 20 40 60 80100
ORF20
FL2-A
Counts
0
NIH3T3
48 h
G0/G1: 57.06
S: 23.73
G2/M: 19.21
Counts
150
0
Counts
0
eGFP
24 h
G0/G1: 55.39
S: 20.21
G2/M: 24.40
Counts
180
0h
G0/G1: 53.55
S: 19.9
G2/M: 26.55
FL2-A
1000
G0/G1: 35.82
S: 17.28
G2/M: 46.9
0
FL2-A
1000
Fig. 3. MHV68 ORF20 induces G2/M arrest
in both murine and human cell lines. NIH3T3
cells infected with lentivirus containing the
eGFP empty vector or ORF20 recombinant
vector were collected at the indicated time
points. Cells were stained with propidium
iodide for cell-cycle analysis by FACS analysis
of DNA content as shown (top and second
rows). The G2/M, G0/G1 and S ratios were
determined using CELLQUEST. A similar experiment with HeLa cells was performed and the
profiles of cells infected with control nonrecombinant and recombinant lentivirus containing ORF20 are shown in the third and
fourth rows, respectively. The percentages of
cells in G0/G1, S and G2/M are given in the
figure. The variation in the percentage of cells
in the S phase between control and recombinant lentivirus-infected cells at time 0 is the
result of day-to-day variation, as each panel of
retrovirus infection represents an independent
experiment performed on different days.
studied. Histone H3 phosphorylation plays an important
role in several cellular functions, including chromosome
condensation and cell division. Mitotic chromosome condensation is always associated with phosphorylation of
histone H3 at Ser-28, making it a frequently used marker of
mitosis.
Lentivirus-infected NIH3T3 cells expressing ORF20 were
stained with anti-phospho-histone H3 at 48 and 72 h after
infection with recombinant lentivirus. Only cells negative for the GFP marker of infection stained positively for
phospho-histone H3, whereas lentivirus-infected cells did
not express phospho-histone H3 (Fig. 5). This experiment
demonstrated that cells infected with ORF20 arrest in G2
and enter apoptosis without entering mitosis.
ORF20 expression results in an increase in Cdc2
phosphorylation
The G2/M transition in mammalian cells is mediated by
the Cdc2–cyclin B complex. The activity of this complex
is positively regulated by the availability of the cyclin B
subunit. Expression of the cyclin B subunit fluctuates
through the cell cycle. In addition, its activity is negatively
regulated by phosphorylation of two amino acids (Thr-14
and Tyr-15) of Cdc2.
To test whether ORF20 was arresting the cell cycle in G2
and acting at the level of the cyclin complex, NIH3T3
cells were infected with lentivirus expressing ORF20 and
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GFP
DAPI
Anti-HA
(b)
Merge
Counts
(a)
80
R. Nascimento and R. M. E. Parkhouse
0
ORF20
72 h
100 101 102 103 104
FL4-H
Apoptosis
eGFP
72 h
Fig. 4. NIH3T3 cells stably expressing MHV68 ORF20 enter apoptosis. (a) NIH3T3 cells infected with control nonrecombinant and ORF20-recombinant lentivirus were seeded onto glass coverslips and at 72 h cells were fixed, permeabilized
and subjected to staining with DAPI to visualize the nuclei (blue). ORF20 expression was assessed using an anti-HA
monoclonal antibody (red) and lentivirus infection by eGFP expression. In the middle row, amplification of the indicated field
reveals the presence of fragmented apoptotic nuclei. (b) NIH3T3 cells were infected with ORF20-recombinant lentivirus (thick
line) and control eGFP lentivirus (thin line) and examined for apoptosis by the TUNEL technique at 72 h p.i.
cyclin B expression was determined by Western blot
analysis with anti-cyclin B antibody. A clearly increased
level of cyclin B was observed in recombinant lentivirusinfected cells from 48 to 60 h p.i., consistent with arrest
at the G2/M phase of the cell cycle (Fig. 6). As the
dephosphorylated form of Cdc2 is required for entry into
mitosis, a similar experiment was performed, but the
Western blots were developed with an antibody specific
for the Tyr-15-phosphorylated form of Cdc2. The experiment revealed an increase in the phosphorylated (inactive) form of Cdc2 relative to the expression of total Cdc2
from 24 to 72 h after infection with recombinant lentivirus
(Fig. 6). These changes were not observed when cells were
transduced with the control, non-recombinant lentivirus.
Similar results were obtained in human 293T cells (data
not shown).
DAPI
48 h
72 h
1450
eGFP
ORF20-mediated G2 arrest is caused by inhibition
of the Cdc2–cyclin B complex
The demonstration that Cdc2 is in its inactive phosphorylated form and the clear increase in levels of cyclin B
indicated that ORF20 blocks the progression of cells to
mitosis by acting at the level of the Cdc2–cyclin B complex.
To confirm the hypothesis that ORF20 induced G2 arrest
by inactivation of the Cdc2–cyclin B complex, we compared the relative kinase activities of Cdc2–cyclin B in control and ORF20-expressing cells. Nocadazol-treated cells,
which are blocked in mitosis, were used as a positive control. Lysates from NIH3T3 cells infected with recombinant lentivirus expressing ORF20 or with lentivirus control
(eGFP) were prepared. Complexes of Cdc2–cyclin B were
precipitated from cell lysates with protein G–Sepharose
Ph-Hist H3
Fig. 5. NIH3T3 cells expressing MHV68
ORF20 arrest in G2. NIH3T3 cells infected
with ORF20-recombinant lentivirus were
seeded onto glass coverslips and, at the
indicated time points, cells were fixed, permeabilized and subjected to staining with DAPI
to visualize nuclei and with anti-phosphohistone H3 antibody as a mitosis marker.
eGFP expression indicates lentivirus-infected
cells. The eGFP-positive transduced cells and
the phospho-histone H3-positive cells are
distinct and indicated with arrows in the middle
and right-hand panels.
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Journal of General Virology 88
MHV68 ORF20 induces G2 cell-cycle arrest
eGFP
0
24
48
ORF20
60
72 h
0
24
48
60
72 h
Ph
Cdc2
Tyr-15_Cdc2
Cyclin B1
a-Tubulin
and an antibody to cyclin B, and their kinase activity
was determined in a kinase assay using histone H1 as the
substrate. The supernatants were examined for phosphorylated histone by SDS-PAGE and autoradiography (Fig. 7a)
and the immunocomplexes bound to protein G were
submitted to SDS-PAGE and Western blot analysis for
anti-Cdc2 (Fig. 7b). Cells expressing ORF20 contained
the higher-molecular-mass phosphorylated Cdc2 (inactive) complexed with cyclin B (Fig. 7b), which was almost
Fig. 6. ORF20 increases Cdc2 phosphorylation and cyclin B expression. NIH3T3 cells
were infected with ORF20 recombinant lentivirus. Cells were lysed at the indicated time
points and immunoblotted for Cdc2, phosphoTyr-15-Cdc2, cyclin B and a-tubulin (righthand column). The same experiment was
performed with ‘empty’ lentivirus (eGFP) (lefthand panel).
completely inactive in the kinase assay (Fig. 7a). In contrast, in the nocadazol positive control, Cdc2 was present as
the active non-phosphorylated form (Fig. 7b) and there
was clear phosphorylation of the histone substrate in the
kinase assay (Fig. 7a). The control, non-recombinant lentivirus (eGFP)-infected cell lysate contained both active and
inactive forms of Cdc2, which catalysed a lower level of
histone phosphorylation.
DISCUSSION
(a)
Noc
eGFP ORF20
Ph-histone H1
(b)
Ph
Cdc2
Fig. 7. ORF20 inhibits the kinase activity of the Cdc2–cyclin B
complex. (a) The Cdc2–cyclin B complex was precipitated with
cyclin B antibody coupled to protein G beads from lysates of
NIH3T3 cells 48 h after infection with ‘empty’ lentivirus (eGFP)
and cells infected with ORF20-expressing lentivirus (ORF20). The
positive control was cells treated for 18 h with nocadazol (Noc).
Immunoprecipitates were incubated with histone H1 and processed for in vitro kinase assays as described in Methods. (b)
Immunocomplexes were denatured and separated on denaturing
polyacrylamide gels for Western blotting with anti-Cdc2 antibody,
which showed the expected correlation with the kinase inhibition
results.
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Manipulation of the host cell cycle is a frequent virus
strategy for host evasion, presumably in order to achieve
a cellular environment favourable for their replication.
Small DNA viruses interact with the host cell-cycle control
mechanism in a way that promotes entry into the S phase
of the cell cycle. In contrast, herpesviruses encode their
own DNA polymerase and accessory factors and do not
require the environment of an S phase for virus replication. At the same time, however, herpesviruses have a
more profound interaction with host cell-cycle machinery, promoting both cell-cycle arrest (Lu & Shenk, 1999;
Song et al., 2000; Sunil-Chandra et al., 1992a) and cellcycle progression (Boldogh et al., 1990; Hobbs & DeLuca,
1999)
The key element in the regulation of the eukaryotic cell
cycle is the periodic synthesis and destruction of cyclins,
the proteins that associate and activate cyclin-dependent
kinases. The related sequential activation and inactivation
of cyclin-dependent kinases, through the periodic synthesis
and destruction of cyclins, provide the basis of cell-cycle
regulation. In addition to the formation of cyclin
complexes, the phosphorylation state of cyclin-dependent
kinases controls the activity of these kinases during the cell
cycle. The key downstream target of G2 arrest is the
mitosis-promoting kinase complex Cdc2–cyclin B. Entry
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R. Nascimento and R. M. E. Parkhouse
into mitosis requires activation of Cdc2 following dephosphorylation of Tyr-15 and Thr-14. Various pathways
converge at this point, shifting the balance towards entry
into mitosis or arrest at G2 (Abraham, 2001; Castedo et al.,
2002; Kastan & Bartek, 2004; Lukas & Bartek, 2004). In this
work, we demonstrated that the non-essential ORF20 gene
of the mouse herpesvirus MHV68 arrests cells in the G2
phase of the cell cycle (Fig. 3) in both human and mouse
cells, followed by induction of apoptosis (Fig. 4). Our
observation that the ORF20 gene of MHV68 induces cellcycle arrest in both human and mouse cells strongly
suggests that this virus gene plays a fundamental role in
herpesvirus strategy for host-cell modification and adaptation, and one that might be exploited for the rational
understanding and control of these important human
infectious agents.
The general mechanism of ORF20 focuses on the inactivation of the Cdc2–cyclin B complex through a block in
the dephosphorylation-dependent activation of the Cdc2
molecule, as indeed occurs with the human papilloma virus
(HPV) E2 protein (Fournier et al., 1999) and the human
immunodeficiency virus (HIV) Vpr protein (He et al.,
1995). Consequently, in cells transduced with ORF20, there
is an increase in Cdc2 phosphorylated at the inhibitory site
(Fig. 6). The exact sequence of events in the inhibition is
still to be elucidated, but the localization of ORF20 to the
nucleus (Fig. 2), as for its herpes simplex virus 1 (HSV-1)
homologue (Pearson & Coen, 2002), suggests a primary
influence on transcription. The HSV-1 homologue of
ORF20, UL24, is not required for growth in cultured cells
(Pearson & Coen, 2002), but, in mice, mutation of the
HSV-1 UL24 gene impairs virus replication in tissue
culture and in mouse eye (Jacobson et al., 1998). The fact
that ORF20 is a virion-associated protein (Bortz et al.,
2003) suggests that an early impact of the virus on cellcycle progression may be a necessary aspect of the function
of ORF20. However, the observation that ORF20 is a late
protein (Ebrahimi et al., 2003) suggests a contributing
requirement, either in cell-cycle control or in some other
function. As mentioned above, several viral proteins induce
G2 arrest. Although the mechanism of action of these viral
proteins has not been elucidated fully, both the E2 gene
from HPV and the Vpr gene from HIV promote G2 arrest
by inactivation of Cdc2–cyclin B1 (Fournier et al., 1999; He
et al., 1995). In the case of Vpr, one suggestion is to maximize viral production by delaying the death of infected cells
and maintaining the host cell in a stage of the cell cycle
(G2) in which transcription of the viral long-terminal
repeat is optimal (Goh et al., 1998; Groschel & Bushman,
2005; Zhu et al., 2001).
Until now, as demonstrated here, cell-cycle arrest promoted by MHV68 or by any of its genes has not been
described. Only recently, a viral D-type cyclin common to
all gamma2-herpesvirus has been characterized in MHV68
(Hoge et al., 2000; Upton et al., 2005) This viral cyclin is
not required for virus replication, either in vitro or in vivo,
but is required for efficient reactivation from latency (Hoge
1452
et al., 2000). Like the MHV68 ORF20 gene, the precise
biological role of this virus strategy for host manipulation is not known.
In conclusion, ORF20 of the mouse gammaherpesvirus
MHV68 is a virion-associated protein with nuclear localization, which has evolved for host cell-cycle arrest at G2
through inactivation of the Cdc2–cyclin B complex. A
more profound understanding of its mechanism and
function provides a fascinating problem for further investigation. For example, whether the observed apoptosis
induced by ORF20 is a default consequence of the G2 arrest
or an independent mechanism will be addressed by deletion mutant analysis. A similar functional analysis of the
homologous human herpesviruses gene UL24 is clearly a
priority.
ACKNOWLEDGEMENTS
This work was supported by Fundação Portuguesa para a Ciência e
Tecnologia (POCI/SAU-MMO/59444/2004). R. N. was a recipient of
a fellowship from Fundação Portuguesa para a Ciência e Tecnologia
(SFRH/BD/4853/2001).
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