JVI Accepted Manuscript Posted Online 18 February 2015 J. Virol. doi:10.1128/JVI.00213-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Adenovirus replaces mitotic checkpoint controls
Roberta L. Turner,a Peter Groitl,b Thomas Dobner,b David A. Ornellesa#
Department of Microbiology and Immunology, Wake Forest School of Medicine, WinstonSalem, North Carolina, USAa; Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germanyb
# Address correspondence to: David A. Ornelles, [email protected]
Running Head: Adenovirus replaces mitotic checkpoints Abstract word count: 197 Manuscript word count: 7892
2 ABSTRACT 8 9
Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins including the E1B-55K and
10
E4orf3 proteins inhibit signaling in response to DNA damage. A fraction of cells infected with
11
an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a
12
mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this
13
state at late times of infection. This arrested state, which displays hallmarks of mitotic
14
catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However,
15
E1B-55K-mutant virus-infected cells became trapped in a mitotic-like state in the presence of the
16
microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or
17
facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The
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E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with
19
the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by
20
possibly mislocalizing and functionally inactivating cyclin B1. When expressed in non-infected
21
cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1
22
variant. IMPORTANCE
23
Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed
24
to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein
25
prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to
26
depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The
27
adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular
28
distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis,
3 29
these adenoviral proteins act to prevent the infected cell from arresting in a mitotic-like state. INTRODUCTION
30
Adenoviral infection and the ensuing replication of the viral double-stranded DNA
31
genome activates the host DNA-damage response (1, 2). Early adenoviral proteins collaborate to
32
dampen this host response (reviewed in 3). The initial phase of the DNA-damage response
33
proceeds through a phosphorylation cascade, while subsequent recruitment of effector proteins
34
also depends on the conjugation of ubiquitin and the related small ubiquitin like modifier SUMO
35
(4). Signals initiated by the three apical kinases or DNA-dependent protein kinase (DNA-PK)
36
(5), ataxia telangiectasia mutated protein (ATM) (6), and ATM- and Rad3-related protein (ATR)
37
(7) trigger downstream consequences of DNA-damage such as DNA repair, cell cycle arrest, and
38
cell death. The tumor suppressor protein p53 is centrally positioned in the cellular response to
39
DNA-damage. Numerous branches of the DNA-damage response are controlled by p53
40
including cell cycle arrest, cell death, senescence, autophagy, and cell proliferation (8). Not
41
surprisingly, viruses that elicit a robust DNA-damage response inevitably target p53. For
42
adenovirus, the transcriptional activity of p53 is suppressed by the E1B-55K protein (9-11), the
43
stability of p53 is decreased by a ubiquitin protein-ligase formed by the E1B-55K and E4orf6
44
protein (12-14) and the expression of p53-responsive genes is epigenetically dampened by the
45
E4orf3 protein (15).
46
Cell cycle arrest mediated by p53 following DNA-damage typically occurs at the G1/S
47
border (16). However p53 also inhibits cell cycle progression immediately before mitosis. p53
48
can prevent entry into mitosis by inhibiting a kinesin involved in the arrangement of condensed
49
chromosomes (17). Polo-like kinase 1 (Plk1) promotes the transition from G2 into mitosis. The
50
inhibition of Plk1 uncovers p53-dependent outcomes in response to mitotic stress. In p53-
4 51
deficient cells, Plk1 inhibitors and microtubule poisons elicit mitotic catastrophe and greater
52
DNA-damage than in p53-proficient cells (18). This may reflect the absence of p53-dependent
53
apoptosis that would normally eliminate cells arrested in mitosis. It has been suggested that p53-
54
dependent cell-cycle-arrest at the G2/M border is the key factor in determining whether a cell
55
undergoes mitotic catastrophe or apoptosis (19).
56
Although progression through the cell cycle can be stopped at many stages, the intricately
57
orchestrated process of mitosis proceeds once the antephase checkpoint has been cleared or
58
bypassed (20), despite the persistence of damaged DNA (21). Mitosis is regulated by the
59
appropriate localization of cellular proteins and their timely degradation by the anaphase-
60
promoting complex or cyclosome (APC/C). During the G2 phase of the cell cycle, levels of
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cyclin B1 rise, which associates with Cdk1 to form the major mitotic kinase (22). Entry into
62
mitosis begins with the activating phosphorylation of the Cdc25C phosphatase and components
63
of the APC/C as well as the inactivating phosphorylation of the Wee1 and Myt1 kinase by polo-
64
like kinases (23). The cyclin B1-Cdk1 complex is believed to shuttle in and out of the nucleus,
65
with hyperphosphorylation of cyclin B1 inhibiting nuclear export of the complex leading to an
66
intranuclear increase in cyclin B1-Cdk1 (24, 25). Within the nucleus, this kinase directs mitotic
67
progression by phosphorylating numerous targets (26) such as the nuclear lamins in order
68
promote nuclear envelope breakdown (27) and condensin II to initiate condensation of the
69
chromosomes (28). Exit from mitosis requires the degradation of proteins ubiquitinated by the
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APC/C (29). Key mitotic targets of the APC/C are cyclin B1, securin, and Plk1. With the
71
degradation of cyclin B1 and securin, separase is free to cleave cohesin from sister chromatids,
72
leading to the precipitous separation of chromatids and progression out of mitosis (30).
73
Cell death by mitotic catastrophe was initially described as a caspase-dependent,
5 74
apoptotic death triggered by aberrant mitosis and the persistence of active mitotic kinases (31). It
75
has been proposed that mitotic catastrophe be considered an oncosuppressive mechanism leading
76
to cell death resulting from the disorder of mitotic machinery that is typified by a period of
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aberrant mitotic arrest (32). Additional hallmarks of mitotic catastrophe include the formation of
78
multipolar spindles and the appearance of a cleaved form of cyclin B1 that is unable to be
79
degraded and is therefore presumed to sustain MPF activity (33, 34). Aberrant mitotic arrest
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requires that cells enter mitosis but halt prior to anaphase. Examples of viruses that arrest cells in
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mitosis and can elicit mitotic catastrophe include adenovirus, adenovirus-associated virus (AAV)
82
and chicken anemia virus. Expression of the adenovirus E4orf4 protein to high levels in H1299
83
cells lead to the accumulation of cells with 4N and greater DNA content followed by cell death
84
characterized as mitotic catastrophe (35). Exposure of p53-deficient osteosarcoma cells to UV-
85
inactivated AAV leads to centriole overduplication and mitotic arrest (19). Another example of
86
mitotic catastrophe occurs in response to apoptin, a viral protein from chicken anemia virus.
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Apoptin directly inhibits the APC/C, thereby preventing exit from mitosis, resulting in mitotic
88
arrest and subsequent apoptosis (36). This arrest was found to be independent of p53 and resulted
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from inhibition of the metaphase-to-anaphase transition (33).
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We show here that the E1B-55K and E4orf3 genes are sufficient to prevent mitotic arrest
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in the adenovirus-infected cell. The E1B-55K protein circumvents entry into mitosis in a p53-
92
dependent manner. The E4orf3 protein circumvents mitotic arrest by facilitating exit from
93
mitosis, perhaps by mislocalizing cyclin B1. These early adenoviral proteins may prevent an
94
untimely death that would occur in response to inappropriate mitotic arrest. MATERIALS AND METHODS
95
Chemicals. Hydroxyurea (HU) from Sigma/Aldrich (St. Louis, MO) was used at a
6 96
concentration of 2 mM from a stock solution of 1 M in water. KaryoMAX Colcemid from
97
Gibco/Invitrogen (Gaithersburg, MD) was used at a concentration of 0.2 μg per mL from a stock
98
solution of 10 μg per mL in HBSS.
99
Cells. All cell culture media, supplements, and sera were obtained from Invitrogen
100
(Gaithersburg, MD) or Lonza (Hopkinton, MA) through the Tissue Culture and Virus Vector
101
Core Laboratory of the Comprehensive Cancer Center of Wake Forest University. HeLa cells
102
were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10%
103
newborn calf serum. H1299 cells were maintained in DMEM supplemented with 10% fetal
104
bovine serum. Both cell lines were cultured in a 5% CO2 atmosphere at 37oC by passaging twice
105
weekly at a 1:10 dilution. For all experiments, cells were plated at a density of 5×104 cells per
106
mL. For high-resolution immunofluorescence microscopy, cells were grown on nitric acid-
107
cleaned, sterilized glass coverslips in 6-well plates or 35-mm dishes. For cell cycle profile
108
analysis or protein lysate collection, cells were grown in 60-mm dishes.
109
Viruses. The phenotypically wild-type virus in this study contains several deletions and
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a substitution within the E3B region. This virus, dl309, displays wild-type characteristics in
111
cultured cells (37). The virus referred to as the double-mutant virus is 3112, a previously
112
described (38) viral recombinant of the dl1520 virus that has a 827 bp deletion in the E1B-55K
113
open reading frame (39) and the dl341 virus that contains an E4orf3 deletion (40). Other E1B-
114
55K deletion mutants include dl338, which bears a 524 bp deletion (41) and dl110, which
115
contains a 472 bp deletion (42). The parental virus for the E1B-55K point mutation mutants
116
listed in Table 2 is H5pg4100, which contains a deletion in the E3 region at nucleotides 28593–
117
30471 and has an added endonuclease restriction site at nucleotide 30955 (BstBI). The
118
adenovirus mutants described in Table 2 were constructed by the method described in (43).
7 119
Briefly, point mutations were introduced into the E1B-55K gene in a shuttle plasmid by site-
120
directed mutagenesis. The appropriate fragment was inserted into the parental plasmid
121
pH5pg4100 and the viral genomes were released from the recombinant plasmids by digestion
122
with PacI. Mutant viruses recovered after transfection of the linear DNA into 293 cells. All
123
viruses were grown in 293 cells and concentrated virus stocks prepared by sequential
124
centrifugation through CsCl gradients as described previously (1).
125
Antibodies. Primary antibodies included a monoclonal mouse antibody against ß-tubulin
126
(Clone TUB 2.1, T4026) from Sigma Aldrich (St. Louis, MO) used at a 1:500 dilution, a
127
monoclonal pantropic mouse antibody against p53 (DO-1) obtained from CalBiochem
128
(Darmstadt, Germany) used at a 1:100 dilution, and a polyclonal rabbit antibody against
129
phospho-histone H3 (Thr11, #9849) from Cell Signaling (Danvers, MA) used at a 1:100 dilution
130
for immunofluorescence. Adenovirus-specific antibodies included the mouse monoclonal
131
antibody Rsa#3 (44) for the E4orf6/7 protein and the rat monoclonal antibody 6A11 (45) used as
132
hybridoma culture supernatant fluid diluted 1:5. Five antibody preparations for cyclin B1 were
133
used for this study. The mouse monoclonal antibody against cyclin B1 (#CC03) was obtained
134
from Oncogene/CalBiochem (Cambridge, MA) and used at a 1:250 dilution for microscopy and
135
1:1000 dilution for western blotting. The remaining cyclin B1 antibodies were obtained from
136
EMD Millipore (Billerica, MA) and included the mouse monoclonal antibody specific for the C-
137
terminus of human cyclin B1 (clone Y106) used at a 1:100 dilution for immunofluorescence and
138
a 1:10,000 dilution in 5% milk for western blotting, a mixture of mouse monoclonal IgGs
139
specific for human cyclin B1 (catalog # 05-373) used at 10 μg per mL for immunofluorescence
140
and 0.33 μg per mL for western blotting, a monoclonal antibody raised against hamster cyclin B1
141
(catalog # MAB3684) used at a 1:100 dilution for immunofluorescence and a 1:5000 dilution in
8 142
5% milk for western blotting and a rabbit monoclonal antibody specific for phosphorylated
143
serine 126 in cyclin B1 (catalog # MABE490) used at a 1:100 dilution for immunofluorescence
144
and a 1:3000 dilution for western blotting. Secondary antibodies used for immunofluorescence
145
microscopy were anti-mouse or anti-rabbit whole IgG conjugated to Alexa Fluor 488 (AF488) or
146
Alexa Fluor 568 (AF568) from Invitrogen used at 2 μg per mL. The secondary antibody used for
147
western blot analysis was an anti-mouse or anti-rabbit whole IgG raised in goats conjugated to
148
horseradish peroxidase from Jackson ImmunoResearch Laboratories (West Grove, PA) and used
149
at a concentration of 0.1 μg per mL.
150
Plasmids and PEI transfection. A plasmid expressing the wild-type human TP53 gene
151
under control of the CMV immediate early promoter and enhancer was generously provided by
152
Guangchao Sui (Wake Forest School of Medicine). Plasmids expressing human cyclin B1 fused
153
to the yellow fluorescent protein Venus (pVenus-N1 Cyclin B1, Addgene plasmid #26062) and
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the degradation-resistant form of Cylin B1 (pVenus Cyclin B1 R42A, Addgene plasmid #39873)
155
were the gift of Jonathon Pines (Gurdon Institute, Cambridge, England). Cells were seeded on
156
glass coverslips in a 6-well culture dish. For each well, 1 μg of DNA was used in a volume of 0.2
157
mL serum-free media with poly(ethylenimine) (PEI) at a 1:5 dilution from a 7.5 mM stock
158
dissolved in deionized water with gentle heating. Transfections were carried out in a 5% CO2
159
atmosphere at 37°C with gentle rocking and regular rotation for 8 h before being replacing the
160
DNA and PEI mixture with growth medium.
161
Indirect immunofluorescence. Cells were washed twice with phosphate-buffered saline
162
(PBS), fixed for 30 min with 2% paraformaldehyde, and permeabilized for 5 min with 0.2%
163
Triton X-100 in PBS at room temperature. All subsequent washes were performed with Tris-
164
buffered saline with BSA, glycine and Tween-20 (TBS-BGT: 0.137 M NaCl, 0.003 M KCl,
9 165
0.025 M Tris-Cl [pH 8.0], 0.0015 M MgCl2, 0.5% bovine serum albumin, 0.1% glycine, 0.05%
166
Tween 20, and 0.02% sodium azide). Antibodies used for immunofluorescence were diluted in
167
TBS-BGT supplemented with 10% normal goat serum (Invitrogen). Samples were stained for 90
168
min with primary antibody and for 30 min with secondary antibody with multiple washes with
169
TBS-BGT between. Samples were mounted with ProLong Gold mounting media (Invitrogen)
170
containing 4′,6-diamidino-2-phenylindole (DAPI).
171
Microscopy. Micrographs were obtained by standard epifluorescence microscopy with a
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Nikon TE300 inverted microscope or by confocal laser scanning microscopy with a Nikon TiE
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inverted microscope fitted with a Nikon C1si system. A 100×/1.4 NA magnification oil-
174
immersion objective was used for all micrographs. Monochromatic images were acquired on the
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Nikon TE300 microscope with a Retiga EX 1350 digital camera (QImaging Corp., Burnaby,
176
British Columbia, Canada). Confocal images were acquired as a series of 5 sections at 0.2 um
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intervals in the center of the nucleus using sequential excitation for each of the three
178
fluorochromes. The confocal images are presented as a maximum intensity projection to reduce
179
the three-dimensional information to two-dimensions. Merged color images were prepared by
180
assigning either red, green or blue to the appropriate fluorochrome. For single channel
181
fluorescent images, a pseudocolor scheme was assigned in order to mimic the visual perception
182
with increased saturation at increased fluorescent intensity. Monochromatic images were
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assigned colors with the open-source program ImageJ (version 1.46). All figures were assembled
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with the open-source vector graphics editor Inkscape (version 0.48) and raster graphics editor
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GIMP (version 2.8.4.)
186 187
Flow cytometry. Cells were harvested by trypsinization. EdU-labeled cells were labeled as indicated below and resuspended in FACS buffer (1% BSA in PBS) with propidium iodide
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provided by the Click-iT EdU kit. All other samples were washed twice with PBS and
189
resuspended in 100 μL PBS. Cells were transferred dropwise into 2.5 mL of 70% ethanol with
190
constant mixing. Samples were stored at -20oC for at least 12 h. Ethanol was then removed and
191
the samples resuspended at a density of 106 cells per mL in PI solution diluted in water from a
192
10X stock (1 M NaCl, 0.36 M sodium citrate, 500 μg per mL propidium iodide, 6% NP-40) with
193
0.1 mg per mL RNase A diluted from a stock solution of 10 mg per mL in 0.01 M sodium acetate
194
with 0.1 M Tris-Cl, pH 7.4. Samples were incubated with PI solution for 30 min in the dark at
195
37oC and then transferred to ice before flow cytometric analysis. A Becton Dickinson FACS
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Calibur instrument was used to acquire the propidium iodide signal in linear mode. Gating on the
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peak width and peak area was used to select single cells for DNA profile and cell cycle analysis.
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Western blotting. Cells were washed in PBS supplemented with protease and
199
phosphatase inhibitors (2 mM EDTA, 1 mM NaF, 1 mM sodium pyrophosphate, 1 mM
200
phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 2 μM leupeptin), harvested by scraping, and
201
suspended in a volume of one-tenth concentrated PBS sufficient to dilute the cells to a density of
202
2×107 cells per mL An equal volume of 2X sodium dodecyl sulfate (SDS) protein sample buffer
203
(4% SDS, 0.135 M Tris [pH 6.8], 20% glycerol, 0.02% bromophenol blue, and 6% β-
204
mercaptoethanol) was added to make the final concentration 107 cells per mL. The cell lysate
205
was heated for 5 min at 95oC and subjected to 3 pulses of sonication lasting 20 sec each. The
206
lysates were separated by SDS-polyacrylamide gel electrophoresis through 10% acrylamide gels.
207
The proteins were then electrophoretically transferred to nitrocellulose (Whatman/GE
208
Healthcare) overnight at 4oC. The nitrocellulose was blocked in TBS-BGT containing 5% nonfat
209
dry milk and sodium azide, stained with primary antibody diluted in TBS-BGT with sodium
210
azide overnight at 4oC, and stained with secondary antibody diluted in TBS-BGT without sodium
11 211
azide for 1 hr at room temperature. The stained protein was visualized by a mixture of
212
SuperSignal West Pico and SuperSignal West Femto chemiluminescence substrate from
213
Pierce/ThermoScientific (Rockford, IL) and X-ray film. The density of the specific signal
214
recorded by the X-ray film was quantified by scanning non-saturated exposures at 16-bit
215
resolution and measuring the optical density with the tools available in ImageJ.
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Click-iT EdU. Cells were pulse-labeled with an appropriate amount of EdU from a 10
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mM stock in DMSO diluted in a small volume of warm growth medium to make a final
218
concentration of 100 μM EdU. At the end of the labeling period, the wells were washed and
219
replaced with pre-warmed growth medium. At time of harvest, samples for DNA profile analysis
220
were harvested with trypsin while samples on glass coverslips were fixed, permeabilized, and
221
processed for immunofluorescence in a humidifying chamber. The samples were fixed with 4%
222
paraformaldehyde in PBS for 15 min, washed with FACS buffer (1% BSA in PBS), and
223
permeabilized with 0.5% Triton X-100 for 20 min at room temperature. The Click-iT reaction
224
cocktail was prepared by mixing Click-iT reaction buffer, CuSO4, fluorescently labeled azide,
225
and reaction buffer additive as indicated by the manufacturer. Samples were washed with FACS
226
buffer and allowed to incubate with the Click-iT reaction cocktail in a humidifying chamber at
227
room temperature for 30 min in the dark. The samples were washed and those on coverslips were
228
mounted on slides with Prolong Gold supplemented with DAPI. Samples for DNA profile
229
analysis were pelleted and resuspended in 500 μL of FACS buffer with 0.2 mg per mL RNase A
230
(from a 20 mg per mL) and 4 μg per mL PI (from a 1 mg per mL stock solution in water).
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Cell synchronization. Mitotic cells were mechanically harvested from subconfluent cells
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grown in 75 cm2 flasks and pelleted gently. The mitosis-enriched pellet was resuspended at a
233
concentration of 2x104 cells per mL in 2 mM HU in growth medium and plated in 60 mm dishes
12 234
for DNA profile analysis or on acid-treated glass coverslips in 35 mm dishes for
235
immunofluorescence. After 1 hour, plates were swirled to dislodge any dead S-phase cells and
236
the media replaced with a fresh solution of 2 mM HU in growth medium. Cells were held in HU
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for 16 h to allow cells to cycle to the G1/S border. All samples were released from HU by
238
washing and replacing with growth medium. At various times post infection, cells were collected
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for DNA profile analysis in order to determine cell cycle distribution in the sample. RESULTS
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Cells infected during early G1 give rise to cells trapped in a mitosis-like state. A
241
subset of cells infected with an adenovirus deleted of the E1B-55K and E4orf3 genes develop
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highly condensed chromatin resembling that of a mitotic cell (46). Because typically fewer than
243
10% of the infected cells develop the highly condensed chromatin, it seems likely that an
244
underlying process or condition limits the number of infected cells that condensed their
245
chromatin and resemble a mitotic cell. Potential processes include the death and loss of cells
246
following mitotic catastrophe, a small probability of entering mitosis, or the existence of a
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limited subset of cells that are competent to enter mitosis. Since the outcome of an infection with
248
the E1B-55K-mutant virus is strongly determined by the stage of the cell cycle at the time of
249
infection (38, 47, 48), we performed two experiments to determine if entry into a mitotic-like
250
state was affected by the stage of the cell cycle at infection. Asynchronously dividing HeLa cells
251
were exposed to the thymidine analog 5-ethynyl-2´-deoxyuridine (EdU) for a 4-h interval in
252
order to identify cells that passed through S phase during this period of time. After returning the
253
cells to EdU-free medium, the cells were maintained for various intervals of time before being
254
infected. At the time of infection, a portion of the cells was collected and EdU-positive cells
255
were identified by the copper(I)-catalyzed reaction between a fluorescent azide and the alkyne in
13 256
EdU. This sample was analyzed by flow cytometry for DNA and EdU in order to determine the
257
stage of the cell cycle that EdU-positive cells had reached at infection. The labeling scheme and
258
associated flow cytometric analyses are shown in Fig. 1. After 72 h, another fraction of the
259
infected cells was stained for EdU and DNA and the number of mitotic-like nuclei were
260
enumerated by fluorescence microscopy. The results of scoring approximately 600 nuclei at each
261
time point show that cells with condensed DNA were seen most frequently among cells infected
262
during G2/M and early G1 (Table 1).
263
Although the EdU-labeling method permits analysis of unperturbed dividing cells, the
264
temporal resolution afforded by a 4 h labeling period was limited. For better resolution,
265
synchronously dividing HeLa cells were prepared as previously described (47, 49) and infected
266
with the E1B-55K/E4orf3 double-mutant virus at hourly intervals from 6 to 14 h following entry
267
into S-phase. A portion of the synchronized cells was collected and analyzed for DNA content by
268
flow cytometry in order to determine the stage of the cell cycle at the time of infection (Fig. 2A).
269
After 72 h of infection, the cells were fixed and the frequency of cells with mitotic-like nuclei
270
was determined (Fig. 2B). These results agree with the findings obtained with asynchronously
271
dividing cells (see Fig. 1) and indicate that cells infected with the E1B-55K/E4orf3 double-
272
mutant virus during a 4 h window in early G1 gave rise to the subset of cells with highly
273
condensed chromatin. These results suggest that the adenoviral E1B-55K and E4orf3 proteins
274
prevent cells infected early in G1 from becoming trapped in a mitotic-like state at late times of
275
infection.
276
Cells infected with the E1B-55K/E4orf3 double-mutant virus show evidence of
277
mitotic distress. Approximately four percent of asynchronously dividing HeLa cells contain
278
condensed chromatin typical of the later stages of mitosis (Fig. 3A, mock and Fig. 3B). The
14 279
mitotic spindle in these cells, visualized by staining for β-tubulin, occurs in a symmetrical
280
bipolar arrangement about the condensed chromatin in metaphase and anaphase cells (Fig. 3A
281
panel a). A fraction of the mitotic cells also contain highly phosphorylated histone H3 or
282
phospho-H3 (Fig. 3A panel b, and Fig. 3B). Phosphorylation of histone H3 initiates late in G2
283
phase, is completed in late prophase and is maintained through metaphase. Histone H3
284
phosphorylation precipitously decreases during anaphase as the cell exits mitosis (50). In
285
contrast to mock-infected cells, none of the cells infected with the wild-type or E4orf3-mutant
286
adenovirus and less than 0.5% of cells infected with the E1B-55K-mutant virus contained
287
condensed chromatin or phospho-H3 when evaluated at 72 hpi (Fig. 3B). This is consistent with
288
previous observations indicating that adenovirus-infected human epithelial cells cease
289
progression through the cell cycle (51). However, in these asynchronously infected cultures, a
290
significant number (>10%) of cells infected with the E1B-55K and E4orf3 double-mutant virus
291
contained chromatin characteristic of a mitotic cell (Fig. 3A, ΔE1B-55K/ΔE4orf3 and Fig. 3B).
292
Some of the double-mutant virus-infected cells also stained for phospho-H3, suggesting that
293
these cells progressed into mitosis. Although these cells superficially resembled mitotic cells,
294
many contained an asymmetric distribution of β-tubulin or even a multipolar spindle. The
295
abundance of aberrant mitotic spindles in these cells makes it seems likely that these cells will
296
fail to produce viable daughter cells. Expression of the small avian virus-derived protein apoptin
297
in human tumor cells has been reported to lead to a similar mixture of cells with apparently
298
normal, asymmetric, and multipolar spindles due to a block in the metaphase to anaphase
299
transition (33).
300 301
Cells infected with the double-mutant virus that appear to arrest in mitosis do so only at late times of infection (Fig. 3C). In some experiments, a transient increase in mitotic-like cells
15 302
occurred at 24 hpi. However, cells with highly condensed DNA do not begin to accumulate until
303
60 hpi. The number of cells apparently arrested in mitosis reached a maximum at 72 hpi. The
304
decrease in cells with mitotic-like nuclei after 72 hpi appears to be due to the death or selective
305
detachment of these cells from the substrate. Consequently, subsequent experiments evaluated
306
cells at 72 hpi. Taken together, these results suggest that the E1B-55K and E4orf3 proteins
307
prevent cells infected during early G1 from becoming trapped in a mitotic-like state at late times
308
of infection.
309
Adenoviral E1B-55K protein prevents entry into mitosis. Because significant
310
numbers of cells were observed to contain condensed DNA only after infection with the double-
311
mutant virus, it seems likely that the E1B-55K and E4orf3 proteins act independently to prevent
312
the infected cell from arresting in a mitotic-like state. Conceptually, these viral oncoproteins
313
could prevent the accumulation of mitotic-like cells by two non-exclusive mechanisms. First, the
314
viral proteins could prevent entry into mitosis. Second, the viral proteins could facilitate exit
315
from mitosis. Cherubini and associates observed that a fraction of E1B-55K-mutant virus-
316
infected cells accumulated highly condensed chromosomes after 12 h of exposure to colcemid
317
(52). By depolymerizing microtubules, colcemid prevents the completion of mitosis thus
318
trapping any cells that entered mitosis during the exposure to colcemid. Since mitotic-like cells
319
did not accumulate in colcemid-treated cells that were infected with the wild-type virus, we
320
reasoned that the E1B-55K protein might prevent the infected cell from entering mitosis. To test
321
this hypothesis, infected cells were treated with colcemid for 12 h prior to fixation and analysis
322
at various times after infection. The number of mitotic-like cells infected with the wild-type or
323
E4orf3-mutant virus did not increase, suggesting that cells infected with the wild-type or E4orf3-
324
mutant virus did not enter into mitosis (Fig. 4). As expected, cells with condensed DNA were
16 325
observed following infection with the double-mutant virus; the rate at which these mitotic-like
326
cells accumulated was the same in the presence (Fig. 4) or absence of colcemid (see Fig. 3C). In
327
contrast to the wild-type or E4orf3-mutant virus, colcemid trapped increasing numbers of
328
mitotic-like cells during the course of infection with the E1B-55K-mutant virus (Fig. 4). These
329
results, which recapitulate the findings reported by Cherubini (52), suggest that some E1B-55K-
330
mutant virus-infected cells enter and exit mitosis at late times of infection. This indicates that
331
another role for the E1B-55K protein is to prevent adenovirus-infected cells from entering into
332
mitosis.
333
E1B-55K prevents entry into mitosis through p53. The E1B-55K protein serves many
334
roles during an infection. The E1B-55K and E4orf6 proteins collaborate to form a novel SCF-
335
type E3 ubiquitin ligase with the scaffold protein Cul5, Elongins B and C, and the RING box
336
protein Rbx1 (12-14). This adenovirus-specific complex targets several cellular proteins for
337
degradation, many of which signal or respond to DNA-damage (see for example 53, 54-58).
338
Acting independently of E4orf6, the E1B-55K protein directs degradation of the cellular
339
transcription factor Daax, mislocalizes the DNA-damage responsive chromatin structure
340
regulator SPOC1, and can directly block p53-mediated transcription (11, 55, 59). In order to
341
identify the activity of the E1B-55K protein that prevents entry into mitosis, HeLa cells were
342
infected with the E1B-55K-mutant adenoviruses described in Table 2. At 60 hpi, the infected
343
cells were treated with colcemid to trap any cells entering mitosis and the cells with condensed
344
DNA were counted by fluorescence microscopy. As expected, cells infected with the three E1B-
345
55K-null adenoviruses as well as the virus bearing four nonsense mutations in the E1B-55K open
346
reading frames (H5pm4149) were trapped in a mitotic-like state by colcemid (Figs. 5A and 5B).
347
Among the viruses bearing missense mutations in the E1B-55K gene, only H5pm4109 was
17 348
associated with a significant increase in mitotic-like cells (Fig. 5B). This virus encodes an E1B-
349
55K protein that is unable to bind p53. All other E1B-55K variants analyzed in Fig. 5B appeared
350
to prevent entry into mitosis as well as the wild-type virus H5pg4100. Because over 20% of non-
351
infected cells were trapped in a mitotic-like state by this treatment (data not shown), the failure
352
to detect mitotic-like cells among virus-infected cells confirms that the cells were uniformly
353
infected. Because cells infected with the mutant virus H5pm4108 were not trapped in a mitotic-
354
like state by colcemid, we conclude that the ability to block entry into mitosis is independent of
355
the E1B-55K/E4orf6 protein complex since H5pm4108 expresses an E1B-55K protein that fails
356
to bind E4orf6 (see 60). These findings suggest that the key property of the E1B-55K protein
357
needed to prevent entry into mitosis is its ability to interact with p53.
358
In many HPV-transformed cells, expression of the integrated E6 gene of HPV directs the
359
continual degradation of p53 protein (61). Without the selective pressure to eliminate p53 gene
360
function, the TP53 gene in HeLa cells has remained intact (62). Wild-type p53 protein
361
accumulates to measurable levels in HeLa cells infected with adenovirus and to a high level in
362
cells infected with adenovirus mutants that fail to direct the degradation of p53. To examine the
363
possibility that p53 contributes to mitotic entry in the infected cell, p53-null H1299 cells were
364
infected with the E1B-55K/E4orf3 double-mutant virus and evaluated. In sharp contrast to HeLa
365
cells, no mitotic-like cells were observed among H1299 cells infected with this virus at any time
366
after infection (data not shown and see Fig. 5C).
367
To test directly a role for p53, H1299 cells were transfected with a p53-expression
368
plasmid and infected after 24 h. At 60 hpi, cells were exposed to colcemid or left untreated. At
369
72 hpi, cells were stained for p53 and DNA then evaluated by fluorescence microscopy.
370
Enforced expression of p53 is acutely toxic to H1299 cells (63). Consequently, transfection of
18 371
the p53-expression plasmid reduced the number of evaluable cells and induced apoptosis in the
372
mock-infected cells after three days (data not shown). It seems likely that the increased number
373
of p53-positive, mock-infected H1299 cells with condensed DNA was due to pyknosis or
374
chromatin condensation from apoptosis rather than mitosis (Fig. 5C, mock). Apoptosis is
375
inhibited in the adenovirus-infected cells because all of the viruses studied here express the anti-
376
apoptotic E1B-19K protein (64). Accordingly, in the absence of colcemid, neither pyknotic nor
377
mitotic-like nuclei were observed among E1B-55K-mutant virus-infected H1299 cells (Fig. 5C).
378
However, colcemid trapped a significant number of p53-positive H1299 cells in a mitotic-like
379
state. Similarly, mitotic-like nuclei were observed only in p53-positive H1299 cells infected with
380
the E1B-55K/E4orf3 double-mutant virus. As noted for HeLa cells, colcemid was not required to
381
trap the mitotic-like double-mutant virus-infected H1299 cells. At least for the HeLa and H1299
382
cell lines, these results are consistent with an unexpected role for p53; if the infected cells with
383
condensed nuclei are indeed mitotic, p53 appears to be necessary for the adenovirus-infected cell
384
to enter mitosis. Furthermore, the E1B-55K protein blocks this activity of p53.
385
E4orf3 targets cyclin B1. Colcemid is required to trap HeLa cells and p53-positive
386
H1299 cells infected with the E1B-55K-mutant virus in a mitotic-like state. By contrast, p53-
387
positive cells infected with the E1B-55K/E4orf3 double-mutant virus are trapped in a mitotic-
388
state without colcemid. If the absence of the E1B-55K protein allows infected p53-positive cells
389
to enter mitosis, this result suggests that the presence of E4orf3 protein may facilitate exit from
390
mitosis. Degradation of the major mitotic cyclin, cyclin B1 is a critical event that precipitates
391
exit from mitosis (65). The complete degradation of cyclin B1 is required for cells to proceed
392
with cytokinesis (66). Previous studies showed an increase in cyclin B1 in wild-type adenovirus-
393
infected WI-38 and A549 cells as well as an S-phase-dependent increase in cyclin B1 in E1B-
19 394
55K-mutant virus-infected cells (67). We therefore compared the nature and abundance of cyclin
395
B1 among HeLa cells infected with wild-type and mutant viruses by immunoblotting. The level
396
of cyclin B1 was indeed higher in virus-infected cells compared to mock-infected cells (Fig. 6)
397
although it seems unlikely that the modest increase in cyclin B1 level in double-mutant virus-
398
infected cells could force an apparent mitotic arrest. Adenovirus-infected cells also contained an
399
additional cyclin B1-related protein of slightly greater electrophoretic mobility than the form in
400
mock-infected cells (Fig. 6A). This product was recognized by four different cyclin B1-specific
401
antibody preparations. Both ~50-kDa forms of cyclin B1 appeared equally abundant when
402
queried by a phosphoserine-126-specific antibody (data not shown). The origin of the two forms
403
of protein of approximately 50-kDa remains unclear. A 35-kDa protein recognized by cyclin B1
404
antibodies was detected in lysates from cells infected with the E1B-55K/E4orf3 double-mutant
405
virus and to a lesser extent, with the E4orf3-mutant virus. This smaller form of cyclin B1 appears
406
to correspond to a cleavage product found during mitotic catastrophe termed cyclin B1Δ (34). It
407
was suggested that cyclin B1Δ acts as a dominant-negative inhibitor of cyclin B1 function and
408
sustains the mitotic block in cells that would otherwise exit mitosis (34). Both the elevated levels
409
of cyclin B1 and the presence of a dominant-negative inhibitor could retard E1B-55K/E4orf3
410
double-mutant virus-infected cells in mitosis. In addition, changes in the localization of cyclin
411
B1 in the infected cells point to another mechanism by which E4orf3 could facilitate exit from
412
mitosis.
413
Because E4orf3 disrupts cell signaling pathways by mislocalizing host proteins (68, 69)
414
we explored the possibility that E4orf3 mislocalizes cyclin B1 in the infected cell. The different
415
localizations of cyclin B1 among mock-infected cells were consistent with fluctuations in the
416
level and movement of cyclin B1 during cell cycle progression. Cells in G2 contained high levels
20 417
of cyclin B1 that was found in a diffuse or speckled pattern in the cytoplasm (Fig. 7A, panel a).
418
Early in mitosis, cells contained high levels of cyclin B1 that was largely coincident with
419
chromatin (Fig. 7A, panel b). Finally, a subset of cells with condensed chromatin was judge to be
420
in late mitosis because of the absence of cyclin B1 staining (Fig. 7A, panel c). A subset of cells
421
infected with the wild-type and E1B-55K-mutant virus contained nuclear cyclin B1; in many of
422
these cells, cyclin B1 was found in large aggregates throughout the nucleus (Fig. 7B panels a-d).
423
A fraction of cells infected with E4orf3-mutant viruses also contained nuclear cyclin B1. In
424
contrast to infected cells containing the E4orf3 protein, cyclin B1 was diffusely distributed in the
425
nucleus of cells infected with the E4orf3-mutant viruses (Fig. 7B, panels e-h). The distribution of
426
cyclin B1 in these infected cells more closely resembled the patterns observed in mitotic or G2
427
mock-infected cells. These results, which are quantified in Fig. 7C, show that the E4orf3 protein
428
alters the distribution of cyclin B1 in the cell nucleus during an adenoviral infection. E4orf3 may
429
functionally inactivate cyclin B1 in order to facilitate exit from mitosis.
430
E4orf3 overcomes metaphase arrest imposed by a non-degradable cyclin B1.
431
Degradation of cyclin B1 during mitosis requires key residues (R42xxL45xxI/V48xN50) in the
432
destruction box (70). Expression of cyclin B1 variants with mutations in the destruction box
433
force cells to accumulate in a mitotic-like state (66, 71). We expressed E4orf3 and the wild-type
434
or degradation-resistant cyclin B1 (CycB1 R42A) by transfection to determine if E4orf3 can
435
overcome the metaphase arrest imposed by CycB1 R42A (71). As expected, enforced expression
436
of wild-type cyclin B1 increased the fraction of cells with condensed DNA (Fig. 8A). Although
437
expression of the E4orf6/7 cDNA reduced the frequency of these mitotic-like cells, the
438
difference was not statistically significant (p=0.09). E4orf3 decreased the frequency of these
439
mitotic-like cells to statistically significant, although modestly reduced levels (7%, p=0.02).
21 440
However, the effect of E4orf3 was pronounced in cells expressing the degradation-resistant
441
R42A cyclin B1. More than half of the cells expressing the R42A variant contained condensed
442
DNA after two days. This number was not affected by E4orf6/7 (p=0.32). By contrast, E4orf3
443
significantly (p=0.01) and substantially reduced the fraction of mitotic-like cells expressing the
444
R42A variant (Fig. 8A). These results indicate that expression of E4orf3 overcomes the mitotic-
445
like state caused by elevated levels of cyclin B1 without forcing the degradation of cyclin B1.
446
Expression of E4orf3 by transfection did not promote the aggregation of cyclin B1 seen
447
in virus-infected cells (see Fig. 7C). We observed no differences in the localization of the cyclin
448
B1 fusion protein among cells expressing E4orf6/7 (Fig. 8B), E4orf3 (Fig. 8C) or no E4
449
construct (data not shown). Occasional aggregates of cyclin B1 were noted irrespective of the co-
450
transfected viral gene such as in Fig. 8B, panel a. However, cyclin B1 appeared to be largely
451
excluded from the nucleus in most of the cells expressing both E4orf3 and the cyclin B1 fusion
452
protein. Since so few productively transfected cells contained detectable levels of endogenous
453
cyclin B1, it was not possible to determine if E4orf3 affected the endogenous protein in a similar
454
manner. Because cells expressing E4orf3 and cyclin B1 R42A did not have significant levels of
455
the cyclin B1 in the nucleus after 24 h, 36 h, and 72 h of transfection (data not shown), it seems
456
likely that E4orf3 precludes entry of cyclin B1 into the nucleus or promotes nuclear export of
457
cyclin B1. DISCUSSION
458
Cells infected by the E1B-55K/E4orf3 double-mutant virus during early G1 are
459
predisposed to arrest in a mitotic-like state. The dependence of this phenomenon on the stage of
460
the cell cycle at the time of infection is unusual but not unexpected. We previously demonstrated
461
that cells infected during S-phase by the E1B-55K-deleted virus support a more productive
22 462
infection (47, 48) and are more rapidly killed than G1-infected cells (51, 72). The findings
463
reported here reinforce the notion that an infection initiated during G1 is restrictive for E1B-
464
55K-mutant adenoviruses.
465
The importance of the E1B-55K and E4orf3 proteins in preventing entry and arrest in
466
mitosis is counterintuitive given that these proteins disable DNA-damage checkpoints. In
467
particular, a dysfunctional G2 checkpoint can allow a cell to enter mitosis inappropriately (73).
468
The G2 checkpoint prevents mitotic entry by blocking activation of the mitotic kinase Cdk1,
469
which depends on activation of the Cdc25C phosphatase and inactivation of the Wee1 and Myt1
470
kinases. Cdc25C and Wee1 are regulated by the ATM (6) and ATR kinases (74). Agents that
471
disable the ATM and ATR pathways thus can force G2 cells to enter mitosis prematurely.
472
Because both E1B-55K and E4orf3 proteins inactivate ATM (75) and ATR (76), the ability of
473
these two adenoviral proteins to prevent cells from entering mitosis or arresting in mitosis is
474
surprising.
475
Insight into adenoviral control of mitotic entry was provided by Cherubini and associates
476
who showed that primary human cells infected with the E1B-55K-mutant virus dl1520
477
accumulated highly condensed chromosomes after 12 h of exposure to colcemid (52). Here we
478
show that additional adenoviruses bearing large deletions in the E1B-55K gene behaved
479
similarly (Fig. 5A). However, among ten E1B-55K-mutant viruses bearing missense mutations
480
(Table 2), all but H5pm4109 prevented cells from entering mitosis (Fig. 5B). We interpret these
481
observations to mean that many E1B-55K properties, including the ability to bind the E4orf6,
482
Daxx and Mre11 proteins, the ability to interact with SUMO-modified proteins, and C-terminal
483
phosphorylation are not critical for the E1B-55K protein to block to entry into mitosis. The
484
H5pm4109 E1B-55K protein contains a histidine in place of alanine at position 260. The
23 485
identical protein expressed by the related virus ONYX-053 is unable to bind both p53 and
486
E4orf6 (60). Because the T255A protein expressed by H5pm4108 fails to bind E4orf6 (60) but
487
prevents entry into mitosis, we conclude that the E1B-55K protein must be able to bind p53 in
488
order to prevent entry into mitosis. Surprisingly, neither the E1B-55K/Eorf3 double-mutant virus
489
nor the combination of colcemid and infection with the E1B-55K single-mutant virus forced the
490
p53-null H1299 cells into mitosis unless p53 was expressed by transfection (Fig. 5C). In addition
491
to the HeLa cells studied in this report, H460 cells, MCF10A cells and hTERT-immortalized
492
retinal pigmented epithelial cells also contain a wild-type p53 gene. A significant number of
493
these cells arrested in mitosis after infection with the double-mutant virus whereas the p53-null
494
H358 and PC3 cell lines failed to show a statistically significant increase in arrested cells after
495
infection (data not shown).
496
The requirement for p53 to promote mitotic entry in the adenovirus-infected cell is at
497
odds with the well-described ability of p53 to enforce cell cycle arrest. For example, p53
498
precludes entry into S-phase by imposing transcriptional and translational blocks to cell cycle
499
progression in response to serum-starvation (77) and prevents inappropriate entry into mitosis by
500
suppressing expression of the chromosome alignment protein Kif23 (17). Perhaps p53 is altered
501
in the adenovirus-infected cell and this altered form of p53 permits cells to slip past the G2
502
checkpoint despite DNA-damage or chromosomal abnormalities. There is ample precedence for
503
the corruption of normal cellular functions by adenovirus proteins. For example, the E1B-55K
504
proteins of different adenoviruses partner with the E4orf6 protein to reprogram ubiquitin-protein
505
ligases of the Skp1/Cul1/F-box (SCF) family (53, 54). SCF complexes orchestrate progression
506
through the cell cycle and activate checkpoint signaling (recently reviewed in 78). It may be
507
significant that the SCF complex associated with the nuclear interaction partner of ALK keeps
24 508
levels of cyclin B1 low and prevents early mitotic entry (79). Furthermore, the physical
509
interaction between the E1B-55K protein and p53 converts p53 from a transcriptional activator
510
to a potent repressor of transcription (11, 80).
511
The apparent need for an interaction between the E1B-55K protein and p53 in order to
512
prevent mitotic arrest may underlie observations suggesting that p53 enables the wild-type virus
513
to elicit greater cytopathic effects than the E1B-55K-mutant virus (81). It was later noted that
514
replication of the E1B-55K-null virus dl1520 was enhanced by expression of the gain-of-
515
function R248W p53 variant that could no longer bind DNA in a site-specific manner (82). It
516
would be of interest to determine if the interaction between the E1B-55K protein and the putative
517
form of p53 responsible for preventing entry into mitosis subverts the transcriptional activity of
518
p53 in a manner that phenocopies gain-of-function p53 variants. Some of these variants promote
519
progression into mitosis in response to genotoxic stress (83). Many gain-of-function mutations in
520
p53 map to the DNA-binding domain (84). Coincidentally, Tip60-dependent acetylation of p53
521
within the DNA-binding domain can determine how p53 governs cell fate in response to DNA-
522
damage (85). Recent evidence shows that Tip60 is targeted for degradation by the E1B-55K
523
protein (54). Although the altered function of p53 is seen irrespective of E1B-55K status, other
524
viral proteins may mimic the action of Tip60, either directly or through the action of redirected
525
cellular proteins.
526
It must be emphasized that the interaction between the E1B-55K protein and p53 cannot
527
simply ablate p53 function, otherwise p53-null cells infected with the E1B-55K/E4orf3 double-
528
mutant virus should have arrested in the mitotic-like state. If a modified form of p53 exists
529
during infection, perhaps the E1B-55K protein allows this form of p53 to reinforce the G2/M
530
checkpoint during the replicative stages of the adenoviral lifecycle.
25 531
The notion of a protein inhibiting the function of another while simultaneously preserving
532
some of the target’s function is not novel. The licensing factor Cdt1 is present during G1 and S
533
phase. During DNA synthesis Cdt1 is expelled from the nucleus, degraded, and inactivated to
534
prevent re-replication. Geminin was initially identified as a mammalian factor that contributed to
535
the inactivation of Cdt1 (86). However, it was later shown that Geminin binds Cdt1 to gain
536
nuclear localization (87) and preserve a portion of Cdt1 during late mitosis (88). Perhaps like
537
Geminin, the E1B-55K protein is able to inactivate most p53 function while retaining a low level
538
of p53 in order to strengthen the G2/M checkpoint.
539
The E1B-55K protein, through its interaction with p53 during an adenovirus infection,
540
acts to prevent entry into mitosis. Because sustained expression of E1B-55K alone does not
541
preclude cell division, this novel viral “checkpoint” must form only in the adenovirus-infected
542
cell. Adenovirus-infected cells that escape this viral G2 checkpoint and enter mitosis may be
543
prone to arrest because of other adenoviral proteins such as the E4orf4 protein. The E4orf4
544
protein inactivates APC/C by reprogramming the activity of protein phosphatase 2A, thereby
545
inducing G2/M arrest (89, 90). Interestingly, when expressed alone, E4orf4 promotes p53-
546
independent, caspase-independent cell death in tumor cells (91-93) while inhibiting apoptosis in
547
normal cells (94). Since prolonged arrest in a mitotic-like state or mitotic catastrophe is often
548
followed by apoptosis or senescence, it would be advantageous for adenovirus to express an
549
activity that facilitates exit from mitosis. Because colcemid fails to trap E4orf3 single-mutant
550
virus-infected cells in a mitotic-like state (Fig. 4), we conclude that E4orf3 does not prevent
551
entry into mitosis. However, because the E4orf3 gene is required to prevent the E1B-55K-mutant
552
virus from arresting in a mitotic-like state (Fig. 3) we suggest that E4orf3 provides an activity
553
that facilitates exit from mitosis.
26 554
Progression through mitosis beyond anaphase requires satisfaction of the spindle
555
assembly checkpoint and activation of the APC/C. Two coactivators of APC/C, Cdc20 and
556
Cdh1, dictate substrate specificity and activity. Cdc20 is sequestered by mitotic checkpoint
557
proteins while unattached kinetochores or perturbations in the mitotic spindle persist. APC/CCdc20
558
initiates anaphase by targeting the cohesins for degradation while APC/CCdh1 promotes the
559
irreversible exit from mitosis into G1 (reviewed in 78). A key target of both APC/CCdc20 and
560
APC/CCdh1 is the mitotic cyclin B1, whose loss leads to the rapid decline in Cdk1 activity (22).
561
Because cyclin B1 accumulates to high levels in adenovirus-infected cells (Fig. 6), E4orf3 does
562
not promote mitotic exit by simply removing cyclin B1. However, the E4orf3 protein may
563
functionally inactivate cyclin B1 by mislocalizing this protein within the infected cell (Fig. 7).
564
Cells infected with E4orf3-mutant viruses also contain a cyclin B-related protein (Fig. 6) similar
565
in size to a cleaved form of cyclin B1 that sustains the mitotic block (34). In the context of an
566
E1B-55K-deletion, E4orf3 may prevent accumulation of this inhibitory product.
567
E4orf3 inactivates many cellular proteins by altering their localization or directing them
568
to the aggresome (95, 96). For example, mislocalization of Nbs1 by the E4orf3 protein is
569
sufficient to prevent ATR activation, thereby crippling the DNA-damage response (76, 97). The
570
activity of cyclin B1 in association with the major mitotic kinase Cdk1 is exquisitely controlled
571
by its intracellular localization, with roles both in the cytoplasm and the nucleus (22). In the
572
infected cell, E4orf3 may promote aggregation of cyclin B1 (Fig. 7). When expressed by
573
transfection, E4orf3 may diminish nuclear levels of cyclin B1 (Fig. 8). E4orf3 may functionally
574
inactivate cyclin B1 to facilitate exit from mitosis. In this manner, E4orf3 may replace a cellular
575
process that was disabled by other adenovirus proteins in order to prevent the untoward
576
consequences of mitotic arrest.
27 577
A remaining question is, why are cells infected in early G1 predisposed to arrest in
578
mitosis after infection with the double-mutant virus? Cellular chromatin in the early G1 cell is
579
distinguished from that in other phases of the cell cycle by the need to reacquire specific
580
proteins. At late stages of mitosis, many proteins, including those that signal DNA-damage are
581
displaced from the condensing chromatin. Consequently G1 cells are able to respond to DNA-
582
damage only after these proteins re-associate with DNA (98, 99). The reacquisition of chromatin
583
proteins can also be regulated by proteins such as the histone variant H2A.Z, which suppresses
584
transcription during mitosis (100). The histone acetyl transferase Tip60 promotes H2A.Z-
585
mediated association of proteins at sites of DNA-damage. Perhaps the ability of the E1B-55K
586
protein to target Tip60 for destruction (54) renders cellular chromatin less able to signal DNA-
587
damage and thus unsuited for further progression through the cell cycle. During late mitosis and
588
early G1 chromatin is also reloaded with replication-critical licensing factors that were displaced
589
or degraded in S phase (101, 102). Early events during the adenovirus infectious cycle may
590
perturb these processes that occur during G1.
591
Studies using somatic cell nuclear transfer to generate cloned animal offspring
592
demonstrate how the unique nature of cells in early G1 can exert consequences over a very long
593
time. Donor cell nuclei derived from serum-starved G0 cells or early G1 cells were transferred
594
into enucleated bovine ova to create embryos. Although embryos derived from G0 nuclei showed
595
greater survival through the blastocyst stage, animals derived from G1 nuclei showed improved
596
birth weights and greater post-natal survival (103). Both the transfer of an early G1 nucleus and
597
the infection of a cell in early G1 have consequences evident much later in time.
598 599
While the properties of early G1 cells that predisposed them to mitotic arrest following infection with adenovirus are unknown, these cells have the potential to enter mitosis despite
28 600
suspension of normal cell cycle progression. Surprisingly, entry into mitosis by the virus-infected
601
cell requires p53, a protein better known for its ability to halt cell cycle progression. The
602
E1B-55K and E4orf3 proteins function independently of one another to prevent arrest in mitosis.
603
Although adenovirus directs the degradation of p53, it appears that a portion of the p53 protein is
604
repurposed by the E1B-55K protein to reinforce the G2/M checkpoint. Through the
605
reorganization of cyclin B1, E4orf3 protein may serve as a failsafe to facilitate exit from mitosis,
606
presumably circumventing cell death associated with prolonged mitotic arrest. ACKNOWLEDGEMENTS
607
Cell culture reagents were provided by the Cell and Viral Vector Core Laboratory,
608
supported by the Comprehensive Cancer Center of Wake Forest University NCI CCSG
609
P30CA012197 grant. Flow cytometry was performed in the Flow Cytometry Core Laboratory,
610
also supported by the Comprehensive Cancer Center of Wake Forest University NCI CCSG
611
P30CA012197 grant. R.L.T. was supported by the Training Program in Immunology and
612
Pathogenesis 5 T32 AI007401 from the National Institutes of Health. This research was
613
supported by Public Health Service grant R01 CA127621 (to D.A.O) from the National Cancer
614
Institute and Deutsche Forschungsgemeinschaft grant Do 343/7-1 (to T.D.)
615
The authors wish to acknowledge the helpful discussion of members of the Parks, Lyles
616
and Barton laboratories of Wake Forest University. We thank Guangchao Sui for kindly
617
providing a p53-expression construct. The content of this report is solely the responsibility of the
618
authors and does not necessarily represent the official views of the respective funding agencies.
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42 FIGURE LEGENDS 904
FIGURE 1. Click-iT EdU labeling protocol and DNA profile analysis of EdU-labeled cells.
905
(A) HeLa cells were labeled with Click-iT EdU for six 4-h periods (indicated by arrows) over the
906
course of 24 h prior to infection. At time 0 h, EdU-labeled cultures were infected with the E1B-
907
55K/E4orf3 double-mutant virus at an MOI of 10 and processed for immunofluorescence at 72
908
hpi or were collected for flow cytometric analysis. Shading indicates the phase of the cell cycle
909
the EdU-labeled cells are expected to have reached at infection (S phase, gray; G2/M, black; G1,
910
white). (B) Samples of cells labeled with Click-iT EdU were collected at the time of infection (0
911
h) and stained with propidium iodide for DNA content and for EdU as described in the Materials
912
and Methods. The shaded density profile represents the DNA profile for the entire population of
913
cells. The unshaded density profile shows the DNA profile of EdU-positive cells.
914
FIGURE 2. Early G1 cells give rise to mitotic-like nuclei after infection with the E1B-
915
55K/E4orf3 double-mutant virus. Synchronously dividing HeLa cells were obtained by mitotic
916
shake and hydroxyurea selection as described in the Materials and Methods. Synchronously
917
dividing cells were either harvested for DNA profile analysis or infected at the indicated time
918
after entering S phase with the E1B-55K/E4orf3 double-mutant virus at an MOI of 10. (A) Cell
919
cycle distribution by DNA profile analysis. (B) At 72 hpi, cells were stained for DNA with DAPI
920
and evaluated by fluorescence microscopy to determine the frequency of cells with condensed
921
DNA. The stage of the cell cycle at the time of infection is indicated below. A representative
922
experiment is shown of three that were performed with overlapping times of infection after
923
entering S phase.
924
FIGURE 3. HeLa cells infected with the E1B-55K/E4orf3 double-mutant virus show
43 925
evidence of mitotic distress. HeLa cells were mock-infected or infected at an MOI of 10 with
926
the indicated viruses and stained at 72 h post infection with DAPI to visualize DNA (blue),
927
antibodies to phospho-H3 as a marker of early mitosis (red), and antibodies to β-tubulin to
928
visualize mitotic spindles (green). (A) Representative images of (a-b) mock, (c) wild-type, and
929
(d-e) E1B-55K/E4orf3-mutant viral infections are shown. (B) The frequency of cells containing
930
mitotic-like condensed DNA, staining for phospho-H3 and asymmetrically stained mitotic
931
spindles was quantified for at least 500 cells for each viral infection. A representative experiment
932
of three is shown. Error bars indicate the 95% exact binomial confidence interval for the
933
representative experiment. (C) HeLa cells were infected with the E1B 55K/E4orf3 double-
934
mutant virus at an MOI of 10. Cells were stained for DNA at the indicated times post infection
935
and the frequency of mitotic-like cells was determined for approximately 500 cells at each time
936
point. The broken symbols for times beyond 72 hpi indicate imprecision due to the probable loss
937
of cells because of death or detachment. The experiment shown is representative of three
938
independent experiments with similar outcomes. Error bars indicate the 95% exact binomial
939
confidence interval for the representative experiment.
940
FIGURE 4. Colcemid traps cells infected with the E1B-55K-mutant virus in a mitotic-like
941
state. HeLa cells were infected at an MOI of 10 with the indicated viruses and treated with
942
colcemid for 12 h at the indicated times post-infection before being fixed and stained with DAPI
943
to visualize DNA. The frequency of mitotic-like cells was determined for approximately 500
944
cells for each virus at each time point.
945
FIGURE 5. The ability to inhibit entry into a mitotic-like state maps to the p53-binding
946
ability of the E1B-55K protein. (A) HeLa cells were infected at an MOI of 10 with the
947
indicated E1B 55K-null viruses and treated with colcemid or vehicle control 12 h prior to
44 948
staining with DAPI at 72 hpi to visualize DNA. The frequency of mitotic-like cells was
949
determined by fluorescence microscopy. A representative experiment of three independent
950
experiments is shown. For each of the E1B-55K-null viruses, colcemid significantly increased
951
the fraction of mitotic-like cells (p-value < 10-6 by Fisher’s exact test). Error bars indicate the
952
95% exact binomial confidence interval for the representative experiment. (B) HeLa cells were
953
infected at an MOI of 10 with the indicated viruses bearing point mutations in the E1B-55K gene
954
(described in Table 2) and treated with colcemid 12 h prior to staining at 72 hpi with DAPI to
955
visualize DNA. The frequency of mitotic-like cells is shown for each mutant infection. The
956
proportion of mitotic-like nuclei was non-randomly distributed among the 11 virus-infected
957
samples exclusive of the E1B-55K-null virus H5pm4149 (p < 0.0001, Chi-squared test). The
958
Chi-squared test was repeated after systematically excluding each sample. The p-value was non-
959
significant (p = 0.56) only when the virus H5pm4109 was excluded from the analysis. This
960
analysis was again repeated by excluding individual samples in the collection that also excluded
961
H5pm4109. The Chi-squared test reported non-significant p-values for each subset lacking
962
H5pm4109 and one other sample. This indicates that among the non-null viruses, only
963
H5pm4109-infected cells exhibited a significant change in the number of condensed nuclei in the
964
presence of colcemid. Results from a representative experiment of three independent
965
experiments with similar outcomes are shown. (C) p53-null H1299 cells were transfected with a
966
plasmid to express p53 24 h before being mock-infected or infected at an MOI of 10 with either
967
the E1B-55K single-mutant or the E1B-55K/E4orf3 double-mutant virus. The cells were either
968
left untreated or treated with 0.2 μg per ml of colcemid 12 h prior to immunostaining for p53
969
and visualizing DNA with DAPI staining at 72 hpi. Cells were classified as either p53-positive or
970
negative and the frequency of mitotic-like cells was determined. As noted in the text, condensed
45 971
nuclei in mock-infected cells resembled pycnotic nuclei of apoptotic cells. The number of p53-
972
positive virus-infected cells with condensed DNA was increased significantly over p53-negative
973
cells (p = 2×10-6 by Fisher’s exact test).
974
FIGURE 6. Cyclin B1 levels are elevated during adenoviral infections. HeLa cells were
975
mock-infected or infected with the indicated viruses at an MOI of 10. Cellular lysates were
976
collected in the presence of protease and phosphatase inhibitors at 72 hpi. Material from identical
977
numbers of infected cells were separated by SDS-PAGE, transferred to a nitrocellulose
978
membrane and immunoblotted for (A) cyclin B1 and (B) β-actin. An overexposed β-actin blot is
979
presented here to permit visualization of the weaker signals. Non-saturated exposures were used
980
for quantitative analyses. The position of a cyclin B1-related product of 35 kDa is indicated by
981
the arrowhead. (C) The optical density of the signal for the intact cyclin B1 products was
982
quantified, normalized to β-actin, and then normalized to the value measured from mock-infected
983
cells in three independent experiments. The mean and standard deviation are plotted on a log-
984
scale. Application of the t-test to log-transformed values shows that levels of cyclin B1 in were
985
significantly greater than mock-infected cells in dl309- and dl1520-infected cells (p80%) cells expressing both
1015
fusion protein and E4orf3 as indicated in panels a and b.
1016
47
TABLES TABLE 1. Edu-positive and mitotic-like double-mutant virus-infected cells EdU-positive cells (%)b
Designationc
Mitotic-like Edu-positive cells (%)
a
Labeling Period
G1
S
G2/M
-2 to +2
27
52
21
mid-S
25.6
-6 to -2
6
15
79
G2/M
69.6
-10 to -6
35
7
58
M, early-G1
50.0
-14 to -10
68
5
27
mid G1
20.0
-18 to -14
61
25
14
late G1
0.0
-22 to -18
56
25
20
G1, early-S
5.8
a
Hours exposed to EdU before being infected at an MOI of 10 at 0 h.
b
Percentage of EdU-positive cells with DNA content characteristic of the indicated stage of the
cell cycle. c
Predominant stage of the cell cycle for EdU-positive cells at 0 h.
48 TABLE 2. E1B-55K mutant viruses and key characteristics Virus
E1B-55K mutation
Likely defect
Reference
dl110
deletion, frame shift
null
(42)
dl1520
stop codon, deletion
null
(39)
dl338
deletion
null
(41)
4149
four stop codons
null for all 55K-related
(104)
4100
none
none
(43)
4108
T255A
E4orf6-binding
(Identical mutation to virus described in 60)
4109
H260A
p53-binding, E4orf6binding
(Identical mutation to virus described in 60)
4127
C454S/C456S
Mre11 binding
(105)
4174
S490A/S491A/T495A
Non-phospho C-term
(10, 106, 107)
4185
P70T/S73A
Unknown (P70T/S73A)
(108)
4197
E472A
Daax interaction 1
(55)
4198
K185A/K187A
Daax interaction 2
(55)
4216
GVVI233-236AAAS
SUMO interaction
Not published
4217
V339A/V341A/I342S
SUMO interaction
Not published
4227
S490D/S491D/T494D
Phosphomimetic C-term
(57, 107)
1
Adenovirus replaces mitotic checkpoint controls
Roberta L. Turner,a Peter Groitl,b Thomas Dobner,b David A. Ornellesa#
Department of Microbiology and Immunology, Wake Forest School of Medicine, WinstonSalem, North Carolina, USAa; Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germanyb
# Address correspondence to: David A. Ornelles, [email protected]
Running Head: Adenovirus replaces mitotic checkpoints Abstract word count: 197 Manuscript word count: 7892
2 ABSTRACT 8 9
Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins including the E1B-55K and
10
E4orf3 proteins inhibit signaling in response to DNA damage. A fraction of cells infected with
11
an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a
12
mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this
13
state at late times of infection. This arrested state, which displays hallmarks of mitotic
14
catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However,
15
E1B-55K-mutant virus-infected cells became trapped in a mitotic-like state in the presence of the
16
microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or
17
facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The
18
E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with
19
the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by
20
possibly mislocalizing and functionally inactivating cyclin B1. When expressed in non-infected
21
cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1
22
variant. IMPORTANCE
23
Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed
24
to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein
25
prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to
26
depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The
27
adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular
28
distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis,
3 29
these adenoviral proteins act to prevent the infected cell from arresting in a mitotic-like state. INTRODUCTION
30
Adenoviral infection and the ensuing replication of the viral double-stranded DNA
31
genome activates the host DNA-damage response (1, 2). Early adenoviral proteins collaborate to
32
dampen this host response (reviewed in 3). The initial phase of the DNA-damage response
33
proceeds through a phosphorylation cascade, while subsequent recruitment of effector proteins
34
also depends on the conjugation of ubiquitin and the related small ubiquitin like modifier SUMO
35
(4). Signals initiated by the three apical kinases or DNA-dependent protein kinase (DNA-PK)
36
(5), ataxia telangiectasia mutated protein (ATM) (6), and ATM- and Rad3-related protein (ATR)
37
(7) trigger downstream consequences of DNA-damage such as DNA repair, cell cycle arrest, and
38
cell death. The tumor suppressor protein p53 is centrally positioned in the cellular response to
39
DNA-damage. Numerous branches of the DNA-damage response are controlled by p53
40
including cell cycle arrest, cell death, senescence, autophagy, and cell proliferation (8). Not
41
surprisingly, viruses that elicit a robust DNA-damage response inevitably target p53. For
42
adenovirus, the transcriptional activity of p53 is suppressed by the E1B-55K protein (9-11), the
43
stability of p53 is decreased by a ubiquitin protein-ligase formed by the E1B-55K and E4orf6
44
protein (12-14) and the expression of p53-responsive genes is epigenetically dampened by the
45
E4orf3 protein (15).
46
Cell cycle arrest mediated by p53 following DNA-damage typically occurs at the G1/S
47
border (16). However p53 also inhibits cell cycle progression immediately before mitosis. p53
48
can prevent entry into mitosis by inhibiting a kinesin involved in the arrangement of condensed
49
chromosomes (17). Polo-like kinase 1 (Plk1) promotes the transition from G2 into mitosis. The
50
inhibition of Plk1 uncovers p53-dependent outcomes in response to mitotic stress. In p53-
4 51
deficient cells, Plk1 inhibitors and microtubule poisons elicit mitotic catastrophe and greater
52
DNA-damage than in p53-proficient cells (18). This may reflect the absence of p53-dependent
53
apoptosis that would normally eliminate cells arrested in mitosis. It has been suggested that p53-
54
dependent cell-cycle-arrest at the G2/M border is the key factor in determining whether a cell
55
undergoes mitotic catastrophe or apoptosis (19).
56
Although progression through the cell cycle can be stopped at many stages, the intricately
57
orchestrated process of mitosis proceeds once the antephase checkpoint has been cleared or
58
bypassed (20), despite the persistence of damaged DNA (21). Mitosis is regulated by the
59
appropriate localization of cellular proteins and their timely degradation by the anaphase-
60
promoting complex or cyclosome (APC/C). During the G2 phase of the cell cycle, levels of
61
cyclin B1 rise, which associates with Cdk1 to form the major mitotic kinase (22). Entry into
62
mitosis begins with the activating phosphorylation of the Cdc25C phosphatase and components
63
of the APC/C as well as the inactivating phosphorylation of the Wee1 and Myt1 kinase by polo-
64
like kinases (23). The cyclin B1-Cdk1 complex is believed to shuttle in and out of the nucleus,
65
with hyperphosphorylation of cyclin B1 inhibiting nuclear export of the complex leading to an
66
intranuclear increase in cyclin B1-Cdk1 (24, 25). Within the nucleus, this kinase directs mitotic
67
progression by phosphorylating numerous targets (26) such as the nuclear lamins in order
68
promote nuclear envelope breakdown (27) and condensin II to initiate condensation of the
69
chromosomes (28). Exit from mitosis requires the degradation of proteins ubiquitinated by the
70
APC/C (29). Key mitotic targets of the APC/C are cyclin B1, securin, and Plk1. With the
71
degradation of cyclin B1 and securin, separase is free to cleave cohesin from sister chromatids,
72
leading to the precipitous separation of chromatids and progression out of mitosis (30).
73
Cell death by mitotic catastrophe was initially described as a caspase-dependent,
5 74
apoptotic death triggered by aberrant mitosis and the persistence of active mitotic kinases (31). It
75
has been proposed that mitotic catastrophe be considered an oncosuppressive mechanism leading
76
to cell death resulting from the disorder of mitotic machinery that is typified by a period of
77
aberrant mitotic arrest (32). Additional hallmarks of mitotic catastrophe include the formation of
78
multipolar spindles and the appearance of a cleaved form of cyclin B1 that is unable to be
79
degraded and is therefore presumed to sustain MPF activity (33, 34). Aberrant mitotic arrest
80
requires that cells enter mitosis but halt prior to anaphase. Examples of viruses that arrest cells in
81
mitosis and can elicit mitotic catastrophe include adenovirus, adenovirus-associated virus (AAV)
82
and chicken anemia virus. Expression of the adenovirus E4orf4 protein to high levels in H1299
83
cells lead to the accumulation of cells with 4N and greater DNA content followed by cell death
84
characterized as mitotic catastrophe (35). Exposure of p53-deficient osteosarcoma cells to UV-
85
inactivated AAV leads to centriole overduplication and mitotic arrest (19). Another example of
86
mitotic catastrophe occurs in response to apoptin, a viral protein from chicken anemia virus.
87
Apoptin directly inhibits the APC/C, thereby preventing exit from mitosis, resulting in mitotic
88
arrest and subsequent apoptosis (36). This arrest was found to be independent of p53 and resulted
89
from inhibition of the metaphase-to-anaphase transition (33).
90
We show here that the E1B-55K and E4orf3 genes are sufficient to prevent mitotic arrest
91
in the adenovirus-infected cell. The E1B-55K protein circumvents entry into mitosis in a p53-
92
dependent manner. The E4orf3 protein circumvents mitotic arrest by facilitating exit from
93
mitosis, perhaps by mislocalizing cyclin B1. These early adenoviral proteins may prevent an
94
untimely death that would occur in response to inappropriate mitotic arrest. MATERIALS AND METHODS
95
Chemicals. Hydroxyurea (HU) from Sigma/Aldrich (St. Louis, MO) was used at a
6 96
concentration of 2 mM from a stock solution of 1 M in water. KaryoMAX Colcemid from
97
Gibco/Invitrogen (Gaithersburg, MD) was used at a concentration of 0.2 μg per mL from a stock
98
solution of 10 μg per mL in HBSS.
99
Cells. All cell culture media, supplements, and sera were obtained from Invitrogen
100
(Gaithersburg, MD) or Lonza (Hopkinton, MA) through the Tissue Culture and Virus Vector
101
Core Laboratory of the Comprehensive Cancer Center of Wake Forest University. HeLa cells
102
were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10%
103
newborn calf serum. H1299 cells were maintained in DMEM supplemented with 10% fetal
104
bovine serum. Both cell lines were cultured in a 5% CO2 atmosphere at 37oC by passaging twice
105
weekly at a 1:10 dilution. For all experiments, cells were plated at a density of 5×104 cells per
106
mL. For high-resolution immunofluorescence microscopy, cells were grown on nitric acid-
107
cleaned, sterilized glass coverslips in 6-well plates or 35-mm dishes. For cell cycle profile
108
analysis or protein lysate collection, cells were grown in 60-mm dishes.
109
Viruses. The phenotypically wild-type virus in this study contains several deletions and
110
a substitution within the E3B region. This virus, dl309, displays wild-type characteristics in
111
cultured cells (37). The virus referred to as the double-mutant virus is 3112, a previously
112
described (38) viral recombinant of the dl1520 virus that has a 827 bp deletion in the E1B-55K
113
open reading frame (39) and the dl341 virus that contains an E4orf3 deletion (40). Other E1B-
114
55K deletion mutants include dl338, which bears a 524 bp deletion (41) and dl110, which
115
contains a 472 bp deletion (42). The parental virus for the E1B-55K point mutation mutants
116
listed in Table 2 is H5pg4100, which contains a deletion in the E3 region at nucleotides 28593–
117
30471 and has an added endonuclease restriction site at nucleotide 30955 (BstBI). The
118
adenovirus mutants described in Table 2 were constructed by the method described in (43).
7 119
Briefly, point mutations were introduced into the E1B-55K gene in a shuttle plasmid by site-
120
directed mutagenesis. The appropriate fragment was inserted into the parental plasmid
121
pH5pg4100 and the viral genomes were released from the recombinant plasmids by digestion
122
with PacI. Mutant viruses recovered after transfection of the linear DNA into 293 cells. All
123
viruses were grown in 293 cells and concentrated virus stocks prepared by sequential
124
centrifugation through CsCl gradients as described previously (1).
125
Antibodies. Primary antibodies included a monoclonal mouse antibody against ß-tubulin
126
(Clone TUB 2.1, T4026) from Sigma Aldrich (St. Louis, MO) used at a 1:500 dilution, a
127
monoclonal pantropic mouse antibody against p53 (DO-1) obtained from CalBiochem
128
(Darmstadt, Germany) used at a 1:100 dilution, and a polyclonal rabbit antibody against
129
phospho-histone H3 (Thr11, #9849) from Cell Signaling (Danvers, MA) used at a 1:100 dilution
130
for immunofluorescence. Adenovirus-specific antibodies included the mouse monoclonal
131
antibody Rsa#3 (44) for the E4orf6/7 protein and the rat monoclonal antibody 6A11 (45) used as
132
hybridoma culture supernatant fluid diluted 1:5. Five antibody preparations for cyclin B1 were
133
used for this study. The mouse monoclonal antibody against cyclin B1 (#CC03) was obtained
134
from Oncogene/CalBiochem (Cambridge, MA) and used at a 1:250 dilution for microscopy and
135
1:1000 dilution for western blotting. The remaining cyclin B1 antibodies were obtained from
136
EMD Millipore (Billerica, MA) and included the mouse monoclonal antibody specific for the C-
137
terminus of human cyclin B1 (clone Y106) used at a 1:100 dilution for immunofluorescence and
138
a 1:10,000 dilution in 5% milk for western blotting, a mixture of mouse monoclonal IgGs
139
specific for human cyclin B1 (catalog # 05-373) used at 10 μg per mL for immunofluorescence
140
and 0.33 μg per mL for western blotting, a monoclonal antibody raised against hamster cyclin B1
141
(catalog # MAB3684) used at a 1:100 dilution for immunofluorescence and a 1:5000 dilution in
8 142
5% milk for western blotting and a rabbit monoclonal antibody specific for phosphorylated
143
serine 126 in cyclin B1 (catalog # MABE490) used at a 1:100 dilution for immunofluorescence
144
and a 1:3000 dilution for western blotting. Secondary antibodies used for immunofluorescence
145
microscopy were anti-mouse or anti-rabbit whole IgG conjugated to Alexa Fluor 488 (AF488) or
146
Alexa Fluor 568 (AF568) from Invitrogen used at 2 μg per mL. The secondary antibody used for
147
western blot analysis was an anti-mouse or anti-rabbit whole IgG raised in goats conjugated to
148
horseradish peroxidase from Jackson ImmunoResearch Laboratories (West Grove, PA) and used
149
at a concentration of 0.1 μg per mL.
150
Plasmids and PEI transfection. A plasmid expressing the wild-type human TP53 gene
151
under control of the CMV immediate early promoter and enhancer was generously provided by
152
Guangchao Sui (Wake Forest School of Medicine). Plasmids expressing human cyclin B1 fused
153
to the yellow fluorescent protein Venus (pVenus-N1 Cyclin B1, Addgene plasmid #26062) and
154
the degradation-resistant form of Cylin B1 (pVenus Cyclin B1 R42A, Addgene plasmid #39873)
155
were the gift of Jonathon Pines (Gurdon Institute, Cambridge, England). Cells were seeded on
156
glass coverslips in a 6-well culture dish. For each well, 1 μg of DNA was used in a volume of 0.2
157
mL serum-free media with poly(ethylenimine) (PEI) at a 1:5 dilution from a 7.5 mM stock
158
dissolved in deionized water with gentle heating. Transfections were carried out in a 5% CO2
159
atmosphere at 37°C with gentle rocking and regular rotation for 8 h before being replacing the
160
DNA and PEI mixture with growth medium.
161
Indirect immunofluorescence. Cells were washed twice with phosphate-buffered saline
162
(PBS), fixed for 30 min with 2% paraformaldehyde, and permeabilized for 5 min with 0.2%
163
Triton X-100 in PBS at room temperature. All subsequent washes were performed with Tris-
164
buffered saline with BSA, glycine and Tween-20 (TBS-BGT: 0.137 M NaCl, 0.003 M KCl,
9 165
0.025 M Tris-Cl [pH 8.0], 0.0015 M MgCl2, 0.5% bovine serum albumin, 0.1% glycine, 0.05%
166
Tween 20, and 0.02% sodium azide). Antibodies used for immunofluorescence were diluted in
167
TBS-BGT supplemented with 10% normal goat serum (Invitrogen). Samples were stained for 90
168
min with primary antibody and for 30 min with secondary antibody with multiple washes with
169
TBS-BGT between. Samples were mounted with ProLong Gold mounting media (Invitrogen)
170
containing 4′,6-diamidino-2-phenylindole (DAPI).
171
Microscopy. Micrographs were obtained by standard epifluorescence microscopy with a
172
Nikon TE300 inverted microscope or by confocal laser scanning microscopy with a Nikon TiE
173
inverted microscope fitted with a Nikon C1si system. A 100×/1.4 NA magnification oil-
174
immersion objective was used for all micrographs. Monochromatic images were acquired on the
175
Nikon TE300 microscope with a Retiga EX 1350 digital camera (QImaging Corp., Burnaby,
176
British Columbia, Canada). Confocal images were acquired as a series of 5 sections at 0.2 um
177
intervals in the center of the nucleus using sequential excitation for each of the three
178
fluorochromes. The confocal images are presented as a maximum intensity projection to reduce
179
the three-dimensional information to two-dimensions. Merged color images were prepared by
180
assigning either red, green or blue to the appropriate fluorochrome. For single channel
181
fluorescent images, a pseudocolor scheme was assigned in order to mimic the visual perception
182
with increased saturation at increased fluorescent intensity. Monochromatic images were
183
assigned colors with the open-source program ImageJ (version 1.46). All figures were assembled
184
with the open-source vector graphics editor Inkscape (version 0.48) and raster graphics editor
185
GIMP (version 2.8.4.)
186 187
Flow cytometry. Cells were harvested by trypsinization. EdU-labeled cells were labeled as indicated below and resuspended in FACS buffer (1% BSA in PBS) with propidium iodide
10 188
provided by the Click-iT EdU kit. All other samples were washed twice with PBS and
189
resuspended in 100 μL PBS. Cells were transferred dropwise into 2.5 mL of 70% ethanol with
190
constant mixing. Samples were stored at -20oC for at least 12 h. Ethanol was then removed and
191
the samples resuspended at a density of 106 cells per mL in PI solution diluted in water from a
192
10X stock (1 M NaCl, 0.36 M sodium citrate, 500 μg per mL propidium iodide, 6% NP-40) with
193
0.1 mg per mL RNase A diluted from a stock solution of 10 mg per mL in 0.01 M sodium acetate
194
with 0.1 M Tris-Cl, pH 7.4. Samples were incubated with PI solution for 30 min in the dark at
195
37oC and then transferred to ice before flow cytometric analysis. A Becton Dickinson FACS
196
Calibur instrument was used to acquire the propidium iodide signal in linear mode. Gating on the
197
peak width and peak area was used to select single cells for DNA profile and cell cycle analysis.
198
Western blotting. Cells were washed in PBS supplemented with protease and
199
phosphatase inhibitors (2 mM EDTA, 1 mM NaF, 1 mM sodium pyrophosphate, 1 mM
200
phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 2 μM leupeptin), harvested by scraping, and
201
suspended in a volume of one-tenth concentrated PBS sufficient to dilute the cells to a density of
202
2×107 cells per mL An equal volume of 2X sodium dodecyl sulfate (SDS) protein sample buffer
203
(4% SDS, 0.135 M Tris [pH 6.8], 20% glycerol, 0.02% bromophenol blue, and 6% β-
204
mercaptoethanol) was added to make the final concentration 107 cells per mL. The cell lysate
205
was heated for 5 min at 95oC and subjected to 3 pulses of sonication lasting 20 sec each. The
206
lysates were separated by SDS-polyacrylamide gel electrophoresis through 10% acrylamide gels.
207
The proteins were then electrophoretically transferred to nitrocellulose (Whatman/GE
208
Healthcare) overnight at 4oC. The nitrocellulose was blocked in TBS-BGT containing 5% nonfat
209
dry milk and sodium azide, stained with primary antibody diluted in TBS-BGT with sodium
210
azide overnight at 4oC, and stained with secondary antibody diluted in TBS-BGT without sodium
11 211
azide for 1 hr at room temperature. The stained protein was visualized by a mixture of
212
SuperSignal West Pico and SuperSignal West Femto chemiluminescence substrate from
213
Pierce/ThermoScientific (Rockford, IL) and X-ray film. The density of the specific signal
214
recorded by the X-ray film was quantified by scanning non-saturated exposures at 16-bit
215
resolution and measuring the optical density with the tools available in ImageJ.
216
Click-iT EdU. Cells were pulse-labeled with an appropriate amount of EdU from a 10
217
mM stock in DMSO diluted in a small volume of warm growth medium to make a final
218
concentration of 100 μM EdU. At the end of the labeling period, the wells were washed and
219
replaced with pre-warmed growth medium. At time of harvest, samples for DNA profile analysis
220
were harvested with trypsin while samples on glass coverslips were fixed, permeabilized, and
221
processed for immunofluorescence in a humidifying chamber. The samples were fixed with 4%
222
paraformaldehyde in PBS for 15 min, washed with FACS buffer (1% BSA in PBS), and
223
permeabilized with 0.5% Triton X-100 for 20 min at room temperature. The Click-iT reaction
224
cocktail was prepared by mixing Click-iT reaction buffer, CuSO4, fluorescently labeled azide,
225
and reaction buffer additive as indicated by the manufacturer. Samples were washed with FACS
226
buffer and allowed to incubate with the Click-iT reaction cocktail in a humidifying chamber at
227
room temperature for 30 min in the dark. The samples were washed and those on coverslips were
228
mounted on slides with Prolong Gold supplemented with DAPI. Samples for DNA profile
229
analysis were pelleted and resuspended in 500 μL of FACS buffer with 0.2 mg per mL RNase A
230
(from a 20 mg per mL) and 4 μg per mL PI (from a 1 mg per mL stock solution in water).
231
Cell synchronization. Mitotic cells were mechanically harvested from subconfluent cells
232
grown in 75 cm2 flasks and pelleted gently. The mitosis-enriched pellet was resuspended at a
233
concentration of 2x104 cells per mL in 2 mM HU in growth medium and plated in 60 mm dishes
12 234
for DNA profile analysis or on acid-treated glass coverslips in 35 mm dishes for
235
immunofluorescence. After 1 hour, plates were swirled to dislodge any dead S-phase cells and
236
the media replaced with a fresh solution of 2 mM HU in growth medium. Cells were held in HU
237
for 16 h to allow cells to cycle to the G1/S border. All samples were released from HU by
238
washing and replacing with growth medium. At various times post infection, cells were collected
239
for DNA profile analysis in order to determine cell cycle distribution in the sample. RESULTS
240
Cells infected during early G1 give rise to cells trapped in a mitosis-like state. A
241
subset of cells infected with an adenovirus deleted of the E1B-55K and E4orf3 genes develop
242
highly condensed chromatin resembling that of a mitotic cell (46). Because typically fewer than
243
10% of the infected cells develop the highly condensed chromatin, it seems likely that an
244
underlying process or condition limits the number of infected cells that condensed their
245
chromatin and resemble a mitotic cell. Potential processes include the death and loss of cells
246
following mitotic catastrophe, a small probability of entering mitosis, or the existence of a
247
limited subset of cells that are competent to enter mitosis. Since the outcome of an infection with
248
the E1B-55K-mutant virus is strongly determined by the stage of the cell cycle at the time of
249
infection (38, 47, 48), we performed two experiments to determine if entry into a mitotic-like
250
state was affected by the stage of the cell cycle at infection. Asynchronously dividing HeLa cells
251
were exposed to the thymidine analog 5-ethynyl-2´-deoxyuridine (EdU) for a 4-h interval in
252
order to identify cells that passed through S phase during this period of time. After returning the
253
cells to EdU-free medium, the cells were maintained for various intervals of time before being
254
infected. At the time of infection, a portion of the cells was collected and EdU-positive cells
255
were identified by the copper(I)-catalyzed reaction between a fluorescent azide and the alkyne in
13 256
EdU. This sample was analyzed by flow cytometry for DNA and EdU in order to determine the
257
stage of the cell cycle that EdU-positive cells had reached at infection. The labeling scheme and
258
associated flow cytometric analyses are shown in Fig. 1. After 72 h, another fraction of the
259
infected cells was stained for EdU and DNA and the number of mitotic-like nuclei were
260
enumerated by fluorescence microscopy. The results of scoring approximately 600 nuclei at each
261
time point show that cells with condensed DNA were seen most frequently among cells infected
262
during G2/M and early G1 (Table 1).
263
Although the EdU-labeling method permits analysis of unperturbed dividing cells, the
264
temporal resolution afforded by a 4 h labeling period was limited. For better resolution,
265
synchronously dividing HeLa cells were prepared as previously described (47, 49) and infected
266
with the E1B-55K/E4orf3 double-mutant virus at hourly intervals from 6 to 14 h following entry
267
into S-phase. A portion of the synchronized cells was collected and analyzed for DNA content by
268
flow cytometry in order to determine the stage of the cell cycle at the time of infection (Fig. 2A).
269
After 72 h of infection, the cells were fixed and the frequency of cells with mitotic-like nuclei
270
was determined (Fig. 2B). These results agree with the findings obtained with asynchronously
271
dividing cells (see Fig. 1) and indicate that cells infected with the E1B-55K/E4orf3 double-
272
mutant virus during a 4 h window in early G1 gave rise to the subset of cells with highly
273
condensed chromatin. These results suggest that the adenoviral E1B-55K and E4orf3 proteins
274
prevent cells infected early in G1 from becoming trapped in a mitotic-like state at late times of
275
infection.
276
Cells infected with the E1B-55K/E4orf3 double-mutant virus show evidence of
277
mitotic distress. Approximately four percent of asynchronously dividing HeLa cells contain
278
condensed chromatin typical of the later stages of mitosis (Fig. 3A, mock and Fig. 3B). The
14 279
mitotic spindle in these cells, visualized by staining for β-tubulin, occurs in a symmetrical
280
bipolar arrangement about the condensed chromatin in metaphase and anaphase cells (Fig. 3A
281
panel a). A fraction of the mitotic cells also contain highly phosphorylated histone H3 or
282
phospho-H3 (Fig. 3A panel b, and Fig. 3B). Phosphorylation of histone H3 initiates late in G2
283
phase, is completed in late prophase and is maintained through metaphase. Histone H3
284
phosphorylation precipitously decreases during anaphase as the cell exits mitosis (50). In
285
contrast to mock-infected cells, none of the cells infected with the wild-type or E4orf3-mutant
286
adenovirus and less than 0.5% of cells infected with the E1B-55K-mutant virus contained
287
condensed chromatin or phospho-H3 when evaluated at 72 hpi (Fig. 3B). This is consistent with
288
previous observations indicating that adenovirus-infected human epithelial cells cease
289
progression through the cell cycle (51). However, in these asynchronously infected cultures, a
290
significant number (>10%) of cells infected with the E1B-55K and E4orf3 double-mutant virus
291
contained chromatin characteristic of a mitotic cell (Fig. 3A, ΔE1B-55K/ΔE4orf3 and Fig. 3B).
292
Some of the double-mutant virus-infected cells also stained for phospho-H3, suggesting that
293
these cells progressed into mitosis. Although these cells superficially resembled mitotic cells,
294
many contained an asymmetric distribution of β-tubulin or even a multipolar spindle. The
295
abundance of aberrant mitotic spindles in these cells makes it seems likely that these cells will
296
fail to produce viable daughter cells. Expression of the small avian virus-derived protein apoptin
297
in human tumor cells has been reported to lead to a similar mixture of cells with apparently
298
normal, asymmetric, and multipolar spindles due to a block in the metaphase to anaphase
299
transition (33).
300 301
Cells infected with the double-mutant virus that appear to arrest in mitosis do so only at late times of infection (Fig. 3C). In some experiments, a transient increase in mitotic-like cells
15 302
occurred at 24 hpi. However, cells with highly condensed DNA do not begin to accumulate until
303
60 hpi. The number of cells apparently arrested in mitosis reached a maximum at 72 hpi. The
304
decrease in cells with mitotic-like nuclei after 72 hpi appears to be due to the death or selective
305
detachment of these cells from the substrate. Consequently, subsequent experiments evaluated
306
cells at 72 hpi. Taken together, these results suggest that the E1B-55K and E4orf3 proteins
307
prevent cells infected during early G1 from becoming trapped in a mitotic-like state at late times
308
of infection.
309
Adenoviral E1B-55K protein prevents entry into mitosis. Because significant
310
numbers of cells were observed to contain condensed DNA only after infection with the double-
311
mutant virus, it seems likely that the E1B-55K and E4orf3 proteins act independently to prevent
312
the infected cell from arresting in a mitotic-like state. Conceptually, these viral oncoproteins
313
could prevent the accumulation of mitotic-like cells by two non-exclusive mechanisms. First, the
314
viral proteins could prevent entry into mitosis. Second, the viral proteins could facilitate exit
315
from mitosis. Cherubini and associates observed that a fraction of E1B-55K-mutant virus-
316
infected cells accumulated highly condensed chromosomes after 12 h of exposure to colcemid
317
(52). By depolymerizing microtubules, colcemid prevents the completion of mitosis thus
318
trapping any cells that entered mitosis during the exposure to colcemid. Since mitotic-like cells
319
did not accumulate in colcemid-treated cells that were infected with the wild-type virus, we
320
reasoned that the E1B-55K protein might prevent the infected cell from entering mitosis. To test
321
this hypothesis, infected cells were treated with colcemid for 12 h prior to fixation and analysis
322
at various times after infection. The number of mitotic-like cells infected with the wild-type or
323
E4orf3-mutant virus did not increase, suggesting that cells infected with the wild-type or E4orf3-
324
mutant virus did not enter into mitosis (Fig. 4). As expected, cells with condensed DNA were
16 325
observed following infection with the double-mutant virus; the rate at which these mitotic-like
326
cells accumulated was the same in the presence (Fig. 4) or absence of colcemid (see Fig. 3C). In
327
contrast to the wild-type or E4orf3-mutant virus, colcemid trapped increasing numbers of
328
mitotic-like cells during the course of infection with the E1B-55K-mutant virus (Fig. 4). These
329
results, which recapitulate the findings reported by Cherubini (52), suggest that some E1B-55K-
330
mutant virus-infected cells enter and exit mitosis at late times of infection. This indicates that
331
another role for the E1B-55K protein is to prevent adenovirus-infected cells from entering into
332
mitosis.
333
E1B-55K prevents entry into mitosis through p53. The E1B-55K protein serves many
334
roles during an infection. The E1B-55K and E4orf6 proteins collaborate to form a novel SCF-
335
type E3 ubiquitin ligase with the scaffold protein Cul5, Elongins B and C, and the RING box
336
protein Rbx1 (12-14). This adenovirus-specific complex targets several cellular proteins for
337
degradation, many of which signal or respond to DNA-damage (see for example 53, 54-58).
338
Acting independently of E4orf6, the E1B-55K protein directs degradation of the cellular
339
transcription factor Daax, mislocalizes the DNA-damage responsive chromatin structure
340
regulator SPOC1, and can directly block p53-mediated transcription (11, 55, 59). In order to
341
identify the activity of the E1B-55K protein that prevents entry into mitosis, HeLa cells were
342
infected with the E1B-55K-mutant adenoviruses described in Table 2. At 60 hpi, the infected
343
cells were treated with colcemid to trap any cells entering mitosis and the cells with condensed
344
DNA were counted by fluorescence microscopy. As expected, cells infected with the three E1B-
345
55K-null adenoviruses as well as the virus bearing four nonsense mutations in the E1B-55K open
346
reading frames (H5pm4149) were trapped in a mitotic-like state by colcemid (Figs. 5A and 5B).
347
Among the viruses bearing missense mutations in the E1B-55K gene, only H5pm4109 was
17 348
associated with a significant increase in mitotic-like cells (Fig. 5B). This virus encodes an E1B-
349
55K protein that is unable to bind p53. All other E1B-55K variants analyzed in Fig. 5B appeared
350
to prevent entry into mitosis as well as the wild-type virus H5pg4100. Because over 20% of non-
351
infected cells were trapped in a mitotic-like state by this treatment (data not shown), the failure
352
to detect mitotic-like cells among virus-infected cells confirms that the cells were uniformly
353
infected. Because cells infected with the mutant virus H5pm4108 were not trapped in a mitotic-
354
like state by colcemid, we conclude that the ability to block entry into mitosis is independent of
355
the E1B-55K/E4orf6 protein complex since H5pm4108 expresses an E1B-55K protein that fails
356
to bind E4orf6 (see 60). These findings suggest that the key property of the E1B-55K protein
357
needed to prevent entry into mitosis is its ability to interact with p53.
358
In many HPV-transformed cells, expression of the integrated E6 gene of HPV directs the
359
continual degradation of p53 protein (61). Without the selective pressure to eliminate p53 gene
360
function, the TP53 gene in HeLa cells has remained intact (62). Wild-type p53 protein
361
accumulates to measurable levels in HeLa cells infected with adenovirus and to a high level in
362
cells infected with adenovirus mutants that fail to direct the degradation of p53. To examine the
363
possibility that p53 contributes to mitotic entry in the infected cell, p53-null H1299 cells were
364
infected with the E1B-55K/E4orf3 double-mutant virus and evaluated. In sharp contrast to HeLa
365
cells, no mitotic-like cells were observed among H1299 cells infected with this virus at any time
366
after infection (data not shown and see Fig. 5C).
367
To test directly a role for p53, H1299 cells were transfected with a p53-expression
368
plasmid and infected after 24 h. At 60 hpi, cells were exposed to colcemid or left untreated. At
369
72 hpi, cells were stained for p53 and DNA then evaluated by fluorescence microscopy.
370
Enforced expression of p53 is acutely toxic to H1299 cells (63). Consequently, transfection of
18 371
the p53-expression plasmid reduced the number of evaluable cells and induced apoptosis in the
372
mock-infected cells after three days (data not shown). It seems likely that the increased number
373
of p53-positive, mock-infected H1299 cells with condensed DNA was due to pyknosis or
374
chromatin condensation from apoptosis rather than mitosis (Fig. 5C, mock). Apoptosis is
375
inhibited in the adenovirus-infected cells because all of the viruses studied here express the anti-
376
apoptotic E1B-19K protein (64). Accordingly, in the absence of colcemid, neither pyknotic nor
377
mitotic-like nuclei were observed among E1B-55K-mutant virus-infected H1299 cells (Fig. 5C).
378
However, colcemid trapped a significant number of p53-positive H1299 cells in a mitotic-like
379
state. Similarly, mitotic-like nuclei were observed only in p53-positive H1299 cells infected with
380
the E1B-55K/E4orf3 double-mutant virus. As noted for HeLa cells, colcemid was not required to
381
trap the mitotic-like double-mutant virus-infected H1299 cells. At least for the HeLa and H1299
382
cell lines, these results are consistent with an unexpected role for p53; if the infected cells with
383
condensed nuclei are indeed mitotic, p53 appears to be necessary for the adenovirus-infected cell
384
to enter mitosis. Furthermore, the E1B-55K protein blocks this activity of p53.
385
E4orf3 targets cyclin B1. Colcemid is required to trap HeLa cells and p53-positive
386
H1299 cells infected with the E1B-55K-mutant virus in a mitotic-like state. By contrast, p53-
387
positive cells infected with the E1B-55K/E4orf3 double-mutant virus are trapped in a mitotic-
388
state without colcemid. If the absence of the E1B-55K protein allows infected p53-positive cells
389
to enter mitosis, this result suggests that the presence of E4orf3 protein may facilitate exit from
390
mitosis. Degradation of the major mitotic cyclin, cyclin B1 is a critical event that precipitates
391
exit from mitosis (65). The complete degradation of cyclin B1 is required for cells to proceed
392
with cytokinesis (66). Previous studies showed an increase in cyclin B1 in wild-type adenovirus-
393
infected WI-38 and A549 cells as well as an S-phase-dependent increase in cyclin B1 in E1B-
19 394
55K-mutant virus-infected cells (67). We therefore compared the nature and abundance of cyclin
395
B1 among HeLa cells infected with wild-type and mutant viruses by immunoblotting. The level
396
of cyclin B1 was indeed higher in virus-infected cells compared to mock-infected cells (Fig. 6)
397
although it seems unlikely that the modest increase in cyclin B1 level in double-mutant virus-
398
infected cells could force an apparent mitotic arrest. Adenovirus-infected cells also contained an
399
additional cyclin B1-related protein of slightly greater electrophoretic mobility than the form in
400
mock-infected cells (Fig. 6A). This product was recognized by four different cyclin B1-specific
401
antibody preparations. Both ~50-kDa forms of cyclin B1 appeared equally abundant when
402
queried by a phosphoserine-126-specific antibody (data not shown). The origin of the two forms
403
of protein of approximately 50-kDa remains unclear. A 35-kDa protein recognized by cyclin B1
404
antibodies was detected in lysates from cells infected with the E1B-55K/E4orf3 double-mutant
405
virus and to a lesser extent, with the E4orf3-mutant virus. This smaller form of cyclin B1 appears
406
to correspond to a cleavage product found during mitotic catastrophe termed cyclin B1Δ (34). It
407
was suggested that cyclin B1Δ acts as a dominant-negative inhibitor of cyclin B1 function and
408
sustains the mitotic block in cells that would otherwise exit mitosis (34). Both the elevated levels
409
of cyclin B1 and the presence of a dominant-negative inhibitor could retard E1B-55K/E4orf3
410
double-mutant virus-infected cells in mitosis. In addition, changes in the localization of cyclin
411
B1 in the infected cells point to another mechanism by which E4orf3 could facilitate exit from
412
mitosis.
413
Because E4orf3 disrupts cell signaling pathways by mislocalizing host proteins (68, 69)
414
we explored the possibility that E4orf3 mislocalizes cyclin B1 in the infected cell. The different
415
localizations of cyclin B1 among mock-infected cells were consistent with fluctuations in the
416
level and movement of cyclin B1 during cell cycle progression. Cells in G2 contained high levels
20 417
of cyclin B1 that was found in a diffuse or speckled pattern in the cytoplasm (Fig. 7A, panel a).
418
Early in mitosis, cells contained high levels of cyclin B1 that was largely coincident with
419
chromatin (Fig. 7A, panel b). Finally, a subset of cells with condensed chromatin was judge to be
420
in late mitosis because of the absence of cyclin B1 staining (Fig. 7A, panel c). A subset of cells
421
infected with the wild-type and E1B-55K-mutant virus contained nuclear cyclin B1; in many of
422
these cells, cyclin B1 was found in large aggregates throughout the nucleus (Fig. 7B panels a-d).
423
A fraction of cells infected with E4orf3-mutant viruses also contained nuclear cyclin B1. In
424
contrast to infected cells containing the E4orf3 protein, cyclin B1 was diffusely distributed in the
425
nucleus of cells infected with the E4orf3-mutant viruses (Fig. 7B, panels e-h). The distribution of
426
cyclin B1 in these infected cells more closely resembled the patterns observed in mitotic or G2
427
mock-infected cells. These results, which are quantified in Fig. 7C, show that the E4orf3 protein
428
alters the distribution of cyclin B1 in the cell nucleus during an adenoviral infection. E4orf3 may
429
functionally inactivate cyclin B1 in order to facilitate exit from mitosis.
430
E4orf3 overcomes metaphase arrest imposed by a non-degradable cyclin B1.
431
Degradation of cyclin B1 during mitosis requires key residues (R42xxL45xxI/V48xN50) in the
432
destruction box (70). Expression of cyclin B1 variants with mutations in the destruction box
433
force cells to accumulate in a mitotic-like state (66, 71). We expressed E4orf3 and the wild-type
434
or degradation-resistant cyclin B1 (CycB1 R42A) by transfection to determine if E4orf3 can
435
overcome the metaphase arrest imposed by CycB1 R42A (71). As expected, enforced expression
436
of wild-type cyclin B1 increased the fraction of cells with condensed DNA (Fig. 8A). Although
437
expression of the E4orf6/7 cDNA reduced the frequency of these mitotic-like cells, the
438
difference was not statistically significant (p=0.09). E4orf3 decreased the frequency of these
439
mitotic-like cells to statistically significant, although modestly reduced levels (7%, p=0.02).
21 440
However, the effect of E4orf3 was pronounced in cells expressing the degradation-resistant
441
R42A cyclin B1. More than half of the cells expressing the R42A variant contained condensed
442
DNA after two days. This number was not affected by E4orf6/7 (p=0.32). By contrast, E4orf3
443
significantly (p=0.01) and substantially reduced the fraction of mitotic-like cells expressing the
444
R42A variant (Fig. 8A). These results indicate that expression of E4orf3 overcomes the mitotic-
445
like state caused by elevated levels of cyclin B1 without forcing the degradation of cyclin B1.
446
Expression of E4orf3 by transfection did not promote the aggregation of cyclin B1 seen
447
in virus-infected cells (see Fig. 7C). We observed no differences in the localization of the cyclin
448
B1 fusion protein among cells expressing E4orf6/7 (Fig. 8B), E4orf3 (Fig. 8C) or no E4
449
construct (data not shown). Occasional aggregates of cyclin B1 were noted irrespective of the co-
450
transfected viral gene such as in Fig. 8B, panel a. However, cyclin B1 appeared to be largely
451
excluded from the nucleus in most of the cells expressing both E4orf3 and the cyclin B1 fusion
452
protein. Since so few productively transfected cells contained detectable levels of endogenous
453
cyclin B1, it was not possible to determine if E4orf3 affected the endogenous protein in a similar
454
manner. Because cells expressing E4orf3 and cyclin B1 R42A did not have significant levels of
455
the cyclin B1 in the nucleus after 24 h, 36 h, and 72 h of transfection (data not shown), it seems
456
likely that E4orf3 precludes entry of cyclin B1 into the nucleus or promotes nuclear export of
457
cyclin B1. DISCUSSION
458
Cells infected by the E1B-55K/E4orf3 double-mutant virus during early G1 are
459
predisposed to arrest in a mitotic-like state. The dependence of this phenomenon on the stage of
460
the cell cycle at the time of infection is unusual but not unexpected. We previously demonstrated
461
that cells infected during S-phase by the E1B-55K-deleted virus support a more productive
22 462
infection (47, 48) and are more rapidly killed than G1-infected cells (51, 72). The findings
463
reported here reinforce the notion that an infection initiated during G1 is restrictive for E1B-
464
55K-mutant adenoviruses.
465
The importance of the E1B-55K and E4orf3 proteins in preventing entry and arrest in
466
mitosis is counterintuitive given that these proteins disable DNA-damage checkpoints. In
467
particular, a dysfunctional G2 checkpoint can allow a cell to enter mitosis inappropriately (73).
468
The G2 checkpoint prevents mitotic entry by blocking activation of the mitotic kinase Cdk1,
469
which depends on activation of the Cdc25C phosphatase and inactivation of the Wee1 and Myt1
470
kinases. Cdc25C and Wee1 are regulated by the ATM (6) and ATR kinases (74). Agents that
471
disable the ATM and ATR pathways thus can force G2 cells to enter mitosis prematurely.
472
Because both E1B-55K and E4orf3 proteins inactivate ATM (75) and ATR (76), the ability of
473
these two adenoviral proteins to prevent cells from entering mitosis or arresting in mitosis is
474
surprising.
475
Insight into adenoviral control of mitotic entry was provided by Cherubini and associates
476
who showed that primary human cells infected with the E1B-55K-mutant virus dl1520
477
accumulated highly condensed chromosomes after 12 h of exposure to colcemid (52). Here we
478
show that additional adenoviruses bearing large deletions in the E1B-55K gene behaved
479
similarly (Fig. 5A). However, among ten E1B-55K-mutant viruses bearing missense mutations
480
(Table 2), all but H5pm4109 prevented cells from entering mitosis (Fig. 5B). We interpret these
481
observations to mean that many E1B-55K properties, including the ability to bind the E4orf6,
482
Daxx and Mre11 proteins, the ability to interact with SUMO-modified proteins, and C-terminal
483
phosphorylation are not critical for the E1B-55K protein to block to entry into mitosis. The
484
H5pm4109 E1B-55K protein contains a histidine in place of alanine at position 260. The
23 485
identical protein expressed by the related virus ONYX-053 is unable to bind both p53 and
486
E4orf6 (60). Because the T255A protein expressed by H5pm4108 fails to bind E4orf6 (60) but
487
prevents entry into mitosis, we conclude that the E1B-55K protein must be able to bind p53 in
488
order to prevent entry into mitosis. Surprisingly, neither the E1B-55K/Eorf3 double-mutant virus
489
nor the combination of colcemid and infection with the E1B-55K single-mutant virus forced the
490
p53-null H1299 cells into mitosis unless p53 was expressed by transfection (Fig. 5C). In addition
491
to the HeLa cells studied in this report, H460 cells, MCF10A cells and hTERT-immortalized
492
retinal pigmented epithelial cells also contain a wild-type p53 gene. A significant number of
493
these cells arrested in mitosis after infection with the double-mutant virus whereas the p53-null
494
H358 and PC3 cell lines failed to show a statistically significant increase in arrested cells after
495
infection (data not shown).
496
The requirement for p53 to promote mitotic entry in the adenovirus-infected cell is at
497
odds with the well-described ability of p53 to enforce cell cycle arrest. For example, p53
498
precludes entry into S-phase by imposing transcriptional and translational blocks to cell cycle
499
progression in response to serum-starvation (77) and prevents inappropriate entry into mitosis by
500
suppressing expression of the chromosome alignment protein Kif23 (17). Perhaps p53 is altered
501
in the adenovirus-infected cell and this altered form of p53 permits cells to slip past the G2
502
checkpoint despite DNA-damage or chromosomal abnormalities. There is ample precedence for
503
the corruption of normal cellular functions by adenovirus proteins. For example, the E1B-55K
504
proteins of different adenoviruses partner with the E4orf6 protein to reprogram ubiquitin-protein
505
ligases of the Skp1/Cul1/F-box (SCF) family (53, 54). SCF complexes orchestrate progression
506
through the cell cycle and activate checkpoint signaling (recently reviewed in 78). It may be
507
significant that the SCF complex associated with the nuclear interaction partner of ALK keeps
24 508
levels of cyclin B1 low and prevents early mitotic entry (79). Furthermore, the physical
509
interaction between the E1B-55K protein and p53 converts p53 from a transcriptional activator
510
to a potent repressor of transcription (11, 80).
511
The apparent need for an interaction between the E1B-55K protein and p53 in order to
512
prevent mitotic arrest may underlie observations suggesting that p53 enables the wild-type virus
513
to elicit greater cytopathic effects than the E1B-55K-mutant virus (81). It was later noted that
514
replication of the E1B-55K-null virus dl1520 was enhanced by expression of the gain-of-
515
function R248W p53 variant that could no longer bind DNA in a site-specific manner (82). It
516
would be of interest to determine if the interaction between the E1B-55K protein and the putative
517
form of p53 responsible for preventing entry into mitosis subverts the transcriptional activity of
518
p53 in a manner that phenocopies gain-of-function p53 variants. Some of these variants promote
519
progression into mitosis in response to genotoxic stress (83). Many gain-of-function mutations in
520
p53 map to the DNA-binding domain (84). Coincidentally, Tip60-dependent acetylation of p53
521
within the DNA-binding domain can determine how p53 governs cell fate in response to DNA-
522
damage (85). Recent evidence shows that Tip60 is targeted for degradation by the E1B-55K
523
protein (54). Although the altered function of p53 is seen irrespective of E1B-55K status, other
524
viral proteins may mimic the action of Tip60, either directly or through the action of redirected
525
cellular proteins.
526
It must be emphasized that the interaction between the E1B-55K protein and p53 cannot
527
simply ablate p53 function, otherwise p53-null cells infected with the E1B-55K/E4orf3 double-
528
mutant virus should have arrested in the mitotic-like state. If a modified form of p53 exists
529
during infection, perhaps the E1B-55K protein allows this form of p53 to reinforce the G2/M
530
checkpoint during the replicative stages of the adenoviral lifecycle.
25 531
The notion of a protein inhibiting the function of another while simultaneously preserving
532
some of the target’s function is not novel. The licensing factor Cdt1 is present during G1 and S
533
phase. During DNA synthesis Cdt1 is expelled from the nucleus, degraded, and inactivated to
534
prevent re-replication. Geminin was initially identified as a mammalian factor that contributed to
535
the inactivation of Cdt1 (86). However, it was later shown that Geminin binds Cdt1 to gain
536
nuclear localization (87) and preserve a portion of Cdt1 during late mitosis (88). Perhaps like
537
Geminin, the E1B-55K protein is able to inactivate most p53 function while retaining a low level
538
of p53 in order to strengthen the G2/M checkpoint.
539
The E1B-55K protein, through its interaction with p53 during an adenovirus infection,
540
acts to prevent entry into mitosis. Because sustained expression of E1B-55K alone does not
541
preclude cell division, this novel viral “checkpoint” must form only in the adenovirus-infected
542
cell. Adenovirus-infected cells that escape this viral G2 checkpoint and enter mitosis may be
543
prone to arrest because of other adenoviral proteins such as the E4orf4 protein. The E4orf4
544
protein inactivates APC/C by reprogramming the activity of protein phosphatase 2A, thereby
545
inducing G2/M arrest (89, 90). Interestingly, when expressed alone, E4orf4 promotes p53-
546
independent, caspase-independent cell death in tumor cells (91-93) while inhibiting apoptosis in
547
normal cells (94). Since prolonged arrest in a mitotic-like state or mitotic catastrophe is often
548
followed by apoptosis or senescence, it would be advantageous for adenovirus to express an
549
activity that facilitates exit from mitosis. Because colcemid fails to trap E4orf3 single-mutant
550
virus-infected cells in a mitotic-like state (Fig. 4), we conclude that E4orf3 does not prevent
551
entry into mitosis. However, because the E4orf3 gene is required to prevent the E1B-55K-mutant
552
virus from arresting in a mitotic-like state (Fig. 3) we suggest that E4orf3 provides an activity
553
that facilitates exit from mitosis.
26 554
Progression through mitosis beyond anaphase requires satisfaction of the spindle
555
assembly checkpoint and activation of the APC/C. Two coactivators of APC/C, Cdc20 and
556
Cdh1, dictate substrate specificity and activity. Cdc20 is sequestered by mitotic checkpoint
557
proteins while unattached kinetochores or perturbations in the mitotic spindle persist. APC/CCdc20
558
initiates anaphase by targeting the cohesins for degradation while APC/CCdh1 promotes the
559
irreversible exit from mitosis into G1 (reviewed in 78). A key target of both APC/CCdc20 and
560
APC/CCdh1 is the mitotic cyclin B1, whose loss leads to the rapid decline in Cdk1 activity (22).
561
Because cyclin B1 accumulates to high levels in adenovirus-infected cells (Fig. 6), E4orf3 does
562
not promote mitotic exit by simply removing cyclin B1. However, the E4orf3 protein may
563
functionally inactivate cyclin B1 by mislocalizing this protein within the infected cell (Fig. 7).
564
Cells infected with E4orf3-mutant viruses also contain a cyclin B-related protein (Fig. 6) similar
565
in size to a cleaved form of cyclin B1 that sustains the mitotic block (34). In the context of an
566
E1B-55K-deletion, E4orf3 may prevent accumulation of this inhibitory product.
567
E4orf3 inactivates many cellular proteins by altering their localization or directing them
568
to the aggresome (95, 96). For example, mislocalization of Nbs1 by the E4orf3 protein is
569
sufficient to prevent ATR activation, thereby crippling the DNA-damage response (76, 97). The
570
activity of cyclin B1 in association with the major mitotic kinase Cdk1 is exquisitely controlled
571
by its intracellular localization, with roles both in the cytoplasm and the nucleus (22). In the
572
infected cell, E4orf3 may promote aggregation of cyclin B1 (Fig. 7). When expressed by
573
transfection, E4orf3 may diminish nuclear levels of cyclin B1 (Fig. 8). E4orf3 may functionally
574
inactivate cyclin B1 to facilitate exit from mitosis. In this manner, E4orf3 may replace a cellular
575
process that was disabled by other adenovirus proteins in order to prevent the untoward
576
consequences of mitotic arrest.
27 577
A remaining question is, why are cells infected in early G1 predisposed to arrest in
578
mitosis after infection with the double-mutant virus? Cellular chromatin in the early G1 cell is
579
distinguished from that in other phases of the cell cycle by the need to reacquire specific
580
proteins. At late stages of mitosis, many proteins, including those that signal DNA-damage are
581
displaced from the condensing chromatin. Consequently G1 cells are able to respond to DNA-
582
damage only after these proteins re-associate with DNA (98, 99). The reacquisition of chromatin
583
proteins can also be regulated by proteins such as the histone variant H2A.Z, which suppresses
584
transcription during mitosis (100). The histone acetyl transferase Tip60 promotes H2A.Z-
585
mediated association of proteins at sites of DNA-damage. Perhaps the ability of the E1B-55K
586
protein to target Tip60 for destruction (54) renders cellular chromatin less able to signal DNA-
587
damage and thus unsuited for further progression through the cell cycle. During late mitosis and
588
early G1 chromatin is also reloaded with replication-critical licensing factors that were displaced
589
or degraded in S phase (101, 102). Early events during the adenovirus infectious cycle may
590
perturb these processes that occur during G1.
591
Studies using somatic cell nuclear transfer to generate cloned animal offspring
592
demonstrate how the unique nature of cells in early G1 can exert consequences over a very long
593
time. Donor cell nuclei derived from serum-starved G0 cells or early G1 cells were transferred
594
into enucleated bovine ova to create embryos. Although embryos derived from G0 nuclei showed
595
greater survival through the blastocyst stage, animals derived from G1 nuclei showed improved
596
birth weights and greater post-natal survival (103). Both the transfer of an early G1 nucleus and
597
the infection of a cell in early G1 have consequences evident much later in time.
598 599
While the properties of early G1 cells that predisposed them to mitotic arrest following infection with adenovirus are unknown, these cells have the potential to enter mitosis despite
28 600
suspension of normal cell cycle progression. Surprisingly, entry into mitosis by the virus-infected
601
cell requires p53, a protein better known for its ability to halt cell cycle progression. The
602
E1B-55K and E4orf3 proteins function independently of one another to prevent arrest in mitosis.
603
Although adenovirus directs the degradation of p53, it appears that a portion of the p53 protein is
604
repurposed by the E1B-55K protein to reinforce the G2/M checkpoint. Through the
605
reorganization of cyclin B1, E4orf3 protein may serve as a failsafe to facilitate exit from mitosis,
606
presumably circumventing cell death associated with prolonged mitotic arrest. ACKNOWLEDGEMENTS
607
Cell culture reagents were provided by the Cell and Viral Vector Core Laboratory,
608
supported by the Comprehensive Cancer Center of Wake Forest University NCI CCSG
609
P30CA012197 grant. Flow cytometry was performed in the Flow Cytometry Core Laboratory,
610
also supported by the Comprehensive Cancer Center of Wake Forest University NCI CCSG
611
P30CA012197 grant. R.L.T. was supported by the Training Program in Immunology and
612
Pathogenesis 5 T32 AI007401 from the National Institutes of Health. This research was
613
supported by Public Health Service grant R01 CA127621 (to D.A.O) from the National Cancer
614
Institute and Deutsche Forschungsgemeinschaft grant Do 343/7-1 (to T.D.)
615
The authors wish to acknowledge the helpful discussion of members of the Parks, Lyles
616
and Barton laboratories of Wake Forest University. We thank Guangchao Sui for kindly
617
providing a p53-expression construct. The content of this report is solely the responsibility of the
618
authors and does not necessarily represent the official views of the respective funding agencies.
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Shtrichman R, Kleinberger T. 1998. Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells. J. Virol. 72:2975-2982.
92.
Marcellus RC, Lavoie JN, Boivin D, Shore GC, Ketner G, Branton PE. 1998. The
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early region 4 orf4 protein of human adenovirus type 5 induces p53-independent cell death
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by apoptosis. J. Virol. 72:7144-7153.
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93.
Lavoie JN, Nguyen M, Marcellus RC, Branton PE, Shore GC. 1998. E4orf4, a novel
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adenovirus death factor that induces p53-independent apoptosis by a pathway that is not
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inhibited by zVAD-fmk. J. Cell Biol. 140:637-645.
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94.
Pechkovsky A, Lahav M, Bitman E, Salzberg A, Kleinberger T. 2013. E4orf4 induces
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PP2A- and Src-dependent cell death in Drosophila melanogaster and at the same time
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inhibits classic apoptosis pathways. Proc. Natl. Acad. Sci. U. S. A. 110:E1724-1733.
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95.
Araujo FD, Stracker TH, Carson CT, Lee DV, Weitzman MD. 2005. Adenovirus type
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5 E4orf3 protein targets the Mre11 complex to cytoplasmic aggresomes. J. Virol.
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79:11382-11391.
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Liu Y, Shevchenko A, Shevchenko A, Berk AJ. 2005. Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex. J. Virol. 79:14004-
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14016. 97.
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adenovirus E4 ORF3 protein is required for viral replication. J. Virol. 79:6207-6215. 98.
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Evans JD, Hearing P. 2005. Relocalization of the Mre11-Rad50-Nbs1 complex by the
Nelson G, Buhmann M, von Zglinicki T. 2009. DNA damage foci in mitosis are devoid of 53BP1. Cell Cycle 8:3379-3383.
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Giunta S, Jackson SP. 2011. Give me a break, but not in mitosis: the mitotic DNA
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damage response marks DNA double-strand breaks with early signaling events. Cell Cycle
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10:1215-1221.
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100. Kelly TK, Miranda TB, Liang G, Berman BP, Lin JC, Tanay A, Jones PA. 2010.
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H2A.Z maintenance during mitosis reveals nucleosome shifting on mitotically silenced
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genes. Mol. Cell 39:901-911.
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101. Tsuyama T, Tada S, Watanabe S, Seki M, Enomoto T. 2005. Licensing for DNA
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replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in
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Xenopus egg extracts. Nucleic Acids Res. 33:765-775.
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102. Symeonidou IE, Taraviras S, Lygerou Z. 2012. Control over DNA replication in time and space. FEBS Lett. 586:2803-2812. 103. Goto Y, Hirayama M, Takeda K, Tukamoto N, Sakata O, Kaeriyama H, Geshi M.
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2013. Effect of synchronization of donor cells in early G1-phase using shake-off method
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on developmental potential of somatic cell nuclear transfer embryos in cattle. Anim Sci J.
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104. Kindsmuller K, Schreiner S, Leinenkugel F, Groitl P, Kremmer E, Dobner T. 2009. A
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49-kilodalton isoform of the adenovirus type 5 early region 1B 55-kilodalton protein is
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sufficient to support virus replication. J. Virol. 83:9045-9056.
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105. Härtl B, Zeller T, Blanchette P, Kremmer E, Dobner T. 2008. Adenovirus type 5 early
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region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism
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independent from blocking p53-activated transcription. Oncogene 27:3673-3684.
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106. Teodoro JG, Halliday T, Whalen SG, Takayesu D, Graham FL, Branton PE. 1994.
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Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B
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protein regulates transforming activity. J. Virol. 68:776-786.
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107. Wimmer P, Blanchette P, Schreiner S, Ching W, Groitl P, Berscheminski J, Branton
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PE, Will H, Dobner T. 2013. Cross-talk between phosphorylation and SUMOylation
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regulates transforming activities of an adenoviral oncoprotein. Oncogene 32:1626-1637.
902 903
108. Koyuncu OO, Dobner T. 2009. Arginine methylation of human adenovirus type 5 L4 100-kilodalton protein is required for efficient virus production. J. Virol. 83:4778-4790.
42 FIGURE LEGENDS 904
FIGURE 1. Click-iT EdU labeling protocol and DNA profile analysis of EdU-labeled cells.
905
(A) HeLa cells were labeled with Click-iT EdU for six 4-h periods (indicated by arrows) over the
906
course of 24 h prior to infection. At time 0 h, EdU-labeled cultures were infected with the E1B-
907
55K/E4orf3 double-mutant virus at an MOI of 10 and processed for immunofluorescence at 72
908
hpi or were collected for flow cytometric analysis. Shading indicates the phase of the cell cycle
909
the EdU-labeled cells are expected to have reached at infection (S phase, gray; G2/M, black; G1,
910
white). (B) Samples of cells labeled with Click-iT EdU were collected at the time of infection (0
911
h) and stained with propidium iodide for DNA content and for EdU as described in the Materials
912
and Methods. The shaded density profile represents the DNA profile for the entire population of
913
cells. The unshaded density profile shows the DNA profile of EdU-positive cells.
914
FIGURE 2. Early G1 cells give rise to mitotic-like nuclei after infection with the E1B-
915
55K/E4orf3 double-mutant virus. Synchronously dividing HeLa cells were obtained by mitotic
916
shake and hydroxyurea selection as described in the Materials and Methods. Synchronously
917
dividing cells were either harvested for DNA profile analysis or infected at the indicated time
918
after entering S phase with the E1B-55K/E4orf3 double-mutant virus at an MOI of 10. (A) Cell
919
cycle distribution by DNA profile analysis. (B) At 72 hpi, cells were stained for DNA with DAPI
920
and evaluated by fluorescence microscopy to determine the frequency of cells with condensed
921
DNA. The stage of the cell cycle at the time of infection is indicated below. A representative
922
experiment is shown of three that were performed with overlapping times of infection after
923
entering S phase.
924
FIGURE 3. HeLa cells infected with the E1B-55K/E4orf3 double-mutant virus show
43 925
evidence of mitotic distress. HeLa cells were mock-infected or infected at an MOI of 10 with
926
the indicated viruses and stained at 72 h post infection with DAPI to visualize DNA (blue),
927
antibodies to phospho-H3 as a marker of early mitosis (red), and antibodies to β-tubulin to
928
visualize mitotic spindles (green). (A) Representative images of (a-b) mock, (c) wild-type, and
929
(d-e) E1B-55K/E4orf3-mutant viral infections are shown. (B) The frequency of cells containing
930
mitotic-like condensed DNA, staining for phospho-H3 and asymmetrically stained mitotic
931
spindles was quantified for at least 500 cells for each viral infection. A representative experiment
932
of three is shown. Error bars indicate the 95% exact binomial confidence interval for the
933
representative experiment. (C) HeLa cells were infected with the E1B 55K/E4orf3 double-
934
mutant virus at an MOI of 10. Cells were stained for DNA at the indicated times post infection
935
and the frequency of mitotic-like cells was determined for approximately 500 cells at each time
936
point. The broken symbols for times beyond 72 hpi indicate imprecision due to the probable loss
937
of cells because of death or detachment. The experiment shown is representative of three
938
independent experiments with similar outcomes. Error bars indicate the 95% exact binomial
939
confidence interval for the representative experiment.
940
FIGURE 4. Colcemid traps cells infected with the E1B-55K-mutant virus in a mitotic-like
941
state. HeLa cells were infected at an MOI of 10 with the indicated viruses and treated with
942
colcemid for 12 h at the indicated times post-infection before being fixed and stained with DAPI
943
to visualize DNA. The frequency of mitotic-like cells was determined for approximately 500
944
cells for each virus at each time point.
945
FIGURE 5. The ability to inhibit entry into a mitotic-like state maps to the p53-binding
946
ability of the E1B-55K protein. (A) HeLa cells were infected at an MOI of 10 with the
947
indicated E1B 55K-null viruses and treated with colcemid or vehicle control 12 h prior to
44 948
staining with DAPI at 72 hpi to visualize DNA. The frequency of mitotic-like cells was
949
determined by fluorescence microscopy. A representative experiment of three independent
950
experiments is shown. For each of the E1B-55K-null viruses, colcemid significantly increased
951
the fraction of mitotic-like cells (p-value < 10-6 by Fisher’s exact test). Error bars indicate the
952
95% exact binomial confidence interval for the representative experiment. (B) HeLa cells were
953
infected at an MOI of 10 with the indicated viruses bearing point mutations in the E1B-55K gene
954
(described in Table 2) and treated with colcemid 12 h prior to staining at 72 hpi with DAPI to
955
visualize DNA. The frequency of mitotic-like cells is shown for each mutant infection. The
956
proportion of mitotic-like nuclei was non-randomly distributed among the 11 virus-infected
957
samples exclusive of the E1B-55K-null virus H5pm4149 (p < 0.0001, Chi-squared test). The
958
Chi-squared test was repeated after systematically excluding each sample. The p-value was non-
959
significant (p = 0.56) only when the virus H5pm4109 was excluded from the analysis. This
960
analysis was again repeated by excluding individual samples in the collection that also excluded
961
H5pm4109. The Chi-squared test reported non-significant p-values for each subset lacking
962
H5pm4109 and one other sample. This indicates that among the non-null viruses, only
963
H5pm4109-infected cells exhibited a significant change in the number of condensed nuclei in the
964
presence of colcemid. Results from a representative experiment of three independent
965
experiments with similar outcomes are shown. (C) p53-null H1299 cells were transfected with a
966
plasmid to express p53 24 h before being mock-infected or infected at an MOI of 10 with either
967
the E1B-55K single-mutant or the E1B-55K/E4orf3 double-mutant virus. The cells were either
968
left untreated or treated with 0.2 μg per ml of colcemid 12 h prior to immunostaining for p53
969
and visualizing DNA with DAPI staining at 72 hpi. Cells were classified as either p53-positive or
970
negative and the frequency of mitotic-like cells was determined. As noted in the text, condensed
45 971
nuclei in mock-infected cells resembled pycnotic nuclei of apoptotic cells. The number of p53-
972
positive virus-infected cells with condensed DNA was increased significantly over p53-negative
973
cells (p = 2×10-6 by Fisher’s exact test).
974
FIGURE 6. Cyclin B1 levels are elevated during adenoviral infections. HeLa cells were
975
mock-infected or infected with the indicated viruses at an MOI of 10. Cellular lysates were
976
collected in the presence of protease and phosphatase inhibitors at 72 hpi. Material from identical
977
numbers of infected cells were separated by SDS-PAGE, transferred to a nitrocellulose
978
membrane and immunoblotted for (A) cyclin B1 and (B) β-actin. An overexposed β-actin blot is
979
presented here to permit visualization of the weaker signals. Non-saturated exposures were used
980
for quantitative analyses. The position of a cyclin B1-related product of 35 kDa is indicated by
981
the arrowhead. (C) The optical density of the signal for the intact cyclin B1 products was
982
quantified, normalized to β-actin, and then normalized to the value measured from mock-infected
983
cells in three independent experiments. The mean and standard deviation are plotted on a log-
984
scale. Application of the t-test to log-transformed values shows that levels of cyclin B1 in were
985
significantly greater than mock-infected cells in dl309- and dl1520-infected cells (p80%) cells expressing both
1015
fusion protein and E4orf3 as indicated in panels a and b.
1016
47
TABLES TABLE 1. Edu-positive and mitotic-like double-mutant virus-infected cells EdU-positive cells (%)b
Designationc
Mitotic-like Edu-positive cells (%)
a
Labeling Period
G1
S
G2/M
-2 to +2
27
52
21
mid-S
25.6
-6 to -2
6
15
79
G2/M
69.6
-10 to -6
35
7
58
M, early-G1
50.0
-14 to -10
68
5
27
mid G1
20.0
-18 to -14
61
25
14
late G1
0.0
-22 to -18
56
25
20
G1, early-S
5.8
a
Hours exposed to EdU before being infected at an MOI of 10 at 0 h.
b
Percentage of EdU-positive cells with DNA content characteristic of the indicated stage of the
cell cycle. c
Predominant stage of the cell cycle for EdU-positive cells at 0 h.
48 TABLE 2. E1B-55K mutant viruses and key characteristics Virus
E1B-55K mutation
Likely defect
Reference
dl110
deletion, frame shift
null
(42)
dl1520
stop codon, deletion
null
(39)
dl338
deletion
null
(41)
4149
four stop codons
null for all 55K-related
(104)
4100
none
none
(43)
4108
T255A
E4orf6-binding
(Identical mutation to virus described in 60)
4109
H260A
p53-binding, E4orf6binding
(Identical mutation to virus described in 60)
4127
C454S/C456S
Mre11 binding
(105)
4174
S490A/S491A/T495A
Non-phospho C-term
(10, 106, 107)
4185
P70T/S73A
Unknown (P70T/S73A)
(108)
4197
E472A
Daax interaction 1
(55)
4198
K185A/K187A
Daax interaction 2
(55)
4216
GVVI233-236AAAS
SUMO interaction
Not published
4217
V339A/V341A/I342S
SUMO interaction
Not published
4227
S490D/S491D/T494D
Phosphomimetic C-term
(57, 107)
